Cancer is a leading cause of death among people living with HIV/AIDS (PLWHA). We conducted a systematic review and meta-analysis to evaluate prevalence of cancer risk factors among Chinese PLWHA based on 102 articles. Random effects meta-analysis was used to calculate the summary prevalence estimate (sPrev) and 95% confidence interval (CI) for each cancer risk factor by demographic group. Overall, the sPrev for each risk factor among Chinese PLWHA was: 41.1% (95% CI: 35.3–46.9%) for current smoking; 30.3% (95% CI: 23.3–37.4%) for current alcohol consumption; 24.4% (95% CI: 14.7–30.2%) for overweight and obesity; 12.5% (95% CI: 10.6–14.3%) for hepatitis B virus infection; 29.1% (95% CI: 23.6–34.5%) for hepatitis C virus infection; 33.9% (95% CI: 24.3–43.5%) for high-risk human papillomavirus infection from cervical samples and 78.6% (95% CI: 69.4–87.7%) from anal samples; 2.7% (95% CI: 0.7–4.7%) for Epstein-Barr virus (EBV) immunoglobulin M (IgM) positivity, 94.7% (95% CI: 90.7–98.8%) for EBV IgG positivity and 25.6% (95% CI: 12.4–38.8%) for EBV DNA positivity; 14.9% (95% CI: 12.4–17.4%) for human herpes virus 8 infection. The prevalence of major cancer risk factors was high among PLWHA in China, suggesting an urgent need for interventions to reduce cancer risk in this high-risk group. For PLWHA, cancer is now one of the leading causes of death with higher incidence of both AIDS-defining cancers (ADCs) and non-AIDS-defining cancers (NADCs) compared with the general population8. In particular, NADCs have shown increasing incidence and account for a rising proportion of all cancers in this population4. China has implemented HAART nationally since 2003. In a Chinese cohort, substantially elevated standardized incidence ratios of NADCS and ADCs were observed, and cancers occurred at a younger age for PLWHA compared to the general population9. Accumulated evidence suggests that HIV-induced immunodeficiency and prevalent risk factors other than HIV infection independently contribute to the increased cancer risk among PLWHA11. The major risk factors associated with leading causes of cancer deaths include tobacco use , alcohol consumption , overweight/obesity , and viral infections 13. Numerous independent studies have reported prevalence of cancer risk factors among PLWHA in China. However, the researches vary in time and geographic areas, and do not provide a comprehensive overview of cancer risk factors epidemics for PLWHA in China. We conducted a meta-analysis among PLWHA in China to describe the prevalence of major cancer risk factors including tobacco smoking, alcohol consumption, overweight and obesity, and cancer-related viral infections such as hepatitis B virus (HBV), hepatitis C virus (HCV), human papillomavirus (HPV), Epstein-Barr virus (EBV) and human herpes virus 8 (HHV8). Prevalence estimates among Chinese PLWHA were compared with the general population or uninfected comparison groups from the same study when available.Highly active antiretroviral therapy (HAART) or combination antiretroviral therapy (cART) has drastically reduced mortality in people living with HIV/AIDS (PLWHA)115. One additional study with unique data on HIV uninfected population was included for comparison analysis116.The process of selection of publications and the numbers of articles for each cancer risk factor are presented in Table 101, 2 retrospective cohort studies79 and 1 intervention trial36 were included. Most studies (99) were based in clinics or hospitals for PLWHA recruitment, 2 were population-based73 and 1 study sampled was recruited from jail75. The 102 eligible studies covered 20 provinces and 4 municipalities in China. The median sample size for PLWHA was 295 [interquartile range (IQR): 178–551]. Seventy-five articles reported the median or mean age, and the median of the median or mean age was 37.2(IQR: 34.0–41.0). Of the 95 articles reporting gender distribution, the median proportion of male was 72.6% (IQR: 61.3–86.9%). Of the 16 articles reporting the proportions of MSM, the median was 79.1% (IQR: 63.9–100.0%). Of the 23 articles reporting the proportions of IDU, the median was 74.8% (IQR: 40.4–100.0%). Of 26 articles reporting the proportions on ART, the median was 67.5% (IQR: 45.3–100.0%).Table Table , I2 = 96.0%) in PLWHA based on the meta-analysis of 21 studies. The sPrev of current smoking was 63.2% (95% CI: 39.7–86.8%) for male PLWHA and was 3.4% (95% CI: 2.2–4.6%) for females, based on the 5 studies with gender-specific prevalence. Overall, the sPrev of ever and former smoking was 51.9% (95% CI: 42.8–61.1%) based on 5 studies and 13.9% (95% CI: 0.3–27.6%) based on 3 studies, respectively  based on the meta-analysis of 25 studies. Gender-specific estimates showed that the sPrev of current alcohol consumption was 26.9%  in male PLWHA based on 5 studies and was 3.3%  in females based on 3 studies. MSM presented a current drinking sPrev of 35.7% (95% CI: 18.9–52.5%) based on 3 studies while IDUs presented a current drinking sPrev of 13.1% (95% CI: 8.3–17.9%) based on 2 studies. Overall, the sPrev of ever and former alcohol drinking was 64.3% (95% CI: 60.8–67.7%) and 17.8% (95% CI: 3.6–32.0%), respectively (Table P = 0.004)  among PLWHA, with a sPrev of 24.4%  , 10.4%  and 13.4%  in overall, female and male groups, respectively. The highest sPrev of HBsAg was among IDUs  (Table P = 0.013) . Male PLWHA showed a sPrev of 47.1%  based on 15 studies, while female PLWHA had a sPrev of 29.9%  based on 16 studies. The highest HCV prevalence was found in IDU group with a sPrev of 83.4%   and 33.9% , respectively. For anal samples, the sPrev among male PLWHA of any type of HPV was 78.6% , and the sPrev of high-risk type was 52.1% . Similar sPrev was observed among HIV positive MSM . IgG was tested in 2 studies, and the sPrev in overall, female and male groups was 94.7% , 92.5%  and 96.8% , respectively. DNA from peripheral blood was tested in 2 studies, and the overall sPrev was 25.6% . One study reported the prevalence of 27.1% (95% CI: 16.7–37.6%) in female and 34.2% (95% CI: 27.4–41.0%) in male PLWHA for EBV DNA positivity  was tested in 4 studies and the sPrev was 2.7% , 12.1% , 17.7%  and 16.3% , respectively. The highest serum HHV8 prevalence was among MSM  . For serum antibody, the sPrev in overall, female, male and IDU groups was 14.9%  and males (63.2% vs. 55.4%), alcohol consumption among males (26.9% vs. 30.2%), overweight and obesity (22.4% vs. 25.8%), and HCV infection among IDUs (83.4% vs. 93.0%).We recalculated each sPrevP-value for Egger test was 0.028, P-value for Begg test was 0.057), the overall sPrev of alcohol consumption , the overall sPrev of HBV infection , and the sPrev of HCV infection overall , in females , and in males .In our meta-analysis, substantial publication bias was significantly observed by the Egger test and/or the Begg test in estimating the overall sPrev of smoking 119 while MSM showed slightly higher sPrev (35.7%) and IDUs showed significantly lower estimate (13.1%). As group I carcinogens defined by IARC, tobacco smoking and alcohol consumption are not only associated with a series of human cancers, but also with other important chronic diseases. Based on our summarized estimates, the high prevalence of tobacco and alcohol use especially among male PLWHA suggested the need for an effective and more targeted intervention in smoking and drinking behaviors as well as a close observation of their roles in HIV and cancer disease progression to reduce morbidity and to improve quality of life in this aging population with prolonged life expectancy.For such two modifiable behavioral risk factors as tobacco and alcohol use, Chinese PLWHA showed high prevalence of tobacco smoking and alcohol drinking overall and especially among males. For example, the smoking prevalence is 53.3% for general Chinese men126, shown by the results from both meta-analyses and independent studies. By demographic group, 10.4–13.4% of PLWHA were HBsAg positive, and 29.1–47.1% were HCV antibody positive. Moreover, the highest prevalence of HBV (32.8%) and HCV (83.4%) infection were both found in IDUs, probably due to the shared transmission route with HIV. For female PLWHA, the sPrev of both any type of HPV (42.0%) and high-risk type of HPV (33.9%) from cervical samples were higher than the prevalence in Chinese general population aged 15–60 years123. Moreover, we found that in anal samples collected from male PLWHA, the HPV prevalence was very high no matter whether they were MSM or not. These observations call attention since HPV infection is also a risk factor for anal cancer. The high seropositivity of EBV-IgG was close to the prevalence reported earlier in the Chinese general population (97.6%)125. Although the latter statistic might be limited to experimental conditions and in need of update, it is believed that EBV infection is very common among adults127. Moreover, several studies suggested higher detection rate of EBV DNA among HIV-infected subjects compared to HIV-negatives, suggesting potentially more active viral application in immune-suppressed population129. Last but not least, the overall seroprevalence of HHV8 infection was 14.9%, higher than the prevalence in the Chinese general population126, with the highest prevalence found in MSM .The prevalence of cancer-related viral infections, including HBV, HCV, HPV, EBV and HHV8 infection, are significantly higher in PLWHA than in general Chinese population119. This is similar with the observations in western countries that the obesity prevalence was lower in HIV population118. We need to further observe the change in weights in PLWHA to have a better understanding and balance in control of both the HIV wasting syndrome and risk for obesity-related cancers and chronic diseases.The overall prevalence of overweight and obesity among PLWHA was 22.4% (95% CI: 14.7–30.2%), lower than the prevalence of 42.6% in Chinese general population118, which, consistent with our analysis, also observed higher prevalence of cancer risk factors among PLWHA than the general population. However, the prevalence level seems to be different between PLWHA in western countries and in China. Compared with PLWHA in western countries, PLWHA in China seemed to have higher prevalence in alcohol consumption (30.3% versus 24.0%), HCV (29.1–47.1% versus 23.0–28.0%) and HBV (10.4–13.4% versus 4.0–5.0%) infection, but lower prevalence in smoking (41.1% versus 54.0%), overweight and obesity (22.4% versus 53.0%) and HPV infection . Thus, the spectrum of cancers among PLWHA in China might be different from that in western countries and further investigations and interventions might be targeted to different high-risk groups.A meta-analysis was recently published on prevalence of risk factors for NADCs among PLWHA in western high-income countriesI2 values larger than 90%. This was expected since heterogeneity could be found in almost all ‘overall’ groups, because we could not know the proportion of genders or behavioral characteristics in these study populations across publications. So was the case in ‘male’ groups, which could have different proportions of heterosexual male, MSM and IDU. Meanwhile, extensive heterogeneity still existed in specific demographic groups such as female, MSMs and IDUs. This could be attributable to many potential factors including study design, sampling method, geographical regions, study period, age, ethnicity, the definition or method of measurement for risk factors, differences in CD4+ and the length of time in ART, etc130. Insufficient number of studies has limited our further stratification analysis. However, random effects models were used to minimize the impact of heterogeneity on precision and provided rational approximations of pooled prevalence estimates in our meta-analyses. Sources of heterogeneity should be further explored in the future with more research for specific subgroups.Substantial heterogeneity across subgroups was significantly observed with 132. The association between behavioral changes such as smoking and alcohol consumption and severity of HIV patients might lead to underestimation of prevalence of cancer risk factors among PLWHA.Several limitations in our study should be noted. First, there might be some information bias since the methods for measuring these risk factors might vary across studies. Most (69.2%) studies did not give a clear definition of smoking habits  or did not have a precise measurement for smoking dose or frequency, while only 50% of studies presented the range of time (past 30 days or a day) for current alcohol drinking. Although we tried to extract information from reference studies using standardized definitions, misclassification might still exist since not every study gave a clear definition for each risk factor. Also, the testable HPV subtypes varied across studies, and therefore, underestimation of HPV prevalence might have occurred in studies with fewer testable HPV subtypes. Second, the representativeness of our meta-analyses is compromised since it covered 24 but not all 31 provinces or municipalities in China, underscoring the need for expanded research and surveillance efforts on cancer risk factors among PLWHA all over the country. Third, comparability was not considered across different sources of data due to time lags among studies. Some risk factors may present a temporal trend, some may not. For those with an increasing or decreasing trend, it may have compromised the comparison. Fourth, as prevalence estimates of cancer risk factors can vary with characteristics including age, lack of matching for age and other factors also limited the validity of the comparisons between PLWHA and the general population. However, consistent results were still observed when stratified roughly by age where possible , Wanfang Data and Chinese Scientific Journals Fulltext Database (CQVIP). Using the Medical Subject Headings (MeSH) terms ‘China’ or ‘Taiwan’ with ‘HIV,’ ‘HIV Infections’ or ‘Acquired Immunodeficiency Syndrome’, the search also included MeSH terms for the specific cancer risk factors as follows: smoking (‘Smoking’ or ‘Tobacco Products’ or ‘Tobacco’ or ‘Tobacco Use’), alcohol consumption (‘Alcohol Drinking’ or ‘Alcoholism’ or ‘Alcoholic Intoxication’), overweight or obesity (‘Overweight’ or ‘Obesity’ or ‘Body Mass Index’ or ‘Body Weight’), HBV (‘Hepatitis B’), HCV (‘Hepatitis C’), HPV (‘Papillomavirus Infections’ or ‘Papillomaviridae’), EBV (‘Epstein-Barr Virus Infections’), and HHV8 . We also searched those MeSH terms and their abbreviations of each risk factor under the condition of ‘all fields’ in each database. Due to the large number of studies published on HBV and HCV infections among PLWHA in China, we restricted published data from January 1, 2013 to February 29, 2016 in order to use the latest published prevalence of HBV and HCV infections. For other cancer risk factors, data published from database inception to February 29, 2016 were included in the analyses.One author (Jin Z-Y) identified relevant articles, excluding those with unrelated titles and abstracts before obtaining full-text references. Two independent authors (Jin Z-Y and Liu X) conducted full-text review. Cross-sectional, case-control, prospective or retrospective cohort and intervention trial studies were included. Studies with sample size greater than 100 were considered to have a sufficiently stable prevalence estimate to be qualified for inclusion in the study. Studies lacking original data for the numerator or denominator of the prevalence estimate were excluded. If information from one study had been published for more than once, we chose the publication with more detailed information such as prevalence estimates stratified by specific demographic groups, studies with a larger sample size, or those most recently updated.Data were extracted and organized using Microsoft Excel . For each risk factor, the number of PLWHA with known status was recorded as the denominator and the number of positive subjects was recorded as the numerator. The participants with unknown status were excluded from the calculation. If an article contained more than one of the mentioned risk factors, we kept all of them. If the PLWHA were stratified into different groups such as females, males, men who have sex with men (MSM) or injection drug users (IDU), we included data from each subgroup for prevalence estimate. The definitions of smoking and drinking status including “ever/never”, “former/current” were based on the descriptions in the original studies when a clear definition is not available. We also extracted prevalence estimates for HIV uninfected comparison groups if data were available from the same study. Other information including the first author’s name, year of publication, study site, original study design, sampling frame, calendar years of research, major information for participants including the mean or median age, percentage of males, MSM, IDU, treatment with antiretroviral therapy (ART), and the measurement of risk factors were also extracted.119. For HBV and HCV infections, prevalence estimates were obtained from a national serosurvey in 2006, of which participants were local residents aged 1–59 years (the median age was in the range from 15 to 59 years for HBV and in the range from 15 to 20 years for HCV) living in 31 provinces with 160 DSPs121. For HPV infection, prevalence estimates were acquired from a nationwide investigation of female subjects aged 15–60 years in 37 Chinese cities in 2012, a review (age ranging from 15 to 59 years) and a population-based study among men aged 25–65 years (the median age was 43 years)124. For EBV infection, the prevalence estimate was extracted from a 1989 study by Liu125. For HHV8 infection, the prevalence estimate was from a systematic review and meta-analysis published in 2012126.We also extracted prevalence estimates for cancer risk factors among the general population from the most representative surveys. For tobacco smoking, alcohol consumption, overweight and obesity, prevalence estimates were extracted from a national survey, which was carried out in Chinese adults (aged 18 years and over with the median age in the range from 45 to 49 years) at 162 disease surveillance points (DSPs) from 31 provinces in 2010P < 0.1 refers to statistically significant heterogeneity) and I2 values140. Random effects models were used because of significant heterogeneity across subgroups142. DerSimonian and Laird method (D-L) was applied to estimate the summary prevalence estimate (sPrev) and its 95% confidence interval (CI) for each risk factor143. If only one study was presented for the specific subgroup, we showed the single prevalence and its 95% CI. In sensitivity analyses, each sPrev was recalculated excluding studies evaluated as having higher potential for bias. Lastly, we performed a publication bias test when the meta-analysis included more than 10 studies145. Publication bias was assessed through the Egger weighted regression and Begg rank correlation method 147. All the analyses were conducted by Stata 12.0 (Stata Corp LP).PLWHA were stratified into overall, female, male, MSM, and IDU demographic groups when such subgroups were available. The ‘Overall’ group represents PLWHA not restricted by gender or HIV transmission category . Similarly, the ‘male’ group implies that PLWHA were not restricted by HIV transmission category. A meta-analysis of prevalence was performed for each risk factor by demographic group if there was more than one study included. Heterogeneity tests across subgroups were examined by Q-test (Supplementary Information


The beneficial effects of traditional herbs of Crete, Greece for the treatment of upper respiratory tract infections have been reported in observational and laboratory studies. Following a published, double blind, randomized, placebo controlled trial, this study aimed to assess the effectiveness of an extract of three Cretan herbs on the treatment of upper respiratory tract infections, upon its market release.An observational study was conducted in Heraklion, Crete, Greece. Participants were patients presenting at selected pharmacies with symptoms of upper respiratory tract infection, choosing to receive the extract for their treatment. Patients’ symptoms  where recorded at three time points within 1 week, using a questionnaire developed based on the Wisconsin Upper Respiratory System Survey. For each patient, symptoms were scored on a 0–7 Likert scale and three indexes were calculated: the score of local symptoms, the score of general symptoms and the total score of symptoms. Effectiveness was assessed by examining the reduction in these indexes over the 1-week observation period.p < 0.0001 for paired differences). Multiple regression models indicated that supplementary medication intake did not seem to affect symptoms’ severity or the day patients reported that their symptoms ceased completely.Mean score of general symptoms was 19.1 (SE: 0.9) in day 1, dropping to 8.6 (SE: 0.6) and 3.1 (SE: 0.4) in days 4 and 7 respectively. Mean score of local symptoms declined from 7.9 (SE: 0.5) in day 1 to 2.3 (SE: 0.3) in day 4 and to 0.5 (SE: 0.1) in day 7. Total score of symptoms reached 27.0 (SE: 1.2) in day 1, decreasing to 10.9 (SE: 0.8) in day 4 and to 3.5 (SE: 0.5) in day 7. The percentage of participants reporting fever was 82.1% at baseline, 8.0% in day 4 and 2.0% in day 7  contains supplementary material, which is available to authorized users. Coridothymus capitatus (L.) Rchb. f. synonym of Thymbra capitata (L.) Cav.), dictamnus (Origanum dictamnus L.) and sage , on the treatment of upper respiratory tract infections. The results indicated amelioration in the severity of symptoms, with 90% of treated patients being symptom-free at the last day of observation [Aromatic plants have been historically used as remedies in the island of Crete, Greece . Anthropervation . The extThe aim of this observational study was to explore the extract’s effectiveness on the treatment of upper respiratory tract infections, after its market release, through a questionnaire-based inquiry of participants. The primary objective was to assess the severity and duration of symptoms upon treatment. Secondary objectives included the investigation of the extract’s effectiveness on the presence of fever and in cases of supplementary medication intake.An observational study was conducted in the prefecture of Heraklion, Crete, Greece (February – June 2016). Sixteen pharmacies, purposively selected, participated as recruiting sites. The preparation of the extract is extensively described elsewhere .Patients presenting at the pharmacies with symptoms of upper respiratory tract infection, who voluntarily requested and choose to purchase the extract as part of their treatment were recruited. Eligibility for inclusion in the study was based on the Jackson criteria for the identification of patients suffering from common cold . In ordeSetting as primary outcome the day patients reported that their symptoms ceased completely and assuming 5% probability of type-I error, a sample size of 185 patients had 90% power to detect an average time of symptoms’ cessation of 6 days, with standard deviation of ±1.2 days and accuracy of 0.17 days.Upon presenting at the pharmacies, eligible patients were invited to complete an anonymous questionnaire  and general . They were scored in a Likert scale, with 0 denoting absence of symptom and 7 denoting maximal severity of the respective symptom. For each patient, three indexes were calculated: The score of local symptoms, the score of general symptoms and the total score of symptoms, defined as the sum of general and local symptoms’ scores. Presence of fever was recorded as a dichotomous variable (yes/no).2), smoking (currently/former/never) and presence of chronic conditions (yes/no). Any supplementary medication received for the treatment of patients’ upper respiratory tract infection was also recorded (type and days of intake). The day patients reported feeling better and the day patients reported that their symptoms ceased completely were recorded as well.Collected socio-demographic and clinical characteristics included age (years), gender , BMI  was computed. Multiple linear regression models were performed with dependent variables: the AuC, the day patients reported feeling better and the day patients reported their symptoms ceased completely. Independent variables were the socio-demographic and clinical characteristics and supplementary medication intake (yes/no). Linear mixed models with dependent variables the total score of symptoms and presence of fever and independent variables the day of observation , supplementary medication intake (by day of observation), socio-demographic and clinical characteristics were used to assess symptoms’ alleviation over time. Confidence level was a = 0.05. Statistical software used were IBM SPSS version 21 and Stata SE version 12.Sample characteristics were summarized using descriptive statistics. For comparisons between categorical variables Pearson’s χ2, 68 (45%) participants were current smokers and 44 (29.1%) reported at least one chronic condition. A total of 85 (56.3%) participants received supplementary medication for the treatment of their upper respiratory tract infection at day 1. The respective percentage for the whole observation period was 64.9% (n = 98).A total of 493 patients with upper respiratory tract infection symptoms visited the selected pharmacies during recruitment. Among those, 315 (63.9%) met the inclusion criteria, 157 (50.2%) agreed to participate and 6 (3.8%) dropped-out. Data from 151 patients were analyzed (81.6% of the estimated target of 185 patients). Participants’ mean age was 43.6 (±17.6) years, while 95 (62.9%) were females. Mean BMI was 26.3 (±5.6) kg/mp < 0.001 for all paired differences). Mean score of local symptoms declined from 7.9 (SE: 0.5) in day 1 to 2.3 (SE: 0.3) in day 4 and to 0.5 (SE: 0.1) in day 7, showing a reduction by 70.9% in day 4 and by 93.7% in day 7 . Mean total score of symptoms reached 27.0 (SE: 1.2) in day 1, falling to 10.9 (SE: 0.8) in day 4 and to 3.5 (SE: 0.5) in day 7, indicating a decrease by 59.6% in day 4 and by 87.0% in day 7 .Table Patients reported feeling better on the third day of extract intake . They also reported that their symptoms had ceased completely by the sixth day of intake . By the end of the observation period 81.7% of our participants reported that their symptoms had ceased completely. The mean AuC for all symptoms was 26.2 (±17.4). Multiple linear regressions did not show any significant predictors (including supplementary medication intake) for the AuC, the day patients reported feeling better or the day patients reported that their symptoms ceased completely.p < 0.0001 for both) and the presence of fever . A borderline positive association between the total score of symptoms and supplementary medication intake was observed , indicating that patients with more severe symptoms tended to consume extra medication. Results indicated no significant interactions between supplementary medication intake and day of observation in the prediction of the total score of symptoms . Post-hoc analyses indicated no significant differences in the total score of symptoms between patients consuming supplementary medication and those who did not .Linear mixed models showed a significant decrease in the total score of symptoms over time  within 10 days of observation [A literature review study reported 16 trials of herbal remedies for respiratory tract infections . Trials  throats  and acut throats , presentervation . In the This study is prone to the limitations of its observational design, including the fact that there was no control group for comparable results. Additionally, information about the daily course of patients’ treatment could not be reported, since data was gathered at distinct time points. The presence of fever was, also, not recorded as exact temperature but rather as a dichotomous variable (yes/no). Almost two out of three patients received supplementary medication for their treatment. In all regression models, supplementary medication intake was modelled as dichotomous on each observation, since types and duration of intake differed for each patient. Thus, the effect of competitive active ingredients and types of medication could not be documented. Moreover, it was not possible to determine the extracts’ effectiveness on specific viral infections, since there was no identification of patients’ viral strains and viral loads. Finally, our sample was confined only to patients who voluntary requested the extract and were able to afford its purchase.Despite these limitations, this study reflected the potential therapeutic effects of the extract, out of the controlled clinical trial setting, in the daily practice scenario where patients consume several medications at different time points for their treatment. In a period were debate regarding the cost-effectiveness of antiviral therapy is ongoing , furtherThe findings of this study affirmed those of a previously published, double blind, randomized, placebo controlled trial, providing additional evidence about the potential therapeutic role of Cretan herbs in cases of upper respiratory tract infections.


However, the role of H2S at spinal dorsal horn level remains elusive in visceral hypersensitivity. The aim of this study is designed to investigate how H2S takes part in visceral hypersensitivity of adult rats with neonatal colonic inflammation (NCI). Visceral hypersensitivity was induced by neonatal colonic injection of diluted acetic acid. Expression of an endogenous H2S synthesizing enzyme cystathionine β-synthetase (CBS) was determined by Western blot. Excitability and synaptic transmission of neurons in the substantia gelatinosa (SG) of spinal cord was recorded by patch clamping. Here, we showed that expression of CBS in the spinal dorsal horn was significantly upregulated in NCI rats. The frequency of glutamatergic synaptic activities in SG was markedly enhanced in NCI rats when compared with control rats. Application of NaHS increased the frequency of both spontaneous and miniature excitatory post-synaptic currents of SG neurons in control rats through a presynaptic mechanism. In contrast, application of AOAA, an inhibitor of CBS, dramatically suppressed the frequency of glutamatergic synaptic activities of SG neurons of NCI rats. Importantly, intrathecal injection of AOAA remarkably attenuated visceral hypersensitivity of NCI rats. These results suggest that H2S modulates pain signaling likely through a presynaptic mechanism in SG of spinal dorsal horn, thus providing a potential therapeutic strategy for treatment for chronic visceral pain in patients with IBS.Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by chronic abdominal pain and alteration of bowel movements. The pathogenesis of visceral hypersensitivity in IBS patients remains largely unknown. Hydrogen sulfide (H Irritable bowel syndrome (IBS) is a functional gastrointestinal disorder affecting approximately 20% of populations worldwide . One of 2S), as one of endogenously occurring gasses, was generated from L-cysteine by endogenous enzymes such as cystathionine β-synthetase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase  and/or supraspinal cord. There is little evidence to show the roles of H2S in the central nerve system. Many primary afferent neurons with unmyelinated and thinly myelinated fibers projects to the substantia gelatinosa  of the dorsal horn of SC  rats, 150∼200 g body weight, were housed in plastic cages and under controlled conditions  with a standard rodent diet and fresh water. Care and handling of these animals were approved by the Institutional Animal Care and Use Committee of the Soochow University and were strictly in accordance with the guidelines of the International Association for the Study of Pain. All efforts were made to minimize the suffering and the number of animals. Visceral hypersensitivity was induced by neonatal colonic injection of diluted acetic acid, as described previously , 2009. I2 and 5% CO2 in a dish. The rats were then killed by exsanguination. The composition of Krebs solution was as follows (in mM): 95 NaCl, 1.8 KCl, 1.2 KH2PO4, 0.5 CaCl2, 7 MgSO4, 26 NaHCO3, 15 glucose and 50 sucrose, at pH of 7.3–7.4 and an osmolarity of 310–320 mOsm. The ventral, dorsal roots and the pia-arachnoid membrane were removed using microscissors and microforceps under a stereo light microscope. The SC was placed in a shallow groove on an agar block, and the agar block was mounted on the vibrating microslicer stage with cyanoacrylate adhesive. Several transverse slices (450 μm thickness) were cut with a Vibratome  while the SC was immersed in cold Krebs solution. The slices were transferred directly to oxygenated Krebs solution at 31°C until use.Acute SC slices were prepared from adult rats as described previously . The exp2PO4, 2.4 CaCl2, 1.3 MgSO4, 26 NaHCO3 and 15 glucose, at pH of 7.3–7.4 and osmolarity of 300–310 mOsm. The substantia gelatinosa  can be indentified as a translucent band across the dorsal horn under the 5x objective (NA 0.10) of an upright microscope . The neurons of SG can be visualized under a 40x magnification water-immersion objective (NA 0.80) with the help of infrared differential interference contrast (IR-DIC) optics. The image of the slice was enhanced with a CCD camera  and was displayed on a computer monitor. The patch pipettes were made by a Puller . The composition of internal solution of the patch pipette was as follows (in mM): 140 K-Gluconate, 3 KCl, 10 HEPES, 0.2 EGTA, 4 NaCl, and 2 Ma-ATP. Whole-cell voltage-clamp recordings were made from SG neurons as previously described  following the instructions. qPCR was conducted according the protocol of TransStart Tip Green qPCR SuperMix kit (Transgen Biotech). Negative control reactions were performed by omitting cDNA temple. The relative expression level for each target gene was normalized via 213-L2) for adult NCI and control rats (6–8 week old) were determined using western blot analyses as described previously hydroxylamine hemihydrochloride  was purchased from Sigma-Aldrich and freshly prepared in NS (0.9% wt/vol NaCl). At the age of 5 week, AOAA was injected intrathecally at 10 μg/kg body weight, one time per day for a consecutive 7 days as described previously , as appropriate. Statistical significance was determined as P < 0.05.A fixed length of trace (5 min) that contains more than 300 events was analyzed using MiniAnalysis program 6.0.3 (Synaptosoft) for frequency and amplitude distributions of miniature and spontaneous EPSCs. After the automatical detection for peaks, each detected event was visually checked to exclude the false data. Three to four times the root mean square value of its background noise was set to be the detection threshold for an event in a set of data. Cumulative fractions were calculated before and after an addition of a drug. Kolmogorov–Smirnov test was used to analyze EPSCs. Data are presented as the means ± SEM. Error bars in the figures stand for SEM. Normality was checked for all data before using the Mann–Whitney test as n = 8) and NCI (n = 8) rats. AWR scores were significantly higher for NCI rats at different distention pressures  when compared with those of age-matched controls . It suggested that NCI significantly induced visceral hypersensitivity in adult rats. This result was consistent with the previous report from our group  at age of 6 week for control . At 6 weeks of the age, relative densitometry of CBS was 1.83 ± 0.26 (n = 5) for control and 3.76 ± 0.37 (n = 8) for NCI group, respectively. It suggested that the expression of CBS was significantly upregulated after NCI . However, the mRNA level of CBS was not significantly altered after NCI . Also, there was no change in the expression of CSE (∼70 kDa) in SC between control and NCI group . The expression of CBS in T7-10 SC  and at L4-6 DRGs  was not altered after NCI. Thus, NCI remarkably increased CBS expression in SC corresponding to the afferents of T13-L2 DRGs when compared with that of age-matched control rats.Since Hthan CSE , is exprn = 109, 70 from control slices, 39 from NCI slices), different discharge patterns were observed, including tonic-firing, delayed-firing, gap-firing, initial-bursting, bursting and phasic firing, as previously reported . The average results were shown in Figure 2B. The number of APs of control SG neurons (n = 17) under 40, 80, and 120 pA current stimulation was 8.23 ± 1.45, 20.53 ± 1.93, and 28.73 ± 2.47, respectively. The number of APs of NCI SG neurons (n = 18) under 40, 80, and 120 pA current stimulation was 17.00 ± 2.04, 37.44 ± 2.48, and 52.12 ± 4.29, respectively. It suggested that the excitability of SG neurons was significantly increased by NCI . In addition, the mean resting membrane potential for CON neurons was -60.71 ± 0.73 mV (n = 59) and -59.36 ± 0.95 mV (n = 33) for NCI neurons. They did not differ significantly . However, the AP threshold for NCI neurons  significantly hyperpolarized when compared with that of the CON neurons . Rheobase reflects the minimal injected current to evoke an AP. The rheobase of SG neurons was 13.89 ± 2.88 pA and 28.00 ± 1.99 pA for NCI and CON group, respectively. It suggested that NCI dramatically decreased the rheobase , which is consistent with the increased excitation of SG neurons by NCI.Whether the intrinsic excitatory property of SG neurons was changed by NCI was examined by whole-cell patch-clamp recordings in acutely prepared transverse lumbar SC slices from control and NCI rats. Among the SG neurons recorded . The average results were also shown in Figure 3B. Mean frequency of sEPSCs averaged to 4.44 ± 0.74 Hz in control group and 7.66 ± 1.17 Hz in NMD group, respectively . In parallel, the mean peak amplitude of sEPSCs averaged to 20.16 ± 1.13 pA in control group and 20.44 ± 1.02 pA in NMD group, respectively . The sEPSCs can be completely blocked by CNQX . It suggested that the AMPA receptor-mediated glutamatergic synaptic activity was significantly enhanced in SG of NCI rats.Since the excitation of SG neurons was enhanced by NCI, the excitatory neurotransmission was presumed to be enhanced by NCI as well. The sEPSCs of SG neurons were then compared between control and NCI group. The representative traces from two typical neurons of control and NCI slices illustrated an increase in frequency of sEPSCs of SG neuron in NCI group  were perfused for 5 min after recording the pretreated data for at least 5 min. The two enlarged traces of a typical current trace illustrated the increase in frequency of sEPSCs in a neuron of SG in control slices . NaHS had no significant effect on the amplitude of sEPSCs . Mean amplitudes averaged to 23.21 ± 1.58 pA before and 24.1 ± 1.73 pA after addition of NaHS . Kolmogorov–Smirnov test proved that NaHS shifted the cumulative fraction of inter-event intervals of sEPSCs toward smaller value  and increased the mean frequency of sEPSC significantly . There was a significant increase in average frequency from 5.53 ± 1.63 Hz before, to 6.31 ± 1.88 Hz after the addition of NaHS. The average increase achieved 123.24 ± 9.13%. It suggested that NaHS application increased the spontaneous glutamatergic synaptic activity of SG neurons in control rats.Since CBS were upregulated and the excitability of SG was increased in NCI group, we presumed that NaHS, a donor of HFigure 5A). NaHS didn’t significantly affect the amplitude of mEPSCs . Mean amplitudes averaged to 25.96 ± 1.87 pA before and 24.53 ± 0.99 pA after addition of NaHS . Kolmogorov–Smirnov test proved that NaHS shifted the cumulative fraction of inter-event intervals of mEPSCs toward smaller value  and significantly increased the mean frequency of mEPSC . There was a significant increase in average frequency from 4.51 ± 0.5 Hz before, to 5.56 ± 0.67 Hz after the addition of NaHS. The average increase achieved 124.14 ± 11.58%. It is well-known that change in frequency of mEPSCs reflects the increase of presynaptic neurotransmitter release, whereas change in amplitude of mEPSCs reflects the changes at the post-synaptic membrane . The typical trace and its two expanded traces of a representative neuron showed that NaHS increased the frequency of mEPSCs recorded in SG neurons of control slices . The average results were also shown in Figure 6B. The mean peak amplitude of sEPSCs averaged to 19.03 ± 1.42 pA in NCI group and 20.33 ± 1.48 pA in AOAA group, respectively . In parallel, mean frequency of sEPSCs averaged to 5.05 ± 0.71 Hz in NCI group and 2.64 ± 0.66 Hz in AOAA group, respectively . It suggested that the glutamatergic synaptic activity was significantly decreased in SG of AOAA-treated NCI rats.Since CBS were upregulated and the excitability of SG was increased in NCI rats, we hypothesized that the increase of excitability of SG in NCI rats might be associated with the upregulation of CBS. Therefore, inhibition of CBS was hypothesized to decrease excitability of SG in NCI rats. AOAA, an inhibitor of CBS, was intrathecally injected in the present study to determine the effect of inhibiting CBS on sEPSCs of SG neurons in NCI slices. Data were compared with the sEPSCs of SG neurons in slices from NS-treated NCI rats. The representative traces from two typical neurons of NS and AOAA treated group illustrated a decrease in frequency of sEPSCs of SG neuron in AOAA group . These data proved that AOAA treatment reversed the visceral hypersensitivity in NCI rats. It is in compliance with the result of our previous study  was intrathecally injected into SC (L5-6) for consecutive 7 days and the behavioral responses were assessed. The AWR scores for all the distension pressure are significantly higher than the corresponding values of NS-treated rats . These data demonstrate that NaHS/H2S induced hyperalgesic effect in healthy rats, which supports the pronociceptive effect of CBS upregulation in NCI rats.Since NaHS/HFigure 1B); (2) NCI significantly increases neuronal excitability and excitatory synaptic transmission in SG of spinal dorsal horn ; (3) Intrathecal injection of AOAA (the antagonist of CBS) not only reversed the visceral hyperalgesia of rats with NCI but also decreased excitatory synaptic transmission in SG ; (4) Intrathecal injection of NaHS (2.5 μM) not only led to visceral pain but also significantly increases excitatory synaptic transmission of SG in control rats . These results indicate that upregulation of CBS expression contributed to enhanced synaptic transmission at SG neurons and visceral hypersensitivity of adult rats with NCI.The pathogenesis of chronic visceral pain in patients with IBS remains largely unknown. The present study demonstrates that hydrogen sulfide signaling at SC level produces visceral hypersensitivity in adult rats with NCI. The conclusion is based on the following observations: (1) NCI leads to a significant increase in expression of CBS in SC where receives input from T13-L2 DRG neurons . The action site of H2S is consistent with that in other tissues, such as neuromuscular junction , indicating CBS might be a major factor contributing to visceral hyperalgesia of NCI rats. However, the mRNA level of CBS was not altered. Reasons for this discrepancy might include: regulation of CBS expression at mRNA and protein levels may not happen at the same time and the CBS protein degradation speed was changed. In the present study, the upregulation of CBS expression at translational level and/or downregulation of protein degradation speed might be the underlying mechanisms. In addition, the protein level of CBS is not equal to endogenous H2S since endogenous H2S in the nervous system is generated from CBS, CSE and 3MST.In the present study, since NaHS or AOAA were injected intrathecally, these drugs may act on SC and/or DRGs. It is therefore difficult to rule out the contribution of DRGs. However, by considering the data obtained by the electrophysiological recordings from the SG neurons of spinal dorsal horn, it is reasonable to hypothesize that SC might be one of the important action sites for Hn the SC . CBS is n the SC . In SC, n the SC . We show2S has multiple molecular targets in peripheral tissues and in the CNS. Spinal and peripheral H2S activates L-type channel (2+ channels (such as Cav3.2 channel) in the primary afferents and/or spinal nociceptive neurons /protein kinase A signaling and/or inhibition of phosphodiesterase . The protocol was approved by the Institutional Animal Care and Use Committee of Soochow University, China.LZ, YX, and R-XW performed experiments, analyzed data and prepared figures and the manuscript. XL and P-AZ performed experiments and analyzed data. C-YH and SY revised the manuscript. G-YX designed experiments, supervised the experiments and finalized the manuscript. All the authors have read and approved the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Contemporary care of repaired tetralogy of Fallot (rTOF) patients is directed toward detection and avoidance of long-term morbidity and mortality, of which arrhythmias play an important role. Increased arrhythmia risk in the rTOF population has been associated with ventricular dysfunction and chamber dilation. In adults without congenital heart disease, obesity has been related to left atrium (LA) dilation and further associated to arrhythmias. The effect of obesity on LA size, and in turn on arrhythmia, in rTOF patients has not been studied. If related, obesity would represent a modifiable risk factor in this population. We hypothesized that LA dilation is associated with obesity in rTOF patients.Patients with rTOF undergoing cardiac magnetic resonance imaging (CMR) performed from 2009-2013 were retrospectively reviewed. Patients were excluded for inadequate image quality and mitral regurgitation graded worse than mild by echocardiography performed within a year of CMR. Left and right ventricular (LV and RV) size and ejection fraction (EF) were measured. LA size was calculated by Simpson's biplane formula using LA area and length in the 2- and 4-chamber views. Body mass index (BMI) was calculated on all patients and dichotomized by BMI percentiles into two categories: normal and overweight/obese. Univariate associations were evaluated using Spearman correlation coefficients for continuous variables and Mann-Whitney U test for dichotomous variables.73 patients were included with a mean age at CMR of 20.2 years (range: 5.7-54.8); 57.5% were female. Average age at repair was 15.8 months. Mean BMI was 23.8 kg/m2. 57.5% had normal weight and 42.5% were overweight/obese. Only 7 patients had confirmed arrhythmia, likely due to the young age of the cohort. Mean indexed RV end-diastolic volume was 140 ml/m2 and RVEF was 52.7%. Mean indexed LV end diastolic volume was 64 mL/m2, and mean LVEF was 63.4%. Mean absolute and indexed LA volumes were 43.2 ml ± 14.0 and 29.7 ml/m2 ± 13.9, respectively. The correlations between absolute and indexed LA volume with BMI were 0.435 and 0.483 (p < 0.001 for both). Patients in the overweight/obese category had significantly increased absolute and indexed LA volume compared with those in the normal weight category . Patients with arrhythmia had increased absolute LA volumes and a trend towards increased indexed LA volumes .Elevated BMI is associated with LA dilation in patients with TOF, which may put these patients at risk for negative cardiovascular outcomes including arrhythmia. Further study is warranted in investigating the complex interaction between obesity and clinical outcomes in congenital heart disease.


Acinetobacterb aumannii (A. baumannii) is an important pathogen in health care associated infections. Quinolone resistance has emerged in this pathogen.gyrA gene and parC genes by Restriction Fragment Length Polymorphism Polymerase Chain Reaction (RFLP-PCR) among clinical isolates of A. baumanii.The aim of the present study was to determine the presence of mutations of A. baumannii. The isolates were subjected to molecular study of mutations of gyrA gene and parC genes by RFLP–PCR beside determination of Minimal Inhibitory Concentration (MIC) by macro dilution tube method.The study was carried out on 140 clinical isolates of A. baumannii were resistant to ciprofloxacine and levofloxacin at MIC >4 µg/ml. The most isolates had MIC >128 µg/ml (42.3%). All resistant strains to ciprofloxacin of A. baumannii had mutations in gyrA and parC. The most frequent mutations were combined mutations in both genes (85.5%) and 5% had single mutation either in gyrA or parC. The most frequently combined mutations were associated with MIC >128 µg/ml (42.3%).The isolates of A. baumannii. The most frequent mutations were present in gyrA and parC. However, mutations in parC alone were not uncommon. Further large scale studies are required to elucidate the resistance pattern of A. baumannii and its molecular mechanisms.From this study we can conclude that resistance to ciprofloxacin was common in clinical isolates of  It is one of the so called “ESKAPE” bacteria known to have high antibiotics resistance  . The discs used for antibiotics susceptibility were ampicillin (2µg), ampicillin/sulbactam (10µg+10µg) cefepime (30µg), cefoxitin (30µg), ceftazidime (10µg), ciprofloxacin (1µg), gentamicin (30µg), imipenem (10µg), ceftriaxone (30 µg), meropenem (10 µg), levofloxacin (5μg) and trimethoprim/sulfamethoxazole (1.25/23.75μg), cefotaxime (30µg) (Oxoid).2.2A. baumanii according to the CLSI instructions. Serial dilutions of ciprofloxacin were prepared using Muller Hinton broth and bacterial isolates with concentration equal to 0.5 McFarland were added to each tube and incubated at 37OC for 18 hours. MIC was defined as the lowest concentration with no visible growth. A. baumannii isolates were considered as intermediate-resistant and full-resistant to ciprofloxacin when the MIC was 2 µg/ml and ≥4 µg/ml, respectively [Broth macrodilution method was used for the determination of MIC for ciprofloxacin among isolated ectively .gyrA and parCRFLP -PCR for Detection of Mutations in Gene’s 2.3A. baumannii were subjected to DNA extraction by boiling method. Briefly colonies were dissolved in 1ml sterile distilled water and boiled for 10 minutes then centrifuged for another 10 minutes. The supernatant was preserved in sterile eppendorf at -20OC until amplification.Identified isolates of 2.41. The programmed cycles for amplifications were described previously by Villa et al., 1995 and Villa et al., 1999 [Amplification procedure was performed by the use of 2 μl of the extracted DNA added to total mixture of 20 μl of amplification mixture supplied by Qiagen. The used primers were summarized in Table l., 1999 , 8.gyrA and parC.Restriction Fragment length Polymorphism for Detection of Mutations in HinfI (Promega) enzyme was used for the digestion of gyrA and parC in the amplified products of A. baumannii. The amplified products were purified by the use of QIA quick PCR purification kit  according to the manufacturer’s protocol. The eluant was incubated at 37°C for 2.5 hours with 10 U of HinfI. The digests were then separated by electrophoresis in 1.5% agarose gel stained with ethidium bromide and photographed with UV illumination. The presence of 291 bp indicated the presence of mutation in gyrA and the presence of 52 bp indicated the presence of mutation in parC.3A. baumannii were recovered from clinical isolates from ICUs during one year duration of the study. The majority of the isolates were recovered from wounds culture 60 (42.9%) followed by blood cultures 50 (35.7%) and urine cultures 30 (21.4%), (Table 2).Total 140 isolates of A. baumannii to antibiotics with resistance to ampicillin (100%), ampicillin/sulbactam (100%), imipenem (100%), ceftazidime (99.3%), cefipime (96.4%). Resistance to ciprofloxacin was 92.3%., (Table 3).There was a high resistance of the isolated A. baumannii with resistance to ciprofloxacine and levofloxacin had MIC >4 µg/ml. The most isolates had MIC >128 µg/ml (42.3%), (Table 4).The isolates of A. baumannii to ciprofloxacin had mutations in gyrA and parC. The most frequent mutations were combined mutations in both genes (85.5%) and 5% had single mutation either in gyrA or parC, (Table 5).All resistant strains of 6).The most frequent combined mutations was associated with MIC >128 µg/ml (42.3%), -based VITEK MS [In the present study VITEK MS .A. baumannii species.For antibiotics susceptibility tests antibiotics discs diffusion method was used for screening of the resistance pattern of the isolated A. baumannii isolates were multidrug resistance according to the definition of Magiorakos & Perl 2008 [A. baumannii to antibiotics with resistance to ampicillin (100%), ampicillin/sulbactam (100%), imipenem (100%), ceftazidime (99.3%), cefipime (96.4%). This pattern of antibiotics resistance is similar to that reported previously from Egypt on limited number of A. baumannii strains [All erl 2008 . Previouerl 2008 . However strains . Therefo strains . The rem strains .A. baumannii to ciprofloxacine has shown marked increase in the last few years in the developing countries with range from 95% up to 97.7% [By disc diffusion and MIC the resistance to ciprofloxacin was 92.3%. This is similar to previous study reported in 2017 and more than another study in Egypt, (75%) [to 97.7% , 19.et al., 2007 [In the present study all isolates of A. resistant by disc diffusion method were resistant by MIC method. Similarly, Stone A. baumannii is mediated mainly by mutation in gyrA and parC genes [Resistance of rC genes , 21, 22.A. baumanii isolates to ciprofloxacin had mutations of gyrA gene and/or parC by PCR-RFLP analysis described by previous studies [In the preset study, all resistant  studies , 8.gyrA and parC mutations at codon 83 and codon 80 with substitution of serine with leucine in gyrA and serine with leucine in parC.This method detects the presence of A. baumanii with MIC to ciprofloxacin more than 128 μg/mL had combined mutations in the gyrA and parC. These findings indicate that combined mutations are associated with high MIC resistance to ciprofloxacin. The combined mutations in these genes are associated with marked decrease in the binding affinity for quinolones in the resistant species [The isolates of  species -27.A. baumannii and similar findings were reported in previous studies [A. baumannii to ciprofloxacin should be clarified in different local epidemiological situations.The results of our study and the previous study from Egypt  suggeste studies , 22, 28.parC and gyrA are reported to be associated with a high-level of resistance to quinolones [parC without mutations in gyrA, suggesting that parC may had a primary role in developing resistance to ciprofloxacin without mutation in gyrA. Similar findings were reported by Ardebili et al., 2015 [Though combined mutations of inolones , four cll., 2015 .A. baumanii resistant to ciprofloxacine were associated with mutations in gyrA and parC. However, other mechanisms can be associated with this resistance such as changes in the efflux systems [A. baumanii.All the isolates of the  systems . FurtherA. baumannii. The most frequent mutations were those present in gyrA and parC genes. However, mutation in parC alone was not uncommon. Further, large scale studies are required to elucidate the resistance pattern of A. baumannii and its molecular mechanisms. From this study we can conclude that resistance to ciprofloxacin is common in clinical isolates of


Evaluation of the MVD  in a series of 79 cases of GISTs diagnosed by the Pathology Service at the HCPA  from January 1993 to December 2009.Seventy nine cases of GISTs were submitted to immunohistochemical analysis for CD31, an endothelial marker, to analyze MVD. Hot spots were identified for each case, and the mean numbers of stained blood vessels collected through Chalkley count, with the use of a 25 point grid, placed onto a scanned image. Images were analysed through an image analysis system. We used a cutoff of six vessels.Our series was composed of 42 males and 37 females and presented an average age of 58.9 years. GISTs were predominately located in the stomach (45.6%) followed by the small intestine (38.0%). Sixty seven GISTs (84.8%) showed an average of less than six vessels stained by CD31 (MVD) and 12 (15.2%) GISTs an average of more than six vessels. A statistically significant difference was observed between survival rate of patients having GISTs with MVD of ≤ 6 vessels  and patients having GISTs with MVD of ≥ 6 vessels , P = 0.001. No association for MVD was observed related to sex, age, histological type, risk category, location and metastasis.Seventy nine cases of GISTs diagnosed at a single center in South Brazil were studied for MVD . There was a statistically significant difference between MVD and the survival rate for these patients. The use of Chalkley method in GISTs may be helpful to evaluate clinical outcome. Gastrointestinal stromal tumors (GISTs) are the most common stromal tumors of the gastrointestinal tract, with an annual incidence of nearly 2/100,000/year . They afThe tyrosine kinase receptor, CD117, is present in 90% to 95% of GISTs, usually with diffuse cytoplasmic expression -16. The Angiogenesis, formation of new blood vessels, plays a central role in cancer survival, local tumor growth and development of distant metastasis -25. TumoSeventy-nine cases of GISTs diagnosed at the Department of Pathology of Hospital de Clinicas de Porto Alegre from January 1993 to December 2009 were submitted to immunohistochemistral analysis for CD31, (1:10 by DAKO), an endothelial marker, for MVD analysis. Procedures were made according to manufacturer’s instructions. For antigenic recovery we used citrate buffer with pH 6.0 and microwave oven. Initially, 3 to 5 microscopic fields (200 x) showing the highest microvascular density (hot spots) were identified, with the use of CD31 antibody . The meaClinical information and follow-up were obtained from medical records of Hospital de Clinicas de Porto Alegre: age, mounth/year of diagnosis, tumor location. In the retrospective follow-up analysis we looked for local recurrence, metastases, site of metastases, use of adjuvant therapy with tyrosine kinase inhibitors, date of the last medical appointment and death as a consequence of disease activity or other causes.The sample composed of 79 cases, 42 males and 37 females presented an average age of 58.9 years ± 13. Forty patients were over 60. All our cases were considered sporadic GISTs. Eleven cases (13.9%) were described as an incidental finding during surgery. GISTs were predominately located in the stomach (45.6%) followed by the small intestine (38.0%) and (26.4%) were Iocated in omentum and mesenterium. We have not identified any case of esophageal GIST.Spindle cell morphology was present in 72.2% of these tumors. Tumor size varied, ranging from 0.5 cm to 25.0 cm, with median of 4.8 cm. According to the NIH classification, 15.4% GIST were classified as very low risk category, 13.8% low risk category, 23.1% intermediate risk category and 47.7% belonged to the high risk category.CD117 was strongly expressed in 78 cases. Only one case was negative for CD117, but strongly positive for CD34, and negative for S-100 protein, desmin and actin (previous data not published). Microvessel density evaluation, through Chalkey method using scanned images, showed an average of less than six vessels (stained by the anti-CD31 antibody reaction) in 67 cases (84.8%), and an average of more than six vessels in 12 cases (15.2%). Out of 12 patients with a mean of MVD ≥ 6 vessels, 3 died (25.0%) and out of 67 patients having a mean of MVD ≤ 6 vessels, 3 died (4.5%). A statistically significant difference was seen when these findings were related to survival rates: MVD ≤ 6 vessels  and MVD ≥ 6 vessels , P = 0.001 . No assoOur patients were followed for a mean time of 2.5 ± 2.8 years (median 1.5 years). Nine patients developed metastasis, five involving liver and four peritoneal cavity. Three patients received adjuvant therapy with tyrosine kinase inhibitors for a period not longer then 3 months.Seventy-nine cases of GISTs from a single center in South Brazil were analysed for MVD to evaluate angiogenesis. Our series was composed of 79 cases, 42 males and 37 females with an average age of 58.9 years ± 13. GISTs were predominately located in the stomach followed by the small intestine, omentum and mesenterium. 15.4% GISTs were classified as very low risk category, 13.8% low risk category, 23.1% intermediate risk category and 47.7% high risk category (NIH classification).Our patients were followed for a mean time of 2.5 ± 2.8 years (median 1.5 year). Many cases were diagnosed and added to this study in the last three years, thus impairing follow-up time. Only nine cases (11.4%) have progressed to metastasis, involving liver and peritoneal cavity. Other GISTs series with a longer follow-up time, observed metastases in 27.0% to 54.0% of their cases. The low incidence of metastasis in our series is probably related to the short follow-up time . Only thA high MVD may indicate poor prognosis in different kinds of human neoplasias such as prostate carcinoma, adult astrocytoma, gastric and breast cancer. These same correlation was not observed for lung and bladder cancers and cerebellar medulloblastoma , 33, 34.MVD appeared to be an important independent factor of poor prognosis by multivariate analysis for adult astrocytomas (P = 0.001) . The stuImamura et al investigating angiogenesis in 95 GISTs, evaluated MVD through CD31 immunochemicalstaining. In his series multivariate analysis identified MVD and tumor grade as being two independent factors of worse prognosis , suggesting that the study of MVD may be a useful predictor of aggressive biologic behavior for GISTs .In our series microvessel density evaluation, through Chalkley method using scanned images, showed an average of less than six vessels (stained by the anti-CD31 antibody reaction) in 67 cases (84.8%) and an average of more than six stained vessels in 15.2%. Out of 12 patients with a mean of MVD ≥ 6 vessels, 3 died (25.0%) and out of 67 patients having a mean of MVD ≤ 6 vessels, 3 died (4.5%). A statistically significant difference was seen when these findings were related to survival rates: MVD ≤ 6 vessels  and MVD ≥ 6 vessels , P = 0.001. Histological quantification of tumor vascularity may be a significant prognosticator in GISTs. No association for MVD was observed related to sex, age, histological type, risk category, location and metastasis in the present work.Angiogenic activity may be measured by MVD, but other factors such as the vascular endothelial growth factor (VEGF), VEGF receptors , cell adhesion molecules, proteases and other cytokines markers may also be involved in the process. Future analysis for more information on the biology of tumor angiogenesis may be necessary , 27, 30.Seventy-nine cases of GISTs diagnosed at a single center in South Brazil were analyzed for MVD . There was a statistically significant difference between MVD and survival rates for these patients. The use of Chalkley method in GISTs may be helpful to evaluate clinical outcome.


Maternal diabetes causes congenital malformations and delays embryonic growth in the offspring. We investigated effects of maternal diabetes on mouse embryos during gastrulation and early organogenesis (ED7.5–11.5). Female mice were made diabetic with streptozotocin, treated with controlled-release insulin implants, and mated. Maternal blood glucose concentrations increased up to embryonic day (ED) 8.5. Maternal hyperglycemia induced severe growth retardation (approx.1 day) in 53% of the embryos on ED8.5, death in most of these embryos on ED9.5, and the termination of pregnancy on ED10.5 in litters with >20% dead embryos. Due to this selection, developmental delays and reduction in litter size were no longer observed thereafter in diabetic pregnancies. Male and female embryos were equally sensitive. High-throughput mRNA sequencing and pathway analysis of differentially expressed genes showed that retarded embryos failed to mount the adaptive suppression of gene expression that characterized non-retarded embryos . We conclude that failure of perigastrulation embryos of diabetic mothers to grow and survive is associated with their failure to shut down pathways that are strongly down-regulated in otherwise similar non-retarded embryos. Embryos that survive the early and generalized adverse effect of maternal diabetes, therefore, appear the subset in which malformations become manifest. Preexisting diabetes affects over 1% of all pregnancies and is rising in prevalence. Diabetic pregnancies are associated with embryonic growth delay3 and increase the risk for congenital malformations in the offspring6.The incidence of diabetes is increasing annually world-wide9, and may result from an adverse effect of high glucose concentrations on the normal development of neural crest cells11. Neural-tube defects range from a wavy neural tube via spina bifida at various locations to exencephaly and craniorhachischisis12, while cardiovascular malformations typically display defects of the outflow tract, atrial septum and atrioventricular valves, and extensive apoptosis13. The frequency of these malformations in several mouse strains is 10–15% of live ED10.5–13.5 embryos14. In rats15, but possibly not in mice16, pregnancies in such preconceptionally diabetic females are accompanied by growth retardation, developmental delay, embryonic death, and increased perfusion of the affected implantation sites17.Rodent models of pregestational diabetes are usually based on alloxan- or streptozotocin (STZ)-induced diabetes in females. Neural-tube and cardiovascular malformations are often seen19. Accordingly, mutant mouse models suggest that perturbations of mesoderm specification and cell migration during and shortly after gastrulation can cause neural-tube and cardiovascular defects20. We reported recently that maternal hyperglycemia impaired expression of genes involved in cell proliferation in non-retarded ED8.5 mouse embryos, and genes involved in cytoskeletal remodeling and oxidative phosphorylation in non-retarded mouse ED9.5 embryos21. A quarter of the differentially regulated genes (mostly down-regulated) were associated with neural-tube or cardiovascular defects if deficient21. Studies of older mouse embryos (ED ≥ 10.5) with manifest diabetic embryopathy showed that maternal hyperglycemia affected the expression of genes involved in apoptosis, proliferation, migration and differentiation during organogenesis in the offspring24. This change in the spectrum of affected genes is compatible with developmental stage-specific adaptation of gene expression.The rodent embryo is most sensitive to the teratogenic effect of maternal hyperglycemia if exposed during the pre- and early somite stages of development (ED7.5–8.5 in the mouse)25. In the present study, therefore, we investigated the developmental progress of embryos from diabetic mothers between ED7.5 and ED11.5 to determine if the manifestation of diabetic embryopathy at and after ED10.5 was preceded by a more general adverse effect on embryonic development. The study was performed in the FVB mouse strain, which is susceptible to malformations induced by maternal hyperglycemia26. Diabetes was induced with streptozotocin and treated with insulin implants, so that severe hyperglycemia only developed after implantation of the embryos7. Pathway analysis after high-throughput mRNA sequencing of one-day retarded experimental embryos revealed that these embryos typically failed to mount the adaptive and generally suppressive response in gene expression that characterized the ED8.5 and ED9.5 experimental embryos that were not retarded.A major difference between our and earlier reported findings is that many ED8.5 and ED9.5 mouse embryos of diabetic mothers  were developmentally retarded, whereas growth retardation and developmental delay were explicitly reported as not being observed in ED10.5 litters of diabetic dams21. All mice treated with STZ became diabetic. Blood glucose concentrations reached steady-state levels (approx. 25 mmol/L) within 3 weeks after inducing diabetes with STZ. After administering the controlled-release insulin implants, glucose levels declined but remained higher (approx. 14 mmol/L) in diabetic than in nondiabetic mice. Due to the fixed release rate of insulin and the increasing demand for insulin with progressing pregnancy7, blood glucose concentrations increased again 1.7-fold in the first week of pregnancy and plateaued after ED8.5. Only pregnant females with blood glucose concentrations exceeding 17 mmol/L at sacrifice were included in the analysis.The details about the efficacy of the STZ treatment protocol can be found in our previous studyFigure 22. Among the abnormal embryos, neural-tube defects  were prevalent, as reported12. Some embryos were also retarded in development. Figure 27. Additionally, the embryo in Fig. 15 and mouse embryos14.An inventory of the visible congenital malformations in 10 litters of ED9.5–11.5 embryos of pregnant diabetic mice yielded 12 abnormal embryos among in total 96 embryos . On ED8.5, 3 litters consisted of retarded embryos only, in 2 litters 60–70% of the embryos were retarded, and in 4 litters 10–40% of the embryos were retarded. Intriguingly, only 14% of the retrieved embryos with maternal hyperglycemia were retarded on ED9.5  and still fewer (3%) on ED10.5 .Since growth retardation appeared a common feature in embryos with maternal hyperglycemia and since ED7-8 was reported as the most sensitive period for the teratogenic effect of hyperglycemia in miceTo track the fate of the retarded embryos, we determined the number of live embryos per litter for each age Fig. . Materna28, we identified the male embryos by determining the presence of the Sry gene in the amniotic and chorionic membranes of ED7.5 and in the tails of older embryos. Table Because the sex of the embryo may determine its sensitivity to the teratogenic effects of maternal hyperglycemia21. To investigate the effects of growth retardation on gene expression, we performed SOLiD SAGE sequencing . In total, 8 ED7.5 embryos and 5 ED8.5 embryos from non-diabetic control mothers, and 7 ED8.5 embryos, 8 ED8.5 one-day delayed embryos (ED8.5-1), and 4 ED9.5 one-day delayed embryos (ED9.5-1) from diabetic mothers were screened. Clusters were generated with generalized linear models (GLM) based on the expression of the top 100 genes ranked by P-value, which separated the retarded from the non-retarded embryos in diabetic pregnancies and from the corresponding embryos in control pregnancies rd column). Only genes involved in antioxidant action of vitamin C were significantly upregulated. These pathways concur largely with those we identified earlier21 on the MetaCoreTM platform . The response in experimental ED8.5-1 of diabetic mothers compared to control ED7.5 embryos was more mixed: genes involved in ILK signaling and antioxidant action of vitamin C were e.g. highly upregulated, whereas NRF2-mediated oxidative stress and EIF2-mediated translation was significantly downregulated (4th column). Unfortunately, we do not have expression profiles of non-retarded experimental ED7.5 embryos. These data suggest that growth-retarded embryos in diabetic pregnancies fail to shut down many pathways that are strongly down-regulated in otherwise comparable non-retarded embryos and that this failure leads to their demise between ED8.5 and ED9.5.To find out whether the discrepancy between retarded and non-retarded embryos of diabetic dams converged on only a few metabolic or signaling pathways or, alternatively, indicated a failure of the retarded embryos to adapt to the maternal diabetic condition we performed pathway analysis of the differentially expressed genes. Figure 8, and develop visible malformations from ED10.5 onwards. Unexpectedly, we observed severe growth retardation in approx. 50% of the embryos of diabetic dams at ED8.5 and a approx. 30% decrease in litter size of these diabetic dams on ED9.5. Because we also observed diabetic dams with only dead embryos in their uterus, we interpret  when cultured in very high-glucose media32. Growth retardation and a temporary reduction in litter size were, however, not noted in diabetic mice16. However, in another study, a 0.5 day developmental delay on ED8.5 and a temporary reduction in litter size of diabetic pregnancies on ED9.5 were found, but the number of pregnancies studied was deemed too small to permit a conclusion33. These findings imply an early, rather general and possibly metabolic effect of maternal hyperglycemia during and immediately after gastrulation that leaves few traces due to the elimination of affected litters between ED9.5 and ED10.5, and a later, teratological effect that has thus far received most interest. If our finding can be extended to the human situation, the early effect would occur in the third week of gestation, so that the consequence, embryonic death, might not even be noted, except possibly for a perceived difficulty to become pregnant.In our experimental model, mouse embryos become sensitive to maternal hyperglycemia on ED7.5, as previously reported8. Our data shows that blood glucose levels in diabetic dams had increased to a plateau by that time21. Euglycemia during gastrulation is apparently crucial for normal embryonic development and further organogenesis (cf ref.20), because nearly half of the embryos of diabetic mice had developed a growth delay on ED8.5. Glycolysis is still the prevailing energy source in ED9.5 mouse embryos34, although studies in rat36 and mouse embryos37 suggest that the production of lactate decreases and mitochondrial activity slowly increases during early organogenesis. We observed that glycolytic activity and capacity had declined substantially in ED9.5 mouse embryos from diabetic mothers, suggesting that embryos of diabetic mothers had an energy problem21. In agreement, exposure of such embryos to a supernormal glucose concentration did not increase glucose oxidation37, but caused swelling of mitochondria38 and a significant decrease in reduced glutathione39.Even a temporary increase in circulating glucose concentration on ED7-8, that is, during gastrulation, suffices to bring about neural tube defects21. It is tempting to speculate that this failure to adapt to maternal hyperglycemia represents or includes a causal factor for the failure to grow and survive. The differential sensitivity of the (mouse) embryos to glucose may develop if the complex downstream effects of maternal hyperglycemia, including glutathione synthesis and oxidation, hexosamine synthesis, p53 upregulation and apoptosis41, are not strictly deterministic, but vary stochastically, so that the outcome is not uniform.These findings raise the question how such a general disturbance of metabolism can result in malformations in a restricted range of organ systems, such as the neural tube and heart. Our present data may explain this paradox, because shortly after becoming sensitive to hyperglycemia, embryos do suffer from a generalized adverse effect. Because embryos that were most severely affected by maternal hyperglycemia did not survive, the embryos with malformations appear to be those that were only relatively mildly affected by the early toxic effect of maternal hyperglycemia. The difference between the 2 groups of embryos appears to reside in their adaptive response in gene expression: the growth-retarded embryos were unable to adapt to the maternal diabetic condition by a pronounced, generally downward change in gene expression that was found in the non-retarded ED8.5 embryos 21. Pathway analysis on one-day delayed ED8.5 experimental embryos revealed a remarkably muted adaptive response in gene expression relative to the non-retarded experimental embryos. Integrin (ILK) signaling, antioxidant action of vitamin C and NRF2-mediated oxidative stress remained most strongly affected in growth-retarded embryos. ILK is the major regulator of Integrin-mediated signaling. Integrins are cell-adhesion molecules that bind to the extracellular matrix (ECM) and forge a connection between the ECM and the actin cytoskeleton42. Oxidative stress emerges as a more prominent factor in the present analysis on the Ingenuity platform than our previous analysis on the MetaCoreTM platform, but also this response was suppressed in the growth-retarded mouse embryos.Non-retarded ED8.5 embryos of diabetic dams suffer mainly from impaired cell proliferation, and ED9.5 embryos from impaired cytoskeletal remodeling and oxidative phosphorylationOur study showed that maternal hyperglycemia has an early and generalized adverse effect on embryonic development between ED7.5 and ED9.5 , whereas the characteristic malformations of the neural tube and cardiac outflow tract become apparent only thereafter. Hyperglycemia-mediated changes in gene expression were markedly absent in growth-retarded ED8.5 (mouse) embryos, while malformations developed in non-retarded, surviving embryos that were able to mount an adaptive response to hyperglycemia on ED8.5.http://labdiet.com/pdf/5020.pdf; production: ABDiets, Woerden, The Netherlands). Mice were kept on a 12-h light/12-h dark cycle at 22 °C with free access to water and food. The study was carried out in accordance with the Dutch Guidelines for the Care and Use of Laboratory Animals and approved by the Ethical Committee for Animal Research of the University of Amsterdam (ALC101225).FVB mice (9–11 weeks old) were obtained from Harlan Sprague Dawley  and fed an irradiated diet that was based on the Purina 9 F diet , freshly dissolved in sodium citrate , was injected intraperitoneally for 5 consecutive days. Hyperglycemia was treated with subcutaneous sustained-release insulin implants  that released approx. 0.1 U/day per implant for at least 30 days. Mice were mated to proven fertile males. Pregnant mice were sacrificed by cervical dislocation and embryos recovered on ED7.5, ED8.5, ED9.5, ED10.5, ED11.5, or ED13.5 (one litter only). Embryos were considered alive when their heart was beating (>ED8.5) or if the RIN number of RNA isolated from them was >9.0. The age of the embryos was verified by comparison with the criteria of Kaufman27.Diabetes was induced in female FVB mice with streptozotocin as recommended by the Animal Models of Diabetic Complications Consortium (AMDCC)44, whereas the reverse was described for mouse embryos45, we studied male and female embryos separately. DNA from the visceral yolk sacs of ED8.5 embryos, or the posterior part  of ED9.5 embryos was isolated with TriPure Isolation Reagent . The tissues were taken from the remaining parts of the embryos after harvesting the anterior part (ED8.5) and heart-containing segment (ED9.5) for mRNA quantification. The sex of the embryos was determined by a multiplex PCR amplification using primers to amplify the male-specific Sry gene and the autosomal Il3 gene for reference46.Because male human embryos were reported to be more sensitive to the diabetic state of their mothers than female embryosED8.5-ED10.5 embryos were fixed in Bouin’s fixative (saturated picric acid (15), formaldehyde (5), glacial acetic acid ) for 2 hours at 4 °C, stored in 70% ethanol, and embedded in paraffin. Serial transversely cut 7 μm-thick sections were stained with hematoxylin and eosin (HE), and photographed with a Leica DMRA2 microscope equipped with a Leica DM300 photo camera. Digital images from serial sections of mouse embryos were used to prepare three-dimensional reconstructions with Amira 5.2 software package .21. In the present study, we compared gene expression in control ED7.5 and ED8.5 embryos with one-day delayed ED8.5 and ED9.5 experimental embryos, respectively. Total RNA was extracted from whole ED7.5 and one-day delayed ED8.5 embryos, and from the anterior half of embryos  control ED8.5 and one-day delayed experimental ED9.5 embryos with the TriPure Isolation Reagent . Embryos were frozen and thawed 3 times using liquid nitrogen. After centrifugation, the aqueous phase of the TriPure extract was re-extracted 3 times with choloroform:isoamyl alcohol (24:1) and precipitated with ethanol after addition of 10 µg glycogen. The quantity of total RNA was determined with a Qubit 1.0 Fluorometer , while the quality was assessed with a Bioanalyser RNA 6000 Nano Chip . The RNA integrity number (RIN) of all samples was between 9.0 and 10.0. The total RNA yield per embryo was 1.0 ± 0.1 µg for one-day delayed ED8.5 embryos and 0.9 ± 0.1 µg for one-day delayed ED9.5 embryos. The relative abundance of individual transcripts was determined by the SOLiD SAGE approach, as in our previous study21. Pathway analysis of the differentially expressed genes was performed using QIAGEN’s Ingenuity Pathway Analysis .In our previous study, we compared gene expression in non-retarded control and experimental ED8.5 or 9.5 embryosBlood glucose concentrations and litter size are expressed as mean ± SEM. Statistical analyses were performed using nonparametric Mann-Whitney U and Kruskal-Wallis tests. The number of normal and retarded embryos was compared with embryonic age and streptozotocin treatment as variables, using contingency-table statistics. Expected values were based on column and row totals or Mendelian expectation (gender). In case of a significant overall Chi-squared value, the contributions to the Chi-squared value were tested to determine the source of the observed difference. A P-value < 0.05 was considered statistically significant.21 are also available in NCBI .The datasets generated during and/or analysed during the current study are available in NCBI . The datasets generated in our earlier studySupplemental Table 1 and 2


Arterial pressure (MAP) and heart rate (HR) were monitored by telemetry in a separate group (n = 8). Data from 5 consecutive days were analyzed for rhythmicity by cosinor analysis. Diurnal electrolyte excretion was assessed by metabolic cages. During lights-off, oxygen levels increased to 105.3 ± 2.1% in cortex and 105.2 ± 3.8% in medulla. MAP was 97.3 ± 1.5 mmHg and HR was 394.0 ± 7.9 bpm during lights-off phase and 93.5 ± 1.3 mmHg and 327.8 ± 8.9 bpm during lights-on. During lights-on, oxygen levels decreased to 94.6 ± 1.4% in cortex and 94.2 ± 8.5% in medulla. There was significant 24-h periodicity in cortex and medulla oxygenation. Potassium excretion  and the distal Na+/K+ exchange (0.72 ± 0.02 vs. 0.59 ± 0.02 P < 0.001) were highest in the lights-off phase, this phase difference was not found for sodium excretion (P = 0.4). It seems that oxygen levels in the kidneys follow the pattern of oxygen delivery, which is known to be determined by renal blood flow and peaks in the active phase (lights-off).Blood pressure, renal hemodynamics, electrolyte, and water excretion all display diurnal oscillation. Disturbance of these patterns is associated with hypertension and chronic kidney disease. Kidney oxygenation is dependent on oxygen delivery and consumption that in turn are determined by renal hemodynamics and metabolism. We hypothesized that kidney oxygenation also demonstrates 24-h periodicity. Telemetric oxygen-sensitive carbon paste electrodes were implanted in Sprague-Dawley rats (250–300 g), either in renal medulla ( Endogenous timing mechanisms have evolved to adapt to environmental changes imposed by alternating periods of light and darkness. Twenty-four hours patterns in sleep, activity, food intake, and excretion are well-known representatives of such physiological homeostatic adaptations. Via the kidneys, these homeostatic mechanisms maintain constancy of the bodies' extracellular fluid compartment throughout the 24-h cycle.In mammals, diurnal variations in urinary volume and electrolyte excretion are the best studied features of 24-h renal rhythm. Urinary excretion of water, sodium, and potassium peak during the active period of the day when intake is also highest  in renal tissue is lowest when sodium reabsorption activity is highest. On the other hand, the increased oxygen use in the kidney may be fully matched by increased oxygen delivery because 24-h variations in blood pressure and renal blood flow coincide with periods of highest excretion  ad libitum. To promote animal welfare and normal physiological activity around the clock, the rats were cohoused. Only during urine collection for electrolyte analysis were the rats housed individually for 24 h.Experiments were conducted in male Sprague Dawley rats . All procedures were approved by the Animal Ethics Committee of University of Utrecht (DEC 2014.II.03.015) and were in accordance with the Dutch Codes of Practice for the Care and Use of Animals for Scientific Purposes. All animals were kept on a 12 h light/dark cycle with lights-on at 6 a.m. (ZT 0), and lights-off at 6 p.m. (ZT 12). Rats had access to water and standard rat chow (contains 0.3% Nan = 7) or medulla (n = 9). The kidney was exposed via laparotomy. The telemeter  was placed in the rat abdomen. After 2 weeks of complete recovery and stabilization of oxygen signal, 24-h oscillations in pO2 were recorded continuously. In a third group, blood pressure telemeters  were implanted in the abdominal aorta (n = 8).The telemetry based technique to measure oxygenation in the kidney has been described in detail . The Cosinor method was applied to determine the amplitude and phase of the oscillation in the pO2 signal  to determine water and food intake and to collect urine. Urine was sampled in epochs of 12 h starting at 6 p.m. (lights-off/active phase) and continued at 6 a.m. (lights-on/resting phase). In these 12-h urine samples sodium and potassium concentrations were determined by flame photometry . Urinary creatinine was determined by DiaSys Kit . Distal sodium/potassium exchange was quantified as kaliuresis/, although this difference was not significant  was increased in the lights-off period  pO2 is mainly the result of a 24 variation in oxygen delivery to the kidneys.Normotensive rats display a significant diurnal rhythmicity in renal oxygenation  and by the Dutch Kidney Foundation (Project KJPB 12.029). BJ is supported by the Dutch Kidney Foundation (Grant 16OI21). This support is gratefully acknowledged.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.