Human papillomavirus (HPV) testing is more sensitive than cytology for detecting cervical cancer and its precursors. This study aimed to analyze the prevalence of high-risk HPV genotypes and evaluate the role of HPV genotyping triage for detecting high-grade squamous intraepithelial lesions, adenocarcinoma in situ and cervical cancer (HSIL+) in HPV-positive women with normal cytology.A retrospective study was performed in women who had undergone co-screening at the China Medical University-affiliated Shengjing Hospital between 2012 and 2014.P = 0.30, P = 0.19, respectively). The odds ratios for histological HSIL+ were 3.26  in women with HPV16/18, 4.21 (95% CI: 2.99–5.93) in those with HPV16/18/31/33, and 5.73 (95% CI: 3.30–9.97) in those with HPV16/18/31/33/52/58. Including HPV31/33 genotyping together with HPV16/18 significantly increased the proportion of HSIL+ detection from 63.2 to 77.5% (P = 0.002) without significantly increasing the colposcopy per HSIL+ detection ratio .Of the 34,587 women, 2665 HPV-positive women with normal cytology who had received colposcopy were eligible for analysis. In HSIL+ groups of 204 women, the common genotypes were HPV16, HPV52, HPV58, HPV33, HPV31 and HPV18 in order of prevalence. The proportion of histological HSIL+ in women infected with HPV33 or HPV31 was not significantly different compared to women infected with HPV16 (HPV genotyping played an important role in managing HPV-positive women with normal cytology. Genotyping for HPV31/33 should be added to the previously recommended HPV16/18 genotyping in triaging HPV-positive women in northeastern China. Cervical cancer is the fourth most common cancer among women worldwide and the leading cause of death from cancer in developing countries . ApproxiRecently, HPV genotyping has been accepted in preference to cytology for detecting cervical cancer and its precursors, due to its higher sensitivity . Based oIntroducing HPV testing for cervical cancer screening is becoming increasingly popular in China. Co-screening, HPV primary screening or cytology primary screening has been used in different districts of China due to unbalanced economic development. It is uncertain which combinations of hrHPV genotyping could provide the optimal triage of HPV-positive women with normal cytology in clinical practice in northeastern China. To address these concerns, a retrospective study was conducted to evaluate the prevalence of HPV, and the association between hrHPV genotypes and the detection of histological HSIL+ in northeastern China. Furthermore, an acceptable triage strategy to reduce the burden of cytological examination and increase the proportion of detecting histological HSIL+ was investigated.We recruited women between 25 and 65 years old who underwent co-screening for cervical cancer when visiting outpatient of the Department of Obstetrics and Gynecology at the China Medical University-affiliated Shengjing Hospital between January 1st 2012 and December 31st 2014. The clinical characteristics and pathological data were obtained from the hospital’s electronic files, including age at diagnosis, cytology results, HPV genotyping results, colposcopy results and histological results. Exclusion criteria for the present study were pregnant women; women who had a hysterectomy; women with a history of cervical cancer; and women who had received previous treatment for any cervical epithelial lesion. The study was approved by the ethics committee of Liaoning Cancer Hospital and Institute (20190971).Cytological testing was performed using ThinPrep® liquid-based cytology . Cell samples were collected using a cytobrush and placed into a tube with transport medium. The first sample from each woman was used for cytology analysis. The second sample was taken for HPV genotyping testing. Slides were screened by two cytotechnologists and diagnosed according to the 2001 Bethesda system.HPV genotyping was identified by the HPV GenoArray test kit . This assay was performed using both DNA amplification by the L1 consensus primer-based polymerase chain reaction (PCR) and a flow-through hybridization technique. A total of 21 genotypes were screened, including13 high-risk genotypes , two probable high-risk genotypes (HPV53 and HPV66) and six low-risk genotypes  . PositivAll women with abnormal cytology or those who tested positive for HPV were referred for colposcopy. A colposcopy-guided biopsy was performed if a suspicious lesion was found. Random cervical biopsy was carried out when colposcopic inspection was inadequate. If colposcopy results were normal upon adequate inspection, women were followed up yearly without biopsy. The grade of the cervical lesion was independently diagnosed by two expert pathologists, according to the 4th edition of the WHO Women’s Reproductive System Tumor Classification . Histolo2) tests were used to compare differences of the proportions of histological HSIL+ in hrHPV positive women with normal cytology who received colposcopy, across all four age groups, and among hrHPV genotypes, as well as between each pairing of two genotypes. Logistic regression analysis, which was adjusted for age, was used to estimate the association between specific hrHPV genotypes and the proportion of detecting histological HSIL+. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Data were analyzed using the SPSS version 22.0 software . A P value of < 0.05 was considered statistically significant.The prevalence of HPV was expressed as a proportion of the number of HPV-positive cases compared to the total number of cases tested for HPV. Multiple HPV infections were defined as those positive for two or more types of HPV. Women with multiple HPV infections were counted more than once for each positive genotype. The prevalence of specific HPV types is presented for the 13 hrHPV types and two probable hrHPV types in women with hrHPV infection, as well as hrHPV positive women with normal cytology and histological HSIL+ women who received colposcopy with hrHPV positivity and normal cytology. Chi-squared  had hrHPV infection and 1839 (5.3%) had abnormal cytology results  women with normal cytology were analyzed in the present study. The mean age of these women was 39.60 ± 8.99 years old, with a median age of 40. The top six hrHPV genotypes were HPV16 , HPV58 , HPV52 , HPV53 , HPV18  and HPV33 . Regarding each hrHPV genotype, the proportion of women with normal cytology was higher in those infected with HPV59 (76.3%), HPV45 (75.8%), HPV53 (75.4%), HPV18 (74.4%) and HPV51 (70.65%) than other hrHPV genotypes  women. A total of 204 women  had HSIL+; 173 women  had low-grade squamous intraepithelial lesions; and 2288 women  had negative histology results or normal colposcopy results. Of the 204 women with HSIL+, 26 women  had ICC , including 14 women  who were FIGO stage IA. In the HSIL+ group, the prevalence of the hrHPV genotypes was HPV16 , HPV52 , HPV58 , HPV33 , HPV31 and HPV18 . The percentage of women infected with multiple hrHPV genotypes was 19.1% (39/204) in the HSIL+ group. In the squamous cervical cancer group, the prevalence of HPV16  was prominent. Of the seven cases of adenocarcinoma in situ and adenocarcinoma, five cases were HPV18-positive and two cases were HPV16-positive, including one case of dual infection with HPV18 and HPV33 (Table P = 0.42). Of the 801 HPV16-positive women with normal cytology who received colposcopy, 119  women were confirmed as HSIL+ by histological diagnosis. The proportion of histological HSIL+ in each genotype was greater than10% for HPV16 (14.9%), HPV33  and HPV31 . The proportion of histological HSIL+ in each genotype was less than 10% for HPV52 , HPV58 , HPV18  and other types. Compared to women infected with HPV16, the proportion of histological HSIL+ was not significantly different in women infected with HPV33 or HPV31 . While HPV58, HPV52, HPV53 and HPV18 were four of the top six most common genotypes in HPV-positive women with normal cytology, the proportion of histological HSIL+ in each of these four HPV genotypes was significantly lower than those in women infected with HPV16 . The proportion of histological HSIL+ in women infected with HPV33 was significantly higher compared to those infected with HPV18 or HPV58 . The differences between the proportion of histological HSIL+ in women infected with HPV18 and those infected with the HPV31, HPV52 or HPV58 were not statistically significant  (Table P = 0.03)  for histological HSIL+ was 3.75 (95% CI = 2.79–5.05) in women with HPV16 infection, compared to women with non-HPV16 infection. In women infected with HPV33, the OR for histological HSIL+ was 1.69 (95% CI = 1.04–2.72). Infection with HPV genotypes 18, 31, 52 or 58 did not increase the risk of HSIL+ . The OR for histological HSIL+ was 3.26 (95% CI = 2.41–4.40) in women with HPV16/18 infection. The OR for histological HSIL+ was 4.21 (95% CI = 2.99–5.93) in women infected with HPV16/18/31/33. The OR for histological HSIL+ was 5.73 (95% CI = 3.30–9.97) in women infected with HPV16/18/31/33/52/58 (Table P = 0.01). Adding the HPV31/33 genotype to the HPV16/18 genotype increased the percentage of HSIL+ detection from 63.2 to 77.5% (P = 0.002) without significantly increasing the colposcopy per HSIL+ detection ratio . (Table HPV16/18 infection was detected in 129 of 204 63.2%) women with histological HSIL+; by contrast, the top six hrHPV genotypes (HPV16/18/31/33/52/58) in the HSIL+ group were detected in 190  women. However, the colposcopy per HSIL+ detection ratio also increased significantly from 7.7 to 10.2  obtained in the present study was lower than that reported in many Chinese cities ; howeverThe oncogenic potential varies with different hrHPV genotypes. A population-based study showed that HPV16, 58, 18, 52 and 33 were most common in persistent infection . AnotherHPV genotyping will enable more precise characterization of cervical disease risk, but genotyping for only HPV16/18 is not sufficient. Although the prevalence and risk of HSIL+ in women with HPV18 didn’t rank high among non-HPV16 types, HPV18 was one of the most common genotypes in adenoepithelial lesions. In the present study, HPV16/18 was positive in 63.2% of women with histological HSIL+. The addition of HPV31/33 genotyping to that of HPV16/18 could detect 14.3% more women with histological HSIL+. The OR for histological HSIL+ in women infected with HPV16/18/31/33 was higher than that in women infected with HPV16/18 (4.21 vs. 3.26). The addition of HPV31/33/52/58 genotyping to that of HPV16/18 could detect 93.1% of histological HSIL+ in the present study. The OR for histological HSIL+ was 5.73 in women infected with HPV16/18/31/33/52/58. A previous population-based, prospective, observational study suggested that HPV16/18/31/33/52/58 infection should be immediately referred for colposcopy . HoweverIn summary, wider hrHPV genotyping provides a better predictive value than HPV16/18 genotyping alone in guiding the clinical management of current cervical cancer screening. In northeastern China, the addition of HPV31/33 genotyping to that of HPV16/18 should be recommended in triaging women with a positive HPV test.


Moreover, limited evidence is available on the contributory role of PM2.5 exposure on the arterial stiffness index.Prior studies have investigated the association of PM2.5 for 35 air pollutant monitoring sites in Beijing from 2014 to 2018 was used to estimate individual exposure by different interpolation methods. Multivariate logistic regression and linear regression were conducted to assess the association of annual average PM2.5 concentration with the incidence of higher baPWV, the progression of ABI, and baPWV, respectively.We used the population data from the Beijing Health Management Cohort and conducted a longitudinal analysis. The annual average concentration of PM2.5 exposure and incidence of higher baPWV was not significant . There was − 0.16% (95% CI: − 0.43-0.11%) decrease in ABI annually and 1.04% (95% CI: 0.72–1.37%) increase in baPWV annually with each increment of 10 μg/m3 average PM2.5 concentration.The association between PM2.5 was associated with the progression of arterial stiffness in Beijing. This study suggests that improvement of air quality may help to prevent arterial stiffness.Long-term exposure to PMThe online version contains supplementary material available at 10.1186/s12940-020-00688-6. Fine padiseases . It has ortality . The potttention .2.5 exposure with baPWV and ABI [2.5 exposure and ABI [Increased arterial stiffness, a measure of vascular dysfunction, is highly relevant to the atherosclerosis process and is also seen as an independent risk factor for cardiovascular events . Ankle-b and ABI –15, of w and ABI , which w and ABI , 16.2.5 pollution in China. The nonlinear relationship between air pollution and health effects may indicate that evidence from other regions may not be suitable to the population in areas with high air pollution in Beijing, China. Moreover, it was reported that racial differences might exist in air pollutants’ cardiovascular effect [Furthermore, few were conducted to investigate the long-term effects in areas with relatively more severe PMr effect .2.5 exposure on the change of arterial stiffness markers, including ABI and baPWV.Therefore, in this study, we used the population data from the Beijing Health Management Cohort (BHMC) and conducted a longitudinal analysis to investigate the long-term effects of PM2.5 exposure on the progression of ABI and baPWV, we retrieved data of participants who underwent at least two examinations of ABI and baPWV (n = 4789) during 2014–2018. The first and last measurements were used. Then those participants with abnormal ABI (ABI < 0.9 or ABI > 1.4) or baPWV (baPWV > 1800 cm/s) at first measurement were excluded (n = 838). Finally, 3951 participants were included in the study. All participants provided written informed consent. This study was approved by the Ethics Committees of Capital Medical University .We conducted this study using data from the BHMC, a large dynamic cohort aiming fixed working environment population in Beijing, of which design has been described in previous studies . ParticiABI and baPWV were measured using an automatic arteriosclerosis analyzer. The ABI is measured as the ratio of systolic blood pressure at the ankle to that at the brachial artery. ABIs were calculated separately for both sides by the highest pressure of the ankle on the side divided by the highest brachial pressure on either side, and the minimum was used for analyses. According to previous studies –21, lowe2.5 concentration data from 2014 to 2018 recorded by 35 monitoring sites from the Beijing Municipal Environmental Monitoring Center’s website (http://zx.bjmemc.com.cn/). The annual average concentration in each monitoring site was calculated. The empirical Bayesian Kriging method was applied to assess individual exposure based on the workplace address using ArcGIS 10.5 software [2.5 data recorded by monitors were not available before 2014, pollution data from 2012 to 2013 was gained from PM2.5 Hindcast Database. PM2.5 Hindcast Database [2.5 in a regular grid of 0.1° × 0.1°, developed and maintained by Tsinghua university based on Moderate Resolution Imaging Spectroradiometer (MODIS) satellite aerosol datasets. In addition, we also used the Inverse Distance Weighting (IDW) interpolation method and data from the nearest monitoring site to estimate individual exposure in the sensitivity analysis. According to prior studies [3) from the baseline year to the last visit year was used as the estimated exposure. Because PM2.5 data from 2014 to 2016 were also available in PM2.5 Hindcast Database, we could conduct linear regression to test the correlation between data from PM2.5 Hindcast Database and those calculated based on records from monitoring sites during 2014 to 2016. The results indicated that data in PM2.5 Hindcast Database were strongly correlated with the estimates of the IDW interpolation method  was calculated according to the formula: t test, Wilcoxon rank test, or χ2 test, as appropriate. Because the number of participants with abnormal ABI incidence was too small , we did not investigate the association of annual average PM2.5 concentration with abnormal ABI incidence. We performed logistic regression to assess the association between annual average PM2.5 concentration and the incidence of high baPWV. The odds ratio (OR) and its 95% Confident interval (CI) were calculated. Then restricted cubic spline was used to depict the dose-response relationship and test whether the nonlinear association exists. Multivariate linear regression was conducted to investigate the relationship of annual average PM2.5 concentration with the progression of ABI and baPWV. We used a step-by-step method to adjust confounding factors in the multivariate models, where the various number of participants were included due to missingness of covariates: (1) crude model (n = 3951); (2) model controlling for age and gender (n = 3951); (3a) model additionally adjusting for BMI, HDL-C, LDL-C, TG, systolic BP, and diabetes (n = 3684); (3b) model adjusting for the same set of covariates with model 3a in the same population with model 4 (n = 1128); (4) model additionally controlling for education, smoking, alcohol consumption and physical activity (n = 1128). Model 4 worked as our final model, and its results were mainly reported.Characteristics of participants were described with the mean  for continuous variables with normal distribution or median  for those with skewed distribution and number (percentage) for categorical variables. Baseline characteristics were compared with 2.5 concentration. To test whether the results were robust, we conducted several sensitivity analyses. Multivariate logistic and linear regression was repeatedly conducted with different methods to assess exposure  and different cut-off values to define higher baPWV [We also conducted subgroup analyses by hypertension status and age group  to see which group is more sensitive to an increase in PMer baPWV , 28 . Two-tailed 2, which fell within the overweight range.The baseline characteristics of study participants are presented in Table 2.5 exposure on the incidence of higher baPWV is depicted in Fig. 2.5 was associated with incidence of higher baPWV . After adjusting age and gender (model 2), participants had a 22% higher risk  for each increment of 10 μg/m3 annual average PM2.5 concentration. The association was still significant in model 3a after controlling for BMI, HDL-C, LDL-C, TG, systolic BP, and diabetes. However, this association was not significant  after further adjusting for education, cigarette smoking, alcohol consumption, and physical activity (model 4). The association was not significant (P = 0.722) in model 3b, which was conducted adjusting for the same set of covariates with model 3a but in the same population as model 4. Besides, we noted a small effect estimate reduction from model 4 to models 3b, but not as large as model 3a, which indicated that model 4 might be mainly influenced by reduced sample size.The effect of PM2.5 concentration with the risk of higher baPWV in the analysis using restricted cubic splines . There was a significant rise in risk with increasing annual average PM2.5 concentrations until the value approached approximately 60 μg/m3.A nonlinear curve was observed to associate the annual average PM2.5 exposure with a relative annual change of ABI and baPWV are presented in Table 2.5 concentration and ABI progression was significantly negative in the crude model (model 1) but turned null after adjusting potential confounding factors (model 2-model 4). However, the effect of annual average PM2.5 concentration on relative annual change of baPWV was always significant from model 1 to model 4. There was a 1.04% (95% CI: 0.72–1.37%) gain in baPWV annually with each increment of 10 μg/m3 average PM2.5 concentration in the full adjusted model.Associations of PM2.5 exposure was marginally significant (P = 0.056) among participants younger than 55 years. Moreover, per 10 μg/m3 elevated PM2.5 concentration was associated with a 1.40% (95% CI: 0.94–1.86%) increase in baPWV annually among younger participants. However, this relationship was not observed among older individuals. Several sensitivity analyses were conducted to show that the results were robust (Supplementary Table S2.5 exposure. It was worth noting that the association turned significant (P = 0.037) with 1700 cm/s as the threshold.Subgroup analyses by age group (age≤55 and age > 55) showed interesting results ; since enrolled participants in our study were organized by their companies. This might reduce the power of the statistical test. Third, pollution data sources were inconsistent from 2012 to 2018 because PM2.5 pollution data recorded by monitors were not available before 2014. Forth, the potential short-term effect of PM2.5 exposure on arterial stiffness was not adjusted in our analysis.According to our knowledge, this is the first study investigating the long-term effects of PM2.5 with baPWV annual change rate, but no association between PM2.5 and ABI annual change rate was found. This study suggests that improvement of air quality may help to prevent arterial stiffness.In conclusion, there is a significantly positive association of PMAdditional file 1: Table S1. Correlation between data from PM2.5 Hindcast Database and those calculated based on records from monitoring sites from 2014 to 2016. Table S2. Subgroup analysis for the association between increased 10 μg/m3 PM2.5 concentration and incidence of higher baPWV. Table S3. Subgroup analysis for the association between increased 10 μg/m3 PM2.5 concentration and relative annual change of ABI and baPWV. Table S4. Sensitivity analysis for the association between increased 10 μg/m3 PM2.5 concentration and relative annual change of ABI and baPWV. Fig. S1. Sensitivity analysis for the association between increased 10 μg/m3 PM2.5 concentration and incidence of higher baPWV. Odds ratios indicated by black boxes were shown along with 95% confident intervals.


Congenital heart disease (CHD) is resulted from the interaction of genetic aberration and environmental factors. Imprinted genes, which are regulated by epigenetic modifications, are essential for the normal embryonic development. However, the role of imprinted genes in the etiology of CHD remains unclear.After the samples were treated with bisulfate salt, imprinted genes methylation were measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. T test and One-way ANOVA were performed to evaluate the differences among groups. Odds ratios (ORs) were performed to evaluate the incidence risk of CHD in relation to methylation levels.GRB10 and MEST and 6 genes with hypomethylation of PEG10, NAP1L5, INPP5F, PLAGL1, NESP and MEG3. Stratified analysis showed that the methylation degree of imprinted genes was different in different types of CHD. Risk analysis showed that 6 imprinted genes, except MEST and NAP1L5, within a specific methylation level range were the risk factors for CHDWe investigated the alterations of imprinted gene germline differential methylation regions (gDMRs) methylation in patients with CHD. Eighteen imprinted genes that are known to affect early embryonic development were selected and the methylation modification genes were detected by massarray in 27 CHD children and 28 healthy children. Altered gDMR methylation level of 8 imprinted genes was found, including 2 imprinted genes with hypermethylation of Altered methylation of imprinted genes is associated with CHD and varies in different types of CHD. Further experiments are warranted to identify the methylation characteristics of imprinted genes in different types of CHD and clarify the etiologies of imprinted genes in CHD. Congenital heart disease (CHD) is the most common type of congenital malformations. The etiology for CHD is complex, due to the interaction of genetic aberration and environmental factors, profoundly influenced by conditions in fetal life . DespiteCITE2, ZIC3, NR2F2 and BRG1 is associated with abnormal methylation [Among the different epigenetic mechanisms, the potential relationship between abnormal DNA methylation and CHD has been increasingly recognized. DNA methylation has been found to be highly dynamic with the feature of demethylation in cardiomyocyte-associated gene sets during cardiomyocyte development . Studieshylation –9.DNA methylation has a major influence on the establishment of imprinting markers . SeveralIGF2 and GRB10 and co-regulated genes of PEG1/MEST, GTL2/MEG3, CDKN1C, PLAGL1 and DLK1 regulates embryonic growth and development by forming a proposed "imprinted gene network" [DLK1-DIO3, encoded ncRNAs, which participate in the commitment of the mesoderm to different subsets of a specific cardiac cell lineage through many DLK1-DIO3 ncRNAs [In placental mammals, the imprinted genes regulate embryonic development. At present, about 94 human imprinted genes have been identified . It has network" . The mos3 ncRNAs . HoweverTherefore, we investigated the alterations of imprinted gene methylation in patients with CHD through methylation modification. We selected 18 imprinted genes that are known to play an important role in early embryonic developments .The study was approved by the Ethics Committee of the Capital Institute of Pediatrics. Written informed consent was obtained from parents of the children. These samples were obtained from children with CHD who visited the Capital Institute of Pediatrics during the biennium 2014–2015, and were diagnosed as CHD by echocardiography and other related tests, ranging in age from 1 to 120 months. According to the International Classification Standard of Diseases, the diagnosis of existing cases was strictly classified, and a complete database of CHD research is established. Moreover, the children with CHD were excluded from other congenital and acquired abnormal diseases, such as congenital mental retardation, genetic metabolic diseases, etc. As control group, 28 controls of blood samples were selected from the physical examination children. They were same age and race and had no developmental deformities or other diseases. Peripheral blood samples were collected from 27 children with CHD and 28 healthy children as controls in Capital Institute of Pediatrics Table . In detaGenomic DNA was extracted from peripheral blood with the Blood and Tissue DNA Kit  according to manufacturer’s instructions. The concentration and purity of the DNA were determined by nanodrop and agarose gel electrophoresis. DNA with an OD260/280 absorbance ratio of 1.8 was used for subsequent work  according to the manufacturer’s instruction. The quality of the bisulfite conversion was controlled by using PCR products that had no methyl group. Sequencing results confirmed that 96.6% of cytosine residues were converted in previous research .http://epidesigner.com/). An additional T7 promoter tag was added to each reverse primer for in vivo transcription, and a 10-mer tag to the forward primer to balance the melting temperature.Eighteen imprinted genes were selected according to WAMZDEX, Gene imprint, Catalogue of Imprinted Genes and other imprinted gene analysis resources  was performed to evaluate the incidence risk of CHD in relation to methylation levels. P < 0.05 was considered significant. GraphPad Prism 7 software  was used to visually display the results of analysis. Data were stored in the EPI 3.1 Database  and analyzed with the SPSS 18.0 software package .GRB10 and MEST, in which GRB10 increased from 43.42% in the normal group to 51.12% (P < 0.01) in the CHD group and MEST increased from 53.22 to 56.6% (P < 0.05). The 6 hypomethylation-imprinted genes were PEG10, NAP1L5, INPP5F, PLAGL1, NESP and MEG3, in which PEG10 decreased from 50.92% in control to 45.17% in CHD group (P < 0.01), NAP1L5 decreased from 68.86 to 62.12% (P < 0.01), INPP5F decreased from 73.17 to 67.02% (P < 0.01), PLAGL1 decreased from 42.80 to 40.97%,(P < 0.05), NESP decreased from 41.12 to 31.31% (P < 0.01), and MEG3 decreased from 45.31 to 39.53%(P < 0.01)  were hypermethylated  hypomethylation  were hypomethylated in all four CHD classifications. NESP hypomethylation occurred in all CHDs except ToF, while GRB10 hypermethylation occurred in all CHDs except valvular malformation.CHD was classified into four groups including atrial and ventricular septal defect (ASD and VSD), valvular malformation , thoracic vascular malformation  and Tetralogy of Fallot (ToF). In ASD and VSD patients, 9 genes showed methylation changes, as compared with control group, whereby 2 imprinted genes . When the methylation levels of PEG10, INPP5F, PLAGL1 and MEG3 were lower than 45.2%, 70.39%, 40.76% and 41.83%, respectively, the risk of CHD increased by 16.8(P < 0.05), 11.06(P < 0.01), 11(P < 0.05) and 12(P < 0.01)times. Due to the small sample size, confidence intervals were wide  in patients with CHD. The methylations of imprinted genes varied with the heterogeneity of CHDs. Six of them were associated with significantly higher risk of CHD except MEST and NAP1L5.It is well known that the epigenetic regulation of imprinted genes is involved in many embryonic developmental processes, including cardiac development , 18. At The etiology of CHD is complex, involving the interaction of environmental and genetic factors, leading to developmental phenotypes that regulate morbidity and severity. The potential relationship between alterations of genomic methylation and CHD is increasingly recognized. The dynamic changes of DNA methylation in cardiomyocyte-related gene sets during cardiac development suggest that DNA methylation modification is essential for the occurrence of cardiac diseases , 4. One GRB10 and MEST as the core molecules [GRB10, as an important growth-limiting factor, has a wide range of effects on embryonic development. Maternal GRB10 knockout results in embryonic overgrowth, and the change of ICR of GRB10 gene results in obvious dysplasia [GRB10 in fetal samples of spontaneous abortion [Ddc gene in mice, involved in the development of trabecular cardiomyocytes of the embryonic and neonatal heart, displays tight conserved linkage with the methylated GRB10 gene [GRB10 may be involved in the development of heart through the interaction with Ddc. Hypermethylation of GRB10 might inhibit its expression and regulate the growth through insulin pathway such as IGF2, potentially leading to cardiac dysplasia. MEST is a maternal imprinted gene and widely expressed throughout the embryonic period [Mest-knockout mice exhibited embryonic and placental growth retardation and postnatal growth inhibition, while loss of the imprinted gene resulted in postnatal weight gain and multiple organ hypertrophy [Mest specifically expressed myocardial trabeculae in developing atria and ventricles of mice. Mest-knockout mice showed subtle changes of myocardial trabeculae, which displayed an increase in thickness and reduction in density of the compact myocardium, similar to that in human heart disease, which hinted MEST is closely related to heart disease [MEST imprinted gene in CHD was also found in our data, especially in ASD and VSD. Potentially, MEST expression disorder via altered methylation of the gene might be involved in the occurrence of CHD by affecting the development of myocardial trabecula.Some imprinted genes have key functions influencing the proper embryonic growth and development by forming "imprinted gene network", including olecules . GRB10, ysplasia , 22. Preabortion . It has B10 gene . In the c period . Mest-knertrophy , 27. Kin disease . The incPEG10, a paternally expressed imprinted gene, is expressed in embryonic tissues and placenta, and participates in cell proliferation, differentiation and apoptosis [Peg10-knockout mice exhibited early embryo death and placental defects [PEG10 in the heart was very low, indicating that trace PEG10 could maintain the normal development of the heart [PEG10 in CHD. Overexpression of PEG10 may be involved in the invasion and metastasis of malignant tumors, such as hepatocellular carcinoma and endometrial cancer etc., through the epithelial mesenchymal transition (EMT) [PEG10 might cause abnormal elevation of expression, and induce abnormal migration of related cells through EMT pathway, potentially leading to abnormal cardiac development.poptosis , 30. The defects . A studyhe heart . On the on (EMT) , 33. TheINPP5F encodes inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase, which is an important functional endogenous regulator. It has been found that INPP5F regulated the size of cardiac myocytes and cardiac stress response, increased the hypertrophy and activation of the fetal gene program in the stress response in Inpp5f-knockout mice, and decreased the hypertrophic response in Inpp5f-overexpressed mice [INPP5F can be used as a negative feedback regulator of insulin signal and downregulation of INPP5F in diabetes mellitus has cardioprotective effect [sed mice , 35. INPe effect . ConsistMEG3 is a maternal-expressed imprinted gene that is expressed in many normal tissues. It inhibits the proliferation of tumor cells in vitro. It also interacts with tumor suppressor p53 to regulate the expression of p53 target genes [MEG3 has an impact on cardiac remodeling induced by pathological hypertrophy by regulating the binding of p53 to other gene promoters [MEG3 participates in the development of myocardial fibrosis and prevents myocardial remodeling by regulating the production of MMP-2 by CFs in vitro and in vivo [MEG3 was reduced and associated with the increased risk of CHD.et genes . Consistromoters . Other s in vivo . In consNAP1L5, PLAGL1 and NESP in CHD. Stratified analysis revealed that different CHDs showed specific methylation patterns, such as hypomethylation of the 3 genes in ASD and VSD, hypomethylation of NAP1L5 and NESP in valvular malformation, hypomethylation of NESP in thoracic vascular malformation, and no significant change in ToF.We also detected hypomethylation of gDMRs of other 3 imprinted genes including As a pilot study, it is subject to a couple of limitations. First, we studied a small and heterogeneous group of patients with CHD. A large sample size is warranted to identify the methylation characteristics and risk factors of imprinted genes in relation to the types of CHD. Second, the causal relationship between methylation alterations of imprinted genes and CHD remains to be explored in further experimental studies to examine their potential impact on RNA expression and CHD development on cellular and organ levels in animal models.Methylation of gDMRs of 8 imprinted genes was altered in white blood cells in patients with CHD. Further experiments are warranted to identify the methylation characteristics of imprinted genes in different types of CHD and clarify the pathogenesis mechanism of imprinted genes in CHD.Additional file 1: Fig. S1. Analysis of methylation level of GRB10. a The mean methylation of gDMR of GRB10 between CHD group and control group. b Methylation level of specific CpG site in GRB10 between CHD group and control group. CpG sites included numbered 1–4 from the 5′ end to the 3′ end. *P < 0.05; **P < 0.01.Additional file 2: Fig. S2. Analysis of methylation level of PEG10. a The mean methylation of gDMR of PEG10 between CHD group and control group. b Methylation level of specific CpG site in PEG10 between CHD group and control group. CpG sites included numbered 1–12 from the 5′ end to the 3′ end. *P < 0.05; **P < 0.01.Additional file 3: Fig. S3. Analysis of methylation level of MEST. a The mean methylation of gDMR of MEST between CHD group and control group. b Methylation level of specific CpG site in MEST between CHD group and control group. CpG sites included numbered 1–26 from the 5′ end to the 3′ end. *P < 0.05; **P < 0.01.Additional file 4: Fig. S4. Analysis of methylation level of NAP1L5. a The mean methylation of gDMR of NAP1L5 between CHD group and control group. b Methylation level of specific CpG site in NAP1L5 between CHD group and control group. CpG sites included numbered 1–7 from the 5′ end to the 3′ end. *P < 0.05; **P < 0.01.Additional file 5: Fig. S5. Analysis of methylation level of INPP5F. a The mean methylation of gDMR of INPP5F between CHD group and control group. b Methylation level of specific CpG site in INPP5F between CHD group and control group. CpG sites included numbered 1–33 from the 5′ end to the 3′ end. *P < 0.05; **P < 0.01.Additional file 6: Fig. S6. Analysis of methylation level of PLAGL1. a The mean methylation of gDMR of PLAGL1 between CHD group and control group. b Methylation level of specific CpG site in PLAGL1 between CHD group and control group. CpG sites included numbered 1–30 from the 5′ end to the 3′ end. *P < 0.05; **P < 0.01.Additional file 7: Fig. S7. Analysis of methylation level of NESP. a The mean methylation of gDMR of NESP between CHD group and control group. b Methylation level of specific CpG site in NESP between CHD group and control group. CpG sites included numbered 1–22 from the 5′ end to the 3′ end. *P < 0.05; **P < 0.01.Additional file 8: Fig. S8. Analysis of methylation level of MEG3. a The mean methylation of gDMR of MEG3 between CHD group and control group. b Methylation level of specific CpG site in MEG3 between CHD group and control group. CpG sites included numbered 1–7 from the 5′ end to the 3′ end. *P < 0.05; **P < 0.01.Additional file 9: Fig. S9. Abnormal methylation level of the imprinted genes between multiple CHDs classification and control groups. a Abnormal methylation level of the imprinted genes between AVSD and control groups. b Abnormal methylation level of the imprinted genes between VHD and control groups. c Abnormal methylation level of the imprinted genes between ToF and control groups. d Abnormal methylation level of the imprinted genes between VM and control groups. AVSD, atrioventricular septal defect. *P < 0.05; **P < 0.01.Additional file 10: Table S1. gDMR sequence information of 18 imprinted genes.Additional file 11: Table S2. Primer sequence information of 18 imprinted genes.Additional file 12: Table S3. The average methylation levels of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 13: Table S4. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 14: Table S5. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 15: Table S6. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 16: Table S7. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 17: Table S8. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 18: Table S9. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 19: Table S10. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 20: Table S11. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 21: Table S12. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 22: Table S13. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 23: Table S14. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 24: Table S15. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 25: Table S16. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 26: Table S17. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 27: Table S18. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 28: Table S19. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 29: Table S20. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.Additional file 30: Table S21. CpG sites methylation level of 18 imprinted genes detected in CHD patients and healthy individuals.


The Gram-positive bacteria are the leading cause of sepsis in both groups: S. epidermidis (35.5%) in pediatric patients and S. aureus (21.7%) in adults. However, statistical significance was detected in pediatric patients (p < 0.05). The number of resistant strains of S. epidermidis (MRSE) in the group of children was 66.7%, while in adults, all S. epidermidis was resistant to azithromycin and cefoxitin (MRSE). S. aureus strains from adult patients and children had a similar picture of antibiotic patterns. The proportion of MRSA in pediatric patients was 16, 6%, and in adult patients, 20%. Enterobacterales (39%) were the second cause of sepsis in adult patients. 62.5% of Enterobacterales strains isolated from adults were phenotypically identified as ESBL, while in pediatric patients, 25% of ESBL producers were isolated. We have noted the resistance to antibiotics that are prescribed according to protocols of treatment of the Republic of Kazakhstan in the strains isolated from the patient's blood.Every year in the world, sepsis occurs in 31.5 million people, and the number of deaths reaches 5.3 million per year. There are not enough studies that describe etiological structure of sepsis pathogens in different groups of population of the Republic of Kazakhstan. In this study, we have investigated difference of local sepsis etiology and antibiotic susceptibility among children and adults. A total 200 blood samples were examined using the standard and express method of identification of bloodstream pathogens. The determination of antimicrobial sensitivity was carried out by the disc-diffusion method according to CLSI guidelines. Overall, 23/90 (25.5%) positive blood cultures were isolated from adult patients and 43/110 (39%) from pediatric patients. It was found that children are statistically more often affected with bacterial bloodstream infection than adults ( Sepsis is a life-threatening organ dysfunction which occurs as a result of a body's response to infection causes . Every yThe primary foci of bloodstream infections are more often the lower respiratory tract in general , the resEscherichia coli and Klebsiella spp.) and Enterococcus spp. [E. coli was the cause of early sepsis in newborns in 45% of cases, followed by group B Streptococcus (36%) [S. aureus and E. faecalis (13%) [Previous studies have found that the leading role in the bloodstream infections in adults belongs to microorganisms of the Enterobacterales family (cus spp. . If we tus (36%) , and a sus (36%) , 10. Othis (13%) . In studis (13%) .There are not much data on sepsis in the countries of the former Soviet Union: the epidemiology of sepsis in Russia can be judged from the Rudnov et al. multicenter study , where tOver the past 10 years, the advent of new technologies in microbiology has improved the diagnosis of septic conditions. The use of the direct detection method, using matrix laser desorption ionization time-of-flight mass spectrometry-MALDI-TOF, has become available for many laboratories. The simplicity of the preparation samples, the high quality of identification, time saving, and the low cost of analysis have made this method popular .The objective of this study is to investigate difference of local sepsis etiology and antibiotic susceptibility among children and adults.The study was conducted on the basis of the shared resource laboratory of the Medical University of Karaganda (Kazakhstan). Material for the study was blood samples from 200 patients diagnosed with sepsis. Patients were divided into 2 groups by age: children from 0 to 18 years old and adults from 18 to 99 years old. The average age of infants was 3.15 ± 2.49 months, and in children, 5.7 ± 5.3 years; in the group of adult patients, the average age was 51.6 ± 17.8 years . PeripheWhen a signal about a positive sample appears, 5 ml of broth is taken into serum gel-separator tubes. Gel-separator tubes were centrifuged on 3000 rpm for 10 minutes to separate media and bacteria from erythrocytes. Then, supernatant is discharged, and a thin white layer above gel is carefully mixed in 1-1, 5 ml ultrapure water, and moved into a 1, 5 ml Eppendorf tube. This sample is being centrifuged for 2 minutes on 13000 rpm. Supernatant is discharged, and the pellet is applied on target  and idenSimultaneously, positive blood culture samples were cultured on blood agar with horse blood for 18–20 hours to exclude mixed cultures and to get isolated colonies for the antibiotic susceptibility test.μg), cefepime (30 μg), imipenem (10 μg), meropenem (10 μg), amikacin (30 μg), gentamycin (10 μg), netilmicin (30 μg), tobramycin (10 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), linezolid (30 μg), clindamycin (2 μg), azithromycin (15 μg), vancomycin (30 μg), teicoplanin (30 μg), fusidic acid (10 μg), and tetracycline (30 μg). Analysis of the results was carried out in accordance with the recommendations of CLSI 2018 [The determination of antimicrobial sensitivity was carried out for the prevailing microorganisms for each group of patients. Susceptibility of bacterial isolates to antibacterial drugs was carried out by the disk diffusion method to following antibiotics: penicillin G (10 u), ampicillin (10 LSI 2018 . DetermiLSI 2018 .S. aureus (MRSA) and S. epidermidis (MRSE) was conducted by cefoxitin disk (30 μg). MRSA positive strains had zone of inhibition ≤23 mm [Phenotypic detection of methicillin resistant n ≤23 mm .μg), amoxicillin/clavulanic acid (20/10 μg), and cefotaxime (30 μg) disks [ESBL producers were determined by using cefuroxime (30 g) disks .Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 (ESBL− and AmpC−), Klebsiella pneumoniae WHO-3 (ESBL +), and Enterobacter cloacae WHO-238 (AmpC + and ESBL−).Internal quality control was carried out on the control strains: The analysis of susceptibility to antibacterial drugs was carried out by calculating the 95% confidence interval using the WHONET 5.6 program. Statistical analysis was carried out in the STATISTICA 7.0 program using the chi square.p < 0.05). From all positive samples, 4/66 (6%) were represented as mixed cultures (the associations of two microorganisms). It is useful to note that three of them were from patients up to 1 month of life. However, there is no statistical significance between frequency of mixed isolates and age of participants (p < 0.05).Overall, a total 200 blood samples were processed. Of these, 23/90 (25.5%) positive blood cultures were isolated from adult patients and 43/110 (39%) from pediatric patients. It was found that bacteremia is statistically more often detected in children with sepsis than in adults  and in pediatric patients, the respiratory system (48.6%) .S. epidermidis (35.5%) and in the group of adult patients, S. aureus (21.7%) (see p < 0.05). In adults, the ratio of Gram-positive and Gram-negative bacteria is not much different, 14 : 9 (60.8% : 39.1%), and the second place after S. aureus in adults belongs to microorganisms of the Enterobacterales family (E. coli (13%), K. pneumonia , and Salmonella sp.)  see . The Gra3%)) see .C. albicans and C. parapsilosis. According to past studies, the role of Candida fungi in the development of BSI has been marked among newborns and children of the first year of live much more often than among adults [The study identified 3 cases of candidemia, and all of them were found in children. The causative agents were g adults , 21.S. epidermidis strains isolated from the blood of pediatric patients were completely susceptible to penicillin, 100% (95% CI 74.7–100), 68.8% (95% CI 41.5–87.9) cases were resistant to azithromycin, 33.3% (95 % CI 13.0–61.3) cases were resistant to clindamycin, 35.7% (95% CI 14.0–64.4) were resistant to ciprofloxacin, and 50% (95% CI 20.1–79.9) were resistant to levofloxacin; to the group of aminoglycosides (gentamicin), 18 were resistant, 8% (95% CI 5.0–46.4)  see . The proS. epidermidis, isolated from an adult group of patients, was characterized by a higher level of resistance to antibacterial drugs, and all strains were resistant to azithromycin and cefoxitin (MRSE). The remaining antibiogram data were similar to those in the group of children. All S. epidermidis strains isolated from adults and children were highly sensitive to vancomycin, linezolid, teicoplanin, and fusidic acid. The data obtained are consistent with the literature: in Amita Jain et al.'s study of catheter-associated bloodstream infections in the pediatric population, 63.1% oxacillin resistant CoNS and high glycopeptide sensitivity were obtained [obtained ; 66.6% oobtained , but theS. aureus (21.7%). S. aureus strains from adult patients and children had a similar picture of antibiotic patterns and were characterized by moderate sensitivity to antimicrobial agents. 1/5 (20%) of the strains from adults were MRSA; from pediatric patients, the proportion of MRSA was 1/6 (16.6%).An important role in the etiology of sepsis in adults in our study was played by Enterobacterales (39%) were the second cause of sepsis in adult patients. 42.9% (95% C.I. 11.8–79.8) strains were resistant to fluoroquinolones (ciprofloxacin) and tetracycline while those from pediatric patients were highly susceptible, 100% (95% CI 0.0–60.4). Colistin (MIC> 2 100% (95% CI 0.0–37.1) and imipenem had high antibacterial activity in both groups. Meropenem was less active in adults, 14.3% of resistant strains. 28.6% (95% CI 5.1–69.8) of strains from adults were resistant to gentamicin, so as in children, 25% (95% CI 1.3–78.1). Other aminoglycosides in pediatric patients were completely susceptible (100%), while 12, 5% (95% CI 0.7–53.3), of isolates from adult patients were resistant to tobramycin and 14, 3% (95% CI 0.8–58.0), to netilmicin see . 62.5% op < 0.05). The most frequently isolated microorganisms in both groups were Gram-positive cocci: in children, S. epidermidis (35.5%); in adults, S. aureus (21.7%). The Gram-positive bacteria in children were detected statistically more often (p < 0.05) than Gram-negative with ratio 37 : 5 (86% : 11.6%). The number of resistant strains of S. epidermidis (MRSE) in the group of children (66.7%) significantly exceeds the number of those in adults, which may be due to the localization of the primary focus of infection and more frequent administration of antistaphylococcal drugs. In adult patients, a large percentage of ESBL (62.5%) relative to children (25%) draws attention, which is associated with the spectrum of prescribed antibacterial drugs according to the protocols of diagnosis and treatment of the Republic of Kazakhstan.In the results of our study, we found that bacteremia is statistically more often detected in children with sepsis than in adults (


Since early diagnosis is very important for treating gastric cancer (GC), we aimed to detect serum small proline-rich protein2A (SPRR2A) to verify its diagnostic value for GC patients.  Serum samples were collected from 200 patients with GC, 100 patients with gastritis, 40 patients with rectal cancer (RC), 50 patients with colon cancer (CC), and 100 healthy controls. An enzyme-linked immunosorbent assay (ELISA) detection kit was applied to measure serum SPRR2A concentration. The correlations between serum SPRR2A and carcinoembryonic antigen (CEA), clinical pathological parameters of GC, and receiver operating characteristic (ROC) curve were also analyzed. P < 0.001). Serum SPRR2A concentration at a cut-off value of 80.7 pg/ml yielded an AUC of 0.851, with 75.7% sensitivity and 74.5% specificity for discriminating GC patients from healthy people. The AUC for the serum SPRR2A concentration combined with the CEA concentration was 0.876, with 79.7% sensitivity and 78.7% specificity. Similarly, serum SPRR2A discriminated GC patients from gastritis patients with an AUC of 0.820, with 90.5% sensitivity and 61.7% specificity. The AUC for the serum SPRR2A concentration combined with the CEA concentration was 0.848, with 87.8% sensitivity and 68.1% specificity. The serum SPRR2A levels in GC patients were associated with lymph node metastasis and the tumor-node-metastasis (TNM) stage (P < 0.05). There was an obvious difference in serum SPRR2A expression between GC patients before and after surgery (P < 0.0001).  The median serum SPRR2A concentration in GC patients was significantly higher than those in healthy controls and gastritis or colorectal cancer patients ( These results suggest that serum SPRR2A can be used as an effective marker for GC. Gastric cancer (GC), an epithelial malignancy, is the third leading cause of cancer-related deaths globally, accounting for 7% of cancer cases and 9% of cancer-related deaths . The oveUpper gastrointestinal endoscopy is a sensitive and specific diagnostic test for gastric cancer. However, endoscopy is an interventional examination, leading to occasional compliance and poor cost-effectiveness . Since pIn a previous study, we screened for GC-associated genes in GC and normal gastric tissues using a gene chip and found that SPRR2A had increased expression in GC tissues as high as 12 times , and 50 with colon cancer (CC). The general clinical information on all 390 subjects is shown in In addition, blood samples from 100 healthy controls were collected at the Department of Medical Examination Center of the Affiliated Hangzhou First People's Hospital of Zhejiang University School of Medicine between January 2017 and May 2017. The healthy controls were confirmed to have no gastritis by gastroscopy and biopsy, and the physical examination report showed that they did not have many other diseases. We selected gastritis including chronic superficial gastritis (48 cases) and chronic atrophic gastritis (52 cases). Among them, 74 gastritis patients were HP positive and 26 were HP negative. Blood from controls was also collected in the morning. The current study was approved by the Institution Ethics Committee of the Affiliated Hangzhou First People's Hospital of Zhejiang University School of Medicine.g for 10 min, and 0.2 ml of serum was subsequently obtained after centrifugation at 10,000g for 5 min. The prepared serum samples were stored at -80°C.3 ml of blood was drawn from each participant in the fasting state in the morning of the first visiting day. 1 ml of serum was separated by centrifuging the blood samples at 3000Serum SPRR2A was measured with an SPRR2A ELISA kit . The standard was diluted with the original standard provided (960 pg/ml), and then the standard dilution was diluted. The standard solutions were prepared at concentrations of 30, 60, 120, 240, 480, and 960 pg/ml, and a standard curve was prepared based on the results.μl of sample buffer into each well, 10 μl of the SPRR2A standard stock solution and a serum sample were added to each well. Subsequently, the plate was covered and incubated at 37°C for 30 min. A SPRR2A detection reagent was added to each well and incubated at 37°C for 30 min. The streptavidin-HRP working solution was added to each well, and then the samples were incubated at 37°C for 15 min. After washing, wells were developed with TMB, and the reaction was stopped by using a stop solution. Finally, the plates were read on a full-wave length microplate reader  at 450 nm.After pipetting 40 Serum CEA quantitative measurements were performed at the Department of Laboratory Medicine of Hangzhou First People's Hospital of Zhejiang University School of Medicine. The concentrations of CEA in the serum samples were determined by using an Elecsys CEA kit  according to the manufacturer's instructions. The tested samples were subsequently assayed on an electrochemiluminescence analyzer . Abnormal reference values for CEA were defined as ≥5.0 ng/ml.U test, and differences between the groups were compared by using the Kruskal-Wallis H test. The predictive ability of SPRR2A, CEA, and the combination of the 2 markers for GC was determined by logistic regression. Receiver operating characteristic (ROC) curve analysis was applied to calculate the area under the ROC curve (AUC), 95% confidence interval (CI), and Youden's index (sensitivity + specificity − 1) for each tumor marker. All the above-mentioned analyses were performed by using IBM SPSS Statistics software, version 21.0 . The corresponding experimental figures were drawn using GraphPad Prism 8.0 software .Comparisons of SPRR2A levels between two groups were performed by using the Mann-Whiney We collected serum samples from 200 untreated patients with GC, 100 patients with gastritis, 40 patients with RC, 50 patients with CC, and 100 healthy controls. The median age of the 200 GC patients was 52 (range 33-81) years, with 126 males and 74 females, and the median absolute serum SPRR2A concentration was 124.10 (82.73-186.05) pg/ml. The median age of the 100 healthy controls was 29 (range 15~60) years, with 54 males and 46 females, and the median absolute serum SPRR2A concentration was 44.93 (28.54-74.27) pg/ml. The median age of the 100 patients with gastritis was 43 (range 23-70) years, with 58 males and 42 females, and the median absolute serum SPRR2A concentration was 50.07 (30.57-87.54) pg/ml. The median age of the patients with RC was 55 (39~72) years, with 22 males and 18 females, and the median absolute serum SPRR2A concentration was 56.58 (41.17-75.20) pg/ml. The median age of the patients with CC was 57 (31~82) years, with 39 males and 11 females, and the median absolute serum SPRR2A concentration was 62.04 (51.56-78.59) pg/ml .P < 0.0001, P > 0.05) in the serum SPRR2A concentration.The median serum SPRR2A concentration in all GC patients was significantly higher than those in healthy controls and gastritis and colorectal cancer patients (P > 0.05) (P < 0.05). The median serum SPRR2A concentrations in patients with lymph node metastasis were significantly higher than those in patients without lymph node metastasis (P = 0.013). As for GC patients, SPRR2A concentration was more likely to be at an advanced stage .Given the significant increase in the level of serum SPRR2A for GC patients, does it relate to the process of GC development? According to the different clinicopathological factors of GC, we classified 200 GC patients. There was no significant difference between SPRR2A level and sex, age, tumor size, histological differentiation, or the Lauren classification in GC patients ( > 0.05) . SignifiSerum SPRR2A discriminated GC patients from healthy people with an AUC of 0.851 (95%CI : 0.785–0.916). At a cut-off value of 80.7 pg/ml, the sensitivity and specificity of detection were 75.7% and 74.5%, respectively. The serum CEA concentration at the cut-off value of 5.0 ng/l yielded an AUC of 0.743 (95%CI : 0.654–0.832), with 23% sensitivity and 98% specificity. Furthermore, the AUC for the combination of SPRR2A and CEA was 0.876 (95%CI : 0.818–0.935), with 79.7% sensitivity and 78.7% specificity. Similarly, serum SPRR2A discriminated GC patients from gastritis patients with an AUC of 0.820 (95%CI : 0.742–0.899), with 90.5% sensitivity and 61.7% specificity. Serum CEA yielded an AUC of 0.763 (95%CI : 0.676–0.850), with 23% sensitivity and 97.9% specificity. The AUC for the serum SPRR2A concentration combined with the CEA concentration was 0.848 (95%CI : 0.777–0.919), with 87.8% sensitivity and 68.1% specificity . Serum SP < 0.001). There was no significant difference in serum SPRR2A expression between GC patients postoperatively and healthy controls (P = 0.1651)  pg/ml, which was significantly lower than that in patients preoperatively (133.46 (88.04-188.01) pg/ml) ( 0.1651) .A tumor marker was defined as a biochemical indicator that was usually found in an abnormal concentration in the presence of a tumor . As discHigh sensitivity is an important index in order to avoid false-negative diagnosis. Therefore, we determine the cut-off value of 80.7 pg/ml with a considerable high sensitivity of 75.7% and specificity of 74.5%. Accumulated data shows that CEA is a convenient indicator for monitoring recurrence and distant metastasis as well as for evaluating the efficacy of chemotherapy and prognosis in GC . TherefoP < 0.001), suggesting that serum SPRR2A may predict the prognosis of GC and aid in the postoperative monitoring of GC patients. Unfortunately, the number of specimens in gastric cancer recurrence was limited, so there is no data to show that SPRR2A is a better tumor marker than CEA in gastric cancer recurrence. In the next step, we will continue to track the prognosis of relevant patients to analyze the recurrence of SPRR2A in tumor monitoring.Serum SPRR2A expression is significantly different in pre- and postoperative GC patients . It was reported that SPRR2A was an important player in cell migration, and this migration characteristic is related to the transcriptional activity of SPRR2A regulating p53 . p53 canAlthough our research shows for the first time that the detection of serum SPRR2A can play a complementary role in the diagnosis of GC, especially when combined with the detection of serum CEA, there are still some limitations of this study: First, this prospective study selected a small number of gastric cancer cases and a relatively simple tissue type (only gastric adenocarcinoma). For the previous study, we found that SPRR2A mRNA in a gene chip is highly expressed in gastric adenocarcinoma. Therefore, the subjects of this study were all selected gastric adenocarcinoma patients. To further verify the role of SPRR2A in the diagnosis of gastric cancer, a retrospective study of multiple centers, large sample sizes, and multiple tissue types is also required . Second, there is no strict restriction on the type of gastritis control cases, and some disease conditions that may affect serum SPRR2A levels, such as infection, ischemia, and diabetes, are not considered. In addition, although our test data shows that the serum SPRR2A level of GC patients is significantly lower than that before surgery, the change in serum SPRR2A concentration may be useful for dynamically monitoring the prognosis of GC patients undergoing surgical intervention. However, due to the small number of cases, we did not assess the prognostic value of serum SPRR2A.In summary, SPRR2A showed a high diagnostic value for patients with GC; the combination of SPRR2A and CEA is a promising novel biomarker for GC diagnosis.


Oryza sativa, five genotypes of Oryza glaberrima, and one genotype of NERICA (New Rice for Africa). The treated rice seeds were sown in plastic boxes under four soil moisture conditions ). The genotypes were categorized into six groups based on growth parameters using hierarchical cluster analysis. Furthermore, emergence properties were investigated by using principal component analysis based on the mean emergence time of control and primed seeds. Seed priming enhanced growth performance under the moderate dry conditions of 10% and 15% soil moisture. Meanwhile, priming efficacy was low in water stress conditions of 5% and 20% soil moisture. There were wide-ranging genotypic differences of priming efficacy under 20% soil moisture condition. Our findings indicate that the anaerobic-tolerant genotypes tend to exhibit priming efficacy under high soil moisture conditions. Furthermore, one group included all upland genotypes of Oryza sativa. This group originally adapted to 10% and 15% of dry conditions, and seed priming improved their features greatly.Seed priming refers to seed enhancement methods that stimulate seed metabolism. This study evaluated the genetic diversity of hydro priming efficacy in 27 different genotypes of rice under dry to wet soil moisture conditions. The genotypes included 21 genotypes of  Althoug3 t ha−1 . Farooq 3 t ha−1  reported3 t ha−1 ,7. As re3 t ha−1 , the ter3 t ha−1  defined 3 t ha−1 ,10. Seve-priming ,12. As r-priming , hydro p-priming ,15,16. R-priming ,18. The Oryza sativa L., O. glaberrima Steud. and interspecific hybrids between O. sativa and O. glaberrima, known as NERICA (New Rice for Africa), were used in the experiment ; in is the number of seeds germinated in time i , and k is the last time of germination [Twenty-seven rice genotypes, including periment . To breaN/2 < nj .t¯  was calculated based on the weight of the completely dried soil. We controlled the soil water via daily spraying and created four different soil moisture contents: 5%, 10%, 15%, and 20% (w/w). Four containers  were prepared for each soil moisture condition. Ten seeds were sown at 1 cm under the soil surface and 270 seeds (27 genotypes × 10 seeds) were sown in one container. Plant emergence was studied using the mean plant emergence time (MET), the time to achieve 50% plant emergence (E50), and plant emergence uniformity (EU). MET and E50 were calculated in the same way as MGT and G50. EU was calculated as the standard deviation of MET, based on the following formula [it is the time between the start of the experiment and the ith observation (day or hour); in is the number of seeds germinated (emerged) in the time i, and k is the last time of germination.For the cultivation experiment, we used plastic pots  in an experimental room (mean temperature 29.0 °C and mean humidity 73.9%.) The pots were filled with soil taken from the White Volta flood plain in the northern region of Ghana  . Regardi formula .(1)St (t-test.We measured plant height (PH) of all plants at seven days after planting and calculated plant height uniformity (PHU) as the coefficient of variation between the plants we observed. We conducted cluster analysis and principal component analysis using JMP version 13  We calculated statistical differences using a O. glaberrima were included in either Group 1 (G1) or Group 3 (G3), and G1 consisted entirely of O. glaberrima. Group 2 (G2) had three genotypes of O. sativa and one NERICA genotype. Group 4 (G4) contained five genotypes from four different origin countries: the Philippines, Japan, Ghana, and Guinea. Group 5 (G5) contained two genotypes from the Philippines. G2 and G5 include genotypes that have anaerobic germination (AG) + Sub1 (Submergence 1) gene . Group 6 (G6) consisted of seven genotypes: two upland genotypes from India, two genotypes originating from Ghana, and three genotypes from the Philippines.We carried out a hierarchical cluster analysis based on G50, MGT, E50, MET, plant EU, PH, and PHU under four soil moisture conditions . Cluster analysis divided the 27 genotypes into six groups . All thet-test (p < 0.05). G50 showed the same tendency as MGT, and the G50 of primed seeds was significantly shorter than that of the control in all groups except G5. In G1, germination took longer for both primed and control seeds.The MGT of the control seed was 51.8, 33.9, 38.5, 37.2, 37.0, and 31.0 in G1, G2, G3, G4, G5, and G6, respectively . The MGTp < 0.05) in the EU of G4 and G6.The E50 of the primed seeds tended to be shorter than that of the control seeds except under the 5% soil moisture condition . There wIn G2, G3, G4, and G6, the PH of primed seeds was significantly higher than that of the control under the 10% soil moisture condition . In G2, p < 0.01) between control and primed seeds under the 10% and 15% soil moisture conditions (p < 0.01) between control and primed seeds in respect to PH, apart from under the 20% soil moisture condition (p < 0.05) and the 15% (p < 0.01) soil moisture conditions. Regarding the correlation diagram of MET, most of the plots were above the dotted line under the 5% soil moisture condition, suggesting that emergence takes much longer for primed seeds than for control seeds  significantly in all cluster groups, apart from the G50 of G5 . Seed prSub1 (Submergence 1) gene . The genotypes in these groups were developed for AG and submergence tolerance (Sub1) and are characterized by rapid germination even when submerged [Conversely, seed priming tends to restrict both emergence and plant growth under the 5% soil moisture condition. In G6, seed priming had significant negative impacts on MET and E50 under the 5% soil moisture condition . There wubmerged . Under sO. glaberrima genotypes . In this group, we did not find significant differences between control and primed seeds in respect of all parameters and under all soil moisture conditions. G1 seeds took much longer to emerge, and the subsequent PH was shortest under the 10%, 15%, and 20% soil moisture conditions  were included in G3, and the MET and PH of G3 were enhanced significantly under the 10% and 15% soil moisture conditions. These findings for G1 and G3 indicated that O. glaberrima has differences in reactivity to seed priming. G5 genotypes also showed a low component-1 in both control and priming, suggesting that they have low emergence ability either with or without priming. However, the IR07F323 of primed seeds was plotted near the 20% axis of MET . Regarding the control seeds of G6, PH was the highest, and E50 and MET were the shortest under the 15% soil moisture condition. Furthermore, seed priming accelerated PH, E50, and MET significantly under the 10% and 15% soil moisture conditions , which adapt well to moderately dry conditions of 10% and 15% of soil moisture, and their growth features were improved greatly by seed priming.This study compared the efficacy of hydro priming of seeds of 27 genotypes of rice under different soil moisture conditions. As with previous reports, seed priming appeared to improve the performance of rice seed sown directly under the moderate dry conditions of 10% and 15% soil moisture. However, priming efficacy was low under 5% and 20% water stress conditions. This study has identified wide genotypic differences in priming efficacy under a 20% soil moisture condition. In particular, two genotypic groups that include anaerobic-tolerant genotypes  tended to show priming efficacy under 20% soil moisture conditions. Conversely, one genotypic group showed good initial growth under wet conditions, but the growth features of the plants were not enhanced by seed priming under wet conditions. Furthermore, our study findings indicate that


Demand on hospital emergency departments for paediatric problems is increasing. However, the volume and nature of paediatric health demands placed on other parts of the urgent care system have not been explored. This understanding is an important first step in developing and improving out-of-hospital care. We aimed to describe the volume, nature, and outcomes of paediatric contacts with out-of-hours general practice (OOH GP). We performed a retrospective service evaluation using data from 12 months of paediatric patient contacts with the Oxfordshire OOH GP service.A database of contacts with the Oxfordshire OOH GP service was created for a 12 month period from December 2014 to November 2015. Descriptive statistics were calculated using SPSS Version 25.27,455 contacts were made by 18,987 individuals during a 12 month period. The majority of these were for children aged under 5. Over 70% of contacts were at the weekend. The peak contact period was between 18:30 and 21:30. Over 40% of contacts resulted in advice only . 19.7% of contacts resulted in an antibiotic prescription, most commonly those linked with ear, chest, and throat infections.Paediatric contacts with the Oxfordshire OOH GP service were predominantly in younger age groups and in the evening, with 19.7% resulting in an antibiotic prescription. Almost half of the contacts had no follow up or prescription, suggesting non-prescribing health care professionals could be involved in providing care in OOH GP. Further research should consider how children and their parents can be best supported to optimise OOH consulting. In. In, [8], . This seUnderstanding the nature and outcomes of contacts with NHS services is key to evaluating and improving these services. Paediatric contacts with NHS services have been described in relation to Emergency Departments (ED) or Urgent Care Centres (UCC) –12 . In To date, there has been no description of paediatric contacts, or the outcome of these contacts, with OOH GP in the UK. This understanding is critical to inform new approaches to managing the rising demand for acute paediatric assessment across urgent and unscheduled care, and ensuring safe and appropriate management. We aimed to characterise the nature, timing, and outcomes of paediatric contacts with the OOH GP using a large dataset of patient contacts with the Oxfordshire OOH service, which provides care to a population of over 600,000 people.A database of all intended patient contacts with the Oxfordshire OOH GP service, which provides care to a population of over 600,000 people, over 1 year from 1 December 2014 to 30 November 2015 was created from the OOH Electronic Record System used by clinicians (Adastra).Service data included contact type, contact outcome, date, clinical codes assigned and prescriptions issued. Demographic data included patient sex, age, and a deprivation index (the Index of Multiple Deprivation (IMD)). Lower IMD scores indicate lower levels of deprivation. Contacts were coded with their final contact type: a ‘telephone consultation’, a ‘base visit’ (patient assessed at OOH base), a ‘home visit’ (patient assessed in their home), or a “111 appointment to book” for patients the 111 service have determined an appointment is needed but cannot be booked . This results in a contact with the OOH GP which may be a base visit, telephone consultation, or home visit.Timings of calls were classified as evening 18:30–23:59 h, night 00:00–07:59 h, and daytime (on weekends and bank holidays) 08:00–18:29 h. The weekend period was classified as 18:30 h Friday until 08:00 h Monday, whilst contacts classified as ‘Bank Holiday’ were those occurring during daytime hours on each Bank Holiday.n = 264, 1%) had “NULL” as their clinical code. These contacts were removed from the dataset.At the end of each consultation, clinicians assign at least one clinical code (for example ‘H05z. Upper respiratory infection. NOS’), which were used in this study to determine clinical presentation for that contact. These clinical codes were validated by two members of the direct care team based on previous coding validity studies . The posClinicians also detail any prescriptions issued during the consultation in up to six fields. Prescriptions were grouped into 21 categories and 79 sub-categories for the purposes of analysis. These are detailed in supplementary Table Each contact is coded with an ‘Outcome’. There were 19 outcome codes, which were condensed into 9 outcome categories. These are detailed in supplementary Table Descriptive statistics were calculated in SPSS version 25. Analyses were undertaken at the contact level unless otherwise stated.Between 1 December 2014 and 30 November 2015, 67,942 patients made 102,876 contacts with the Oxfordshire OOH GP service. Of these, 27,455 contacts (26.69%) made by 18,987 individuals (29.95%) were for children under the age of 18. Of these individuals, 13,833 (72.9%) had one contact, 3341 (17.6%) had two contacts, and 1813 (9.5%) had three or more contacts. See Fig. The median age of children contacting the OOH GP service was 3.1 (IQR 1.1–7.0). In the twelve month period, 6136 contacts (22.3%) were for children under the age of one, and 11,852 (43.2%) of contacts were for children aged between 1 and 4 years. Children aged 5–11 account for 6118 contacts (22.3%), whilst there were 3349 contacts (12.2%) for children aged 12 and over. The median index of multiple deprivation (IMD) score was 10.79 (IQR 6.15–18.24).48.7% of contacts were for female patients, and of these the median age in years was 3.5 IQR 1.2–8.1). The median age of male patients was 2.8 (IQR 1.1–6.0). Figure –8.1. The). 19,260 (70.2%) contacts were at the weekend and 870 (3.2%) were on a Bank Holiday. 9103 (33.2%) contacts were on a Saturday and 8048 (29.3%) were made on a Sunday. During the week, Tuesdays and Wednesdays had the lowest proportion of contacts (6.4% in both cases), followed by Thursday (7.0%), Monday (8.8%) and Friday (9.0%).The month with the greatest number of contacts was December, and July had the lowest number of contacts Table . 19,260 There were 20,692 (75.4%) base visits whilst 6603 (24.1%) contacts were managed purely by telephone. There were 123 (0.4%) home visits and 37 (0.1%) “111 appt to book contacts” (contacts for which appointments cannot be booked within the time frame dictated by 111 and are passed onto the OOH GP service separately).Figure n = 5627, 20.5%), ‘Digestive’ (Chapter D) , ‘Skin’ (Chapter S) , and ‘Ear’ (Chapter H) . 472 (1.7%) of contacts were coded with a musculoskeletal code and this was most prevalent in children aged 12 and older. 82 (2.4%) contacts for children aged 12 and over were coded with a psychiatric condition. Ninety-four of the 101 of children aged less than 1 with a psychological code had problems feeding.Table The most common ICPC Codes were Upper Respiratory Tract Infection R74) (14.3% of contacts), Viral disease other/NOS (A77) (7.7%), Fever (A03) (7.1%), Tonsillitis Acute (R76) (4.9%), and Acute otitis media/myringitis (H71) (4.9%)  contacts there was no documented requirement for follow up. After 11,359 (41.4%) contacts there was no documented requirement for follow up and no prescription issued, considered as ‘advice only’ appointments. In 8394 (30.6%) of contacts the patient was advised to contact their own GP, and there was a referral to secondary care in 1754 (6.4%) of contacts. Table There were a total of 9633 prescriptions issued at 7840 (28.6%) contacts. In total, 5598 (29.5%) individual children received prescriptions. 5656 (58.7%) prescriptions were for antibiotics, and these were issued in 5420 (19.7%) of contacts. Of contacts resulting in antibiotic prescription, 2278 of these contacts (42.0%) had Respiratory codes (R), 1215 (22.4%) had Ear codes (H) and 778 (14.4%) had Skin codes (S) (Table Prescriptions for asthma were the next most common with 852 (8.8%) prescriptions for inhaled asthma medication and devices issued in 567 (2.1%) contacts (Table Over a quarter (26.69%) of contacts with the Oxfordshire OOH GP service between December 2014 and November 2015 were for children under the age of 18. Of these, the majority (65.5%) were for children aged under 5 years. There were more male than female patients amongst the under 5 s, but amongst teenagers almost two thirds of the contacts were for female patients.In over 40% of contacts there was no onward referral, requirement for GP follow up, or prescriptions, indicating that the primary clinical output of the contact was advice or reassurance. The proportion of these contacts was highest amongst the youngest age group (47.9%) and fell in each subsequent age group. Antibiotics were prescribed in 19.7% of contacts, most commonly linked with ear, respiratory, and skin conditions.During the week, the majority of contacts were in the evening, with a peak between 18:30 and 21:30. At the weekend, there was an early peak of contacts at 09:00 and a second, higher peak between the hours of 19:00 and 21:00.Children presented most commonly with respiratory conditions. The most common ICPC code was an upper respiratory tract infection. Other common codes were unspecified viral diseases, tonsillitis and acute otitis media. There were few contacts with musculoskeletal or psychological complaints.In the UK research into paediatric use of NHS services has focused on unscheduled urgent care in Urgent Care Centres (UCCs) and the Emergency Department (ED). Use of health services is highest amongst the youngest children , 17. In In Europe the proportion of paediatric contacts with OOH GP ranged from 1.9% in Switzerland to 33.3% in Denmark . In BelgThere is limited research into the outcomes of paediatric consultations with OOH GP. One study of consultations for fever amongst children consulting with an OOH GP service in the Netherlands found that 92% of contacts were managed without referral to secondary care . In our Antibiotics were prescribed in 19.7% of contacts in this study, primarily for respiratory, ear, and skin conditions. Antibiotic prescribing has previously been associated with 15% of all contacts with the UK OOH GP service, and 18.0% of contacts for individuals under the age of 18 [To our knowledge this is the first study to explore in detail paediatric demand on a UK OOH GP service. It uses a large dataset of 27,455 contacts. However, as the dataset is limited to Oxfordshire, our results may not be generalisable to the UK population as a whole. Oxfordshire is, on the whole, less deprived than the UK, and our methods should be replicated in other regions with different populations and care models. These data also precede recent extended access schemes which may alter how people navigate the health services.We found that 41.4% of contacts were ‘advice only’. Whilst these contacts may have been appropriate, this highlights the need for research exploring the content and main function of these consultations, and reviewing whether alternative sources of advice could offer a safe substitute. This also suggests that nurse practitioners and paramedics who do not prescribe could be part of the team offering paediatric assessment with appropriate training.Antibiotics were prescribed in 19.7% of contacts. UK OOH GP is associated with a disproportionately high rate of antibiotic prescribing , represeFinally, our study took a clinician perspective on contacts with OOH GP linking the reason for contact with the clinical codes applied during the consultation. Qualitative research exploring the process by which parents make decisions to consult OOH GP specifically will deepen our understanding of these consultations and how patients can be best supported. This is of specific interest for general and respiratory illnesses amongst younger children.We found that paediatric contacts with the Oxfordshire OOH GP service were predominantly in younger age groups and in the early evening with 19.7% resulting in an antibiotic prescription. Almost half of the contacts were advice-only appointments, and further research should consider how patients making these appointments can be best supported.Additional file 1.Additional file 2: Supplementary Table 2. Prescription CategorisationAdditional file 3: Supplementary Table 3. Outcome codes from the dataset and their reported category.Additional file 4.


Campylobacter spp. At the next stage of the research the frequency of occurrence of selected virulence genes, i.e. cadF, flaA and iam as well as genes responsible for the formation the cytolethal distending toxin (CDT), i.e. cdtA, cdtB and cdtC was determined. The isolates obtained in the research were tested for their resistance to selected antibiotics: ciprofloxacin (CIP), enrofloxacin (EF), erythromycin (E) and tetracycline (TE). Campylobacter spp. was found in 63 (12.6%) out of a total number of 500 isolates. 61 (12.2%) isolates were identified as C. jejuni. The number of C. jejuni isolates found in the younger animals was smaller (p <0.05) than in the older ones. The frequency of occurrence of virulence genes and the genes responsible for the formation of CDT was significantly (p <0.05) higher in the older dogs. A comparison of the effect of antibiotics showed that the isolates obtained from both age groups exhibited low resistance to erythrosine (13.5% in the group aged under 1 year and 8.6% in the group aged over 1 year). Both groups exhibited the highest resistance to ciprofloxacin and enrofloxacin.The aim of the study was to determine the role of dogs as a potential reservoir of  They can also be found in water and soil contaminated with animal faeces [st century [Campylobacter spp. isolates from humans are increasingly often resistant to quinolone chemotherapeutics [Campylobacter spp. [cdtA, cdtB and cdtC. This cytotoxin causes cell cycle arrest at the G2/M phase [fla, cad, rac, vir, cia, pld, iam. The fla genes (flaA and flaB) are responsible for bacterial motility. They encode flagellin – the ciliaryThermotolerant bacteria of the l faeces . As thes century , 3. The  century . In 2015 century . The mos century . Accordi century , each yeapeutics , aminoglapeutics . The devter spp. . The bac/M phase . Other vCampylobacter spp. cells to move. The cadF gene encodes the fibronectin binding protein of enterocytes, which participate in adherence. Many researchers believe that this gene, which is necessary to induce symptoms of campylobacteriosis, is a conservative gene in C. jejuni and C. coli. The vir gene, which can be found in the Campylobacter spp. plasmid , also encodes proteins responsible for pathogenicity. The iam sequence, which is responsible for adhesion and invasiveness, is found in C. coli more often than in C. jejuni [protein which enables . jejuni . In consCampylobacter bacteria in dogs, to identify species within the Campylobacter genus, selected virulence genes in isolated strains and genes determining the occurrence of CDT, and to determine resistance to selected antibiotics.The aim of the study was to determine the frequency of occurrence of 22.1n = 250 dogs younger than 1 year and n = 250 dogs older than 1 year) from shelters located in the Wielkopolska region were used as material for the tests. No dog showed gastrointestinal symptoms. The samples were collected on three consecutive days and transported to a laboratory within 6 hours while stored at 4°C.Swab samples  collected from the rectum of 500 dogs . Next, they were incubated in a CampyGen anaerostat  at 41.5 °C/24 h . After incubation the bacterial cultures were centrifuged (4.000 x g/15 min), the supernatant was poured and the sediment was added to 1 cm3 of Karmali agar. 200 μL of the suspension was spread with a spreader on a selective Karmali agar  and incubated in an anaerostat at 41.5 °C/48 h. An API Campy biochemical test, product No. 20800 (Biomérieux) was used to confirm the presence of Campylobacter spp. The identity of the colonies grown on the agar was determined by observation of their morphological features and motility . The following tests were carried out: oxidase , catalase  and the hydrolysis capacity of hippurate and indoxyl acetate . A selective mCCDA medium (Oxoid) was used for a confirmatory test, where colonies grown after incubation at 41.5 °C/48 h were counted. Campylobacter spp. was also distinguished from other gram-negative bacteria by means of an O.B.I.S. Campy test . Real-time PCR (Biorad) with the BAX System Real-Time PCR Assay for Campylobacter  was used for species identification.Swab samples were initially placed in 3 cm2.3cdtA, cdtB, and cdtC genes. A 25μL reaction mixture was used. It was composed of: 2.5 μL 10 x PCR buffer, 6 x 1 μL primers (5 μM), 1 μL dNTP (2.5 mM), 0.2μL Taq polymerase, 1.8 μL DNA, 13.5 μL water. The thermal conditions of the reaction were as follows: initial denaturation at 94 °C/2 min. It was followed by 30 cycles, each of which consisted of: denaturation (94 °C/0.5 min), primer attachment (50 °C/0.5 min) and extension (72 °C/1 min and 72 °C/5 min). The resulting products were analysed by means of electrophoresis in 1.5% agarose gel.The primers shown in 2.4cadF, flaA, and iam genes were identified by means of the primers shown in 2 (25 mM), 1 μL dNTP (2.5 mM), 1 μL primers (5 μM), 0.2 μL (1U ) U TaqDNA polymerase (Promega Corporation), 2 μL DNA, 15, 5 μL water. The thermal conditions of the reaction were as follows: initial denaturation (94 °C/1 min). It was followed by 30 cycles, each of which consisted of: denaturation (94 °C/0.5 min), primer attachment (45 °C/1 min), extension (72 °C/2 min and 72 °C/5 min). The resulting products were analysed by means of electrophoresis in 1.5% agarose gel.2.5Campylobacter bacterial strains were tested for sensitivity to antibiotics by means of the Kirby-Bauer disc diffusion test and E-test. The method was compatible with the EUCAST [The isolated e EUCAST  Disc DifC. jejuni ATCC 33291 and C. coli ATCC 33559 (DSMZ Germany) were used as reference strains in the study.2.6The counts of microorganisms measured in the investigations were analysed statistically using the GLM procedure of the SAS program. The significance of differences was verified with Duncan’s test (α=0.05).33.1Campylobacter spp. was found in 63 (12.6%) out of the total number of 500 isolates. 61 (12.2%) isolates were identified as C. jejuni  than the number of positive isolates found in the dogs aged over 1 year . The analysis of the species composition of the isolates  showed that all of them belonged to C. jejuni. There were 26 (10.4%) C. jejuni isolates found in the dogs aged under 1 year. This number was lower than in the group of dogs aged over 1 year . The differences were statistically significant (p <0.05).. jejuni . There w3.2cdtA gene. The cdtB gene was found in 2 dogs aged under 1 year (7.7%) and 17 dogs aged over 1 year (48.6%). The cdtC gene was found in 4 dogs aged under 1 year (15.3%) and 20 dogs aged over 1 year (57.1%). The differences were statistically significant (p <0.05). The frequency of occurrence of the genes determining CDT occurrence was higher in the animals aged over 1 year (There were 3 dogs aged under 1 year (11.5%) and 15 dogs aged over 1 year (42.8%) with the r 1 year .3.3cadF gene. The flaA gene was found in 15 dogs aged under 1 year (57.7%) and 35 dogs aged over 1 year (100%). The iam gene was found in 9 dogs aged under 1 year (34.6%) and 23 dogs aged over 1 year (65.7%). The differences were statistically significant (p <0.05). The frequency of occurrence of the sequences of virulence genes was higher in the dogs aged over 1 year (There were 16 dogs aged under 1 year (61.5%) and 35 dogs aged over 1 year (100%) with the r 1 year .3.4Campylobacter jejuni sensitivity (S) and resistance (R) to the antibiotics applied in the experiment. The E Test and disc diffusion test were two methods compared in the study. Enrofloxacin was the only antibiotic which resulted in statistically significant differences (p <0.05) between the methods, both in the group of dogs aged under 1 year and in the group of older animals. The isolates from the younger animals exhibited the highest resistance to ciprofloxacin (46%) and enrofloxacin (30.7%). The analysis of resistance of the isolates obtained from the older animals showed that they also exhibited high sensitivity to ciprofloxacin (50%) and enrofloxacin (22.9%) (this was observed only in the disc diffusion test). Both the isolates from the younger and older dogs exhibited low resistance to erythromycin (13.5% and 8.6%) and tetracycline (7.7% and 8.6%).4C. coli and C. jejuni in research on Campylobacter spp. Particular attention was paid to the ability of C. jejuni to hydrolyse sodium hippurate. Since the description of hippurate-negative C. jejuni strains it has seemed more reliable to use the PCR method and the Real-Time PCR system with complementary primers [Campylobacter spp. in the group of dogs (n = 500), i.e. 12.6%. This result stood in opposition to our earlier studies [n = 70) of healthy dogs and found bacteria of the Campylobacter genus in 58% of the samples. Likewise, the authors did not find C. coli or C. lari, whereas C. jejuni was detected in 7% (n = 5) of the positive samples. Differences in the results of studies conducted by various authors may have been caused by many factors, e.g. the sampling method, the culturing conditions in a laboratory (in vitro) or high species diversity within the Campylobacter genus [Campylobacter spp. [Campylobacter spp. The authors observed that the incidence of these bacteria increased from 60% in young animals (3 months old) to 100% in animals aged 12 months, but it then decreased to 67% in dogs aged 24 months.Initially, biochemical tests were used to detect species such as  primers . Our stu studies  and resedogs n = 00, i.e.  studies . Chaban  studies  analyseder genus . Other ater spp. . Hald etter spp.  noticed C. jejuni genome WCTC 11168, sequenced in 2000, revealed the presence of genes encoding a protein with potential toxic properties [cdt genes encoding the CDT protein . These were two genes encoding proteins with haemolytic domains and one phospholipase gene. CDT was the first described genotoxin that damages the genetic material of sensitive eukaryotic cells by stopping the cell cycle and causing the death of cells as a consequence [cdtA, cdtB and cdtC, which are usually located on the chromosome [cdtB subunit is responsible for the nucleolytic effect. In our study the occurrence of this gene in older dogs was higher than in the younger animals, i.e. 48.6% vs. 7.7%. Other authors have observed higher values. Andrzejewska et al. [cdtB was as high as 72.7% or even 100% [cdtA and cdtC subunits should not be neglected either, because they may transport the cdtB subunit into the target cell  [The operties . The strsequence . A CDT lromosome . All thrromosome  recordeda et al.  researchven 100% , whereasven 100%  noted 97 et al.) .fla – the motility gene, cadF – the adhesion gene and iam – the invasiveness gene [cadF, flaA genes and iam sequence in both groups of dogs. Rodrigues et al. [cadF and flaA genes in dogs and noted that these genes may act as reporters in research on the mechanisms of C. jejuni pathogenicity.The pathogenesis of campylobacteriosis may be directly related with factors such as: motility, chemotaxis  as well as adhesion and invasiveness. Many genes and the effects of their expression are thought to be responsible for pathogenicity, e.g. ess gene , 32. Ours et al.  observedCampylobacter spp. strains isolated from animals and food exhibited the highest resistance to ciprofloxacin and tetracycline [Campylobacter spp. is the resistance of these bacteria to aminoglycosides (gentamicin), macrolides (erythromycin) and quinolones (ciprofloxacin), which are commonly used to treat campylobacteriosis [Campylobacter jejuni which were resistant to ciprofloxacin was very high, i.e. 58% [Campylobacter spp. strains isolated from poultry were resistant to one or more antibiotics. 99.5% of C. jejuni strains and 96.3% of C. coli strains were resistant to tetracycline. Research conducted in Slovenia also showed a high percentage of C. jejuni isolates resistant to enrofloxacin (58.2%). The strains were less resistant to erythromycin (14.5%) and tetracycline (12.7%) [The progressive increase in bacterial resistance is closely correlated with the misuse of chemotherapeutics. Apart from that, as a result of the widespread application of antibiotics to animals, often without medical indications, bacteria become selected towards antibiotic resistance. In consequence, they develop multi-drug resistance . Campyloacycline . The maiteriosis . Researci.e. 58% . Researc (12.7%) . These r (12.7%)  observed (12.7%)  revealed5Campylobacter spp. and were found to be vectors carrying and transmitting Campylobacter infections to humans. It is necessary to conduct further research on these bacteria as risk factors to better understand the epidemiology of Campylobacter spp. and to emphasise the dangers of the unreasonable use of antibiotics in both human and animal therapy.To sum up, the dogs were identified as asymptomatic carriers of