Hemi-ablation of prostate cancer (PCa) requires an accurate prediction of laterality. Recently, multi-parametric magnetic resonance imaging (mpMRI) has recently been increasingly used to enhance clinical staging and characterization of tumor foci. Thus, we tried to investigate the real-life accuracy of combinatory approach of using both transrectal ultrasound (TRUS)-guided prostatic biopsy and mpMRI in predicting the laterality of PCa. We reviewed the records of 335 men who were suspected of having unilateral PCa on multi (≥ 12)-core TRUS-guided biopsy and preoperative mpMRI and subsequently had undergone radical prostatectomy (RP) at our institution. Based on the analysis of pathologic outcomes from RP, the performance of the combinatory approach in predicting the pathological laterality of PCa was evaluated. Pathology was classified to be unfavorable when showing a Gleason pattern of 4/5 or pT3/N1 features. Significant cancer was defined as non-organ-confined disease, having a Gleason pattern of 4/5, or showing a cancer volume of ≥ 0.5 mL. Among the 335 unilateral lobes not suspected to harbor tumor from either the TRUS biopsy or mpMRI, the actual absence rate of malignancy was only 13.7% from a pathologic analysis of RP specimens. Even among the 115 D’Amico low-risk group, the absence rate of malignancy was only 26.1% in unilateral lobes not suspected to harbor tumor. Among the 335 lobes, unfavorable pathology and significant cancer were not observed in 36.1% and 30.7%, respectively. The absence rates of unfavorable pathology and significant cancer among the D’Amico low risk group were 56.5% and 47.8%, respectively. Meanwhile, the absence rate of dominant Gleason pattern 4 or 5 was 74.9% among the 335 total subjects. Our real-life clinical experience showed that the combination of multi-core TRUS-guided biopsy and mpMRI did not provide reliable accuracy in the prediction of true unilaterality of PCa. A widespread implementation of prostate-specific antigen (PSA) screening has contributed to earlier detection of prostate cancer (PCa) at lower stages, lower grades, and smaller tumor volumes . MoreoveMeanwhile, a proper selection of candidates for focal therapy remains a major challenge in actual clinical practice. The success of focal therapy heavily depends on an accurate localization of significant tumor foci within the prostate. For hemi-ablation of PCa, accurate prediction of laterality of PCa is crucial. Traditionally, transrectal ultrasound (TRUS)-guided prostatic biopsy has been widely used to assess the laterality of tumor foci; however, the method was less than satisfactory with respect to its accuracy. To overcome such a limitation, multi-parametric magnetic resonance imaging (mpMRI) has been increasingly used to enhance clinical staging and characterization of tumor foci . RecentlThis retrospective study was approved by the Institutional Review Board (IRB) of Seoul National University Bundang Hospital . The approval number is B1706/402-115. The requirement for informed consent was waived. Personal identifiers were all removed and the data were anonymously analyzed.Between January 2010 and July 2015, a total of 730 patients were diagnosed with localized PCa  via extended multi (≥ 12)-core TRUS-guided biopsy, underwent preoperative mpMRI with ADC mapping, and subsequently received radical prostatectomy (RP) at a single tertiary center. Of these 730 patients, 335 men-who were suspected of having unilateral localized prostate cancer from preoperative clinical staging onmulti (≥ 12)-core TRUS-guided biopsy and mpMRI, separately, and eventually underwent RP without any prior treatment were finally included in the present study. The pathological analyses including histopathologic mapping by quadri-mount section were performed in all 335 patients following RP. Predictive performance of the combination of pretreatment clinical parameters (multi (≥ 12)-core TRUS-guided biopsy, and mpMRI) in predicting the pathological laterality of PCa was evaluated.MRIs were performed using a 3.0-T MR system  equipped with phased-array cardiac 6-channel coil. Thirty minutes prior to imaging, all patients were injected with 20 mg butylscopolamine  intramuscularly to suppress bowel peristalsis. Axial diffusion-weighted (DW) images were acquired using single-shot echo planar imaging. The scan parameters were as follows: TR, 2,500–3,000 ms; TE, 56–65 ms; slice thickness, 3 mm; interslice gap, 1 mm; field of view, 180 mm×180 mm; matrix, 92×90; and number of excitations, 10. Diffusion encoding gradients were applied as a bipolar pair at b-values 0 and 1,000 s/mm2. ADC maps were automatically generated on a pixel-by-pixel basis. MRI examinations were conducted at least 2 weeks after prostate biopsy.Two experienced uro-radiologists with more than 10 years of mpMRI experience, who were blinded to all clinical variables including pathological outcome, graded the level of suspicion for PCa from mpMRI using the Prostate Imaging Reporting and Data System Version 2 (PI-RADS V2) scale from 1 to 5 in following manner: grade 1, highly unlikely to be present; grade 2, unlikely to be present; grade 3, equivocal; grade 4, likely to be present; and grade 5, highly likely to be present) . Th. Th12]. Pathology was classified to be unfavorable when showing a Gleason pattern 4/5, or pT3/N1 features. . Signifi® SPSS® version 22.0, IBM, Armonk, New York, USA). The p values were considered statistically significant when <0.01.We first assessed the clinical characteristics of the cohort and the pathological outcome of the unilateral lobe not suspected to harbor tumor. The pathological outcomes were compared between the clinically low-risk group and high-risk group. Sensitivity analysis was performed to evaluate the accuracy of the combination approach in predicting the pathological outcome of each prostate lobe. Chi-square test was used for the comparison of pathological outcome and predictive performance. All statistical analyses were performed using SPSS 22.0 for Windows  at our institution between January 2010 and July 2015, there were 355 (45.9%) potential candidates for hemi-ablation who were suspected of having tumor only in the unilateral prostatic lobe from preoperative clinical staging , as shown in In this study, we observed that only about 14% of the potential candidates for hemi-ablation actually had true unilateral disease on final pathologic analysis of RP specimens. Moreover, although not many, some candidates even harbored locally advanced disease. Even when the analysis was limited to subjects in the low-risk group , only 26.1% were found to have pathologically-confirmed unilateral disease among the potential candidates for hemi-ablation. Even if the goal of hemi-ablation was only to remove of significant cancer or unfavorable pathology, such a goal would have only been achieved in about one third of our potential candidates for hemi-ablation. Moreover, even when limited to only those in the D’Amico low risk group, about half of the potential candidates for hemi-ablation were found to harbor unfavorable pathology or significant cancer in the unilateral lobe not suspected to harbor tumor preoperatively. Overall, the current study demonstrated that the combination of extended TRUS-guided prostate biopsy and mpMRI did not provide reliable accuracy in the prediction of unilateral PCa, which is essential in the identification of appropriate candidates for hemi-ablative focal therapy.Despite the appeal of performing organ-sparing treatment for PCa, concerns regarding the risk of failing to detect the significant tumor foci in untreated prostatic region largely limit the more widespread use of focal therapy. As widely known, PCa is often multifocal with incidence ranging from 67% to 87% . On the Several recent reports have offered promising findings on the accuracy of MRI in the depiction and localization of tumor foci in the prostate –20. OtheOn the other hand, not all published reports have offered such promising data on the accuracy of mpMRI. Though not on mpMRI, Jeong et al investigated the accuracy of prostate biopsy and conventional MRI in predicting the laterality of PCa, and found only a fair correlation between the combination approach  and laterality of PCa . In theiSeveral factors may have contributed to such a discrepancy. First, most of the published studies on mpMRI in men who underwent RP included only a small number of highly selected subjects , 23. In Our findings from real-life clinical practice revealed that the combination of contemporary extended TRUS-guided biopsy and mpMRI did not predict unilaterality of PCa with reliable accuracy. Therefore, the benefit of performing a repeat mpMRI-guided biopsy after the initial TRUS-guided biopsy, for the purpose of improving the accuracy of laterality prediction, such as in-bore biopsy, cognitive fusion or MRI/US fusion biopsy, might be limited. The mpMRI-guided biopsy before the initial biopsy could improve accuracy; however, to the best of our best knowledge, there is no published study on the specific subject. At this time point, a more accurate biopsy scheme is needed for the proper selection of candidates for hemi-ablative focal therapy. Although transperineal template-guided mapping biopsy and saturation biopsy approach may be more costly and likely to increase the level of patients’ burden and discomfort, such a scheme provides more precise representation of the intra-prostatic lesion. These new biopsy strategies should be thoroughly evaluated and refined to improve efficiency and safety for a wide application.In the light of recent studies, mpMRI appears to be a promising modality for localization of tumor foci. However, real-time monitoring of focal therapy seems to be technically challenging. For real-time monitoring of focal therapy, newer multiparametric ultrasonography, such as shear wave elastography and contrast-enhanced ultrasound, may well be more appropriate in the future. The present study has some limitations to consider. The retrospective design may be a limiting factor to our data. Moreover, only patients who underwent definitive treatment of radical prostatectomy were included in the study, which may have introduced selection bias. However, for a series on preoperative mpMRI in a RP cohort, the number of subjects in our study is relatively larger than or comparable to other similar series. Meanwhile, clinicopathologic profiles of our subjects appeared to be relatively more aggressive  than those reported from western series. The rate of PSA screening in Asia is still not as high as in Western countries . AlthougIn this study, we observed that the combination of multi-core TRUS-guided prostate biopsy and mpMRI did not provide reliable accuracy in predicting the true unilaterality of PCa in patients who underwent prostate biopsy, preoperative mpMRI, and RP. This suggests that improvement on biopsy and MRI technique is warranted for an accurate localization of tumor foci, which is crucial for the appropriate selection of candidates for focal therapy.


Background: Endothelial progenitor (EPC) and mesenchymal stromal cells (MSC) can regenerate damaged endothelium and thereby improve pulmonary endothelial dysfunction. We do not know, how extracorporeal membrane oxygenation (ECMO) might affect EPC- and MSC-mediated regenerative pathways in patients with congenital diaphragmatic hernia (CDH). Therefore, we investigated, if ECMO support impacts EPC and MSC numbers in CDH patients.Methods: Peripheral blood mononuclear cells from newborns with ECMO-dependent (n = 18) and ECMO-independent CDH (n = 12) and from healthy controls (n = 12) were isolated. The numbers of EPC and MSC were identified by flowcytometry. Serum levels of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-2 were determined.Results: EPC and MSC were elevated in newborns with CDH. ECMO-dependent infants had higher EPC subpopulation counts  before treatment compared to ECMO-independent infants. In the disease course, EPC and MSC subpopulation counts in ECMO-dependent infants were lower than before ECMO initiation. During ECMO, VEGF serum levels were significantly reduced (by 90.5%) and Ang2 levels significantly increased (by 74.8%).Conclusions: Our data suggest that ECMO might be associated with a rather impaired mobilization of EPC and MSC and with a depression of VEGF serum levels in newborns with CDH. Severe congenital diaphragmatic hernia (CDH) in newborns is accompanied by pronounced hypoplasia of both pulmonary tissue and the pulmonary vasculature . Thus, tTo improve survival and outcome of severe forms of CDH, therapies are needed that resolve pulmonary hypertension and the underlying hypoplasia of pulmonary tissue, such as therapeutic measures to promote growth of the pulmonary vasculature. In this respect, endothelial progenitor cells (EPC) and mesenchymal stromal cells (MSC) appear to be interesting potential research targets, since they have been demonstrated to promote angiogenesis and endothelial cell regeneration in several diseases with underlying endothelial dysfunction and damage in numerous experimental and pre-clinical studies , 7. WithIn this study, we hypothesized that ECMO in newborns with CDH upregulates the mobilization of EPC and MSC as well as serum levels of VEGF and Ang2 as potent progenitor cell mobilizers.n = 18) or no ECMO (n = 12) were selected from the neonatal intensive care unit (NICU) of the Department of Neonatology of the University Children Hospital Mannheim, University of Heidelberg directly after birth. In our manuscript, patients suffering from severe CDH with the need for and application of ECMO will be referred to as “ECMO-dependent.” Patients suffering from CDH but had not undergone ECMO will be referred to as “ECMO-independent.” The indication for and the allocation to ECMO were performed based on the recommendations made by the CDH EURO Consortium Consensus Team (2015 Update)  count to maintain adequate arterial oxygen saturation, elevated lactate, resistant systemic hypotension, and prolonged oxygenation index of more than 40 for at least 3 h (n = 12) at day 3 post-partum served as healthy controls. This study was approved by the local ethics committee of the Medical Faculty Mannheim of the University of Heidelberg and informed consent was obtained from the parents of all study subjects.Patient recruitment took place between 10/2010 and 04/2016. Newborns with CDH receiving ECMO  was obtained from the central venous catheter of ECMO-dependent newborns before connection to the ECMO system (day 0). Cannulation for ECMO in all study patients of the ECMO-dependent group was performed within 24 h after birth. Further blood sampling was performed at day 1, 3, and 7 during ECMO support, as well as directly before cannulation and at day 7 and 14 after decannulation or, respectively, on the day of discharge from the ICU, if discharged earlier than 14 days after decannulation. Blood samples from ECMO-independent newborns were obtained from the central venous catheter at the day of birth (day 0) and on day 3, 7, and 14 or, respectively, on the day of discharge from the ICU, if discharged earlier than 14 days after admission. In healthy controls, 2 mL of blood were collected in tubes containing sodium citrate (0.105 M) as anticoagulant by insertion of a 24-gauge cannula intravenously. All blood samples were processed within 4 h after collection. To reflect the respective course of the disease, we averaged all obtained blood samples for each patient of each group. The data generated by that measure are referred to as “disease-course”-data in the ongoing text. 6 cells) were incubated with 20 μL of FcR-blocking reagent  for 10 min to inhibit non-specific bindings. Thereafter, the cells were incubated at 4°C for 30 min with either 10 μL of PE-conjugated anti-human CD133 monoclonal antibody , 10 μL of FITC-conjugated anti-human CD34 monoclonal antibody , 5 μL of PerCP-conjugated anti-human CD45 monoclonal antibody and 10 μL of APC-conjugated anti-human CD31 monoclonal antibody or 5 μL of PE-conjugated anti-human CD90 monoclonal antibody , 5 μL of FITC-conjugated anti-human CD29 monoclonal antibody , 2 μL of PerCP-conjugated anti-human CD34 monoclonal antibody and 5 μL of APC-conjugated anti-human CD73 monoclonal antibody. Titration experiments have been performed for all antibodies. Isotype-matched immunoglobulin G1 and immunoglobulin G2a antibodies  served as negative controls and were used for each patient and measurement. The cells were washed three times to remove unbound antibodies and were finally resuspended in 400 μL of FACS Cellfix solution . FACS analysis was performed on a FACSCalibur flow cytometer (BD Biosciences), and the data were analyzed using WinMDI 2.8 software . A minimum of 500,000 events were collected. FACS analysis of each probe was performed in triplicates. The frequency of the expression of surface antigens was determined by a two-dimensional side-scatter/fluorescence dot-plot analysis of the samples after appropriate gating. EPC subpopulations were defined according to current protocols as CD45dim/CD34+, CD45dim/CD34+/CD133+, and CD45dim/CD34+/CD133+/CD31+  were prepared by density gradient centrifugation using Ficoll-Hypaque . The expression of cell-surface antigens was determined by immunofluorescence staining as described previously . One hun3+/CD31+ , 17 and 3+/CD31+ —were def9+/CD90+ . EPC andThe serum concentrations of vascular endothelial growth factor (VEGF) and Angiopoietin 2 (Ang2) were assessed using enzyme-linked immunosorbent assay kits  in triplicate samples obtained from 1 mL of serum. The enzyme-linked immunosorbent assays were performed according to the manufacturer's instructions.p < 0.05 was considered statistically significant for all results. All analyses were performed using the SAS system release 9.3 .All quantitative data are presented as median (range) and mean ± SD. Both parametric and non-parametric methods were used, as appropriate. All variables were examined for normal and non-Gaussian distribution by the Kolmogorov-Smirnov test. For comparison among normally distributed groups, one-way ANOVA, followed by pairwise multiple comparison (Student-Newman-Keuls method), was used. For non-normally distributed data, the non-parametric Kruskal-Wallis test followed by an all pairwise multiple comparison (Dunn's test) was used. Correlation analyses (according to Pearson or Spearman) were considered for all target variables that were considered statistically significant. Forty-two newborns  were included in our study. The ECMO-dependent group consisted of 18 and the ECMO-independent group of 12 CDH patients. The healthy control group comprised 12 healthy newborns. In total, six patients died, resulting in an overall in-hospital mortality of 20%; five cases of death in the ECMO-dependent group (28%); and one case of death in the ECMO-independent group (8%). For characteristics of the study population, see p = 0,619, At day 0 there were no significant differences in the basic blood cell counts including total leukocyte count (LC), hemoglobin (Hb), hematocrit (Hct), creatinine (Crea), and c-reactive protein (CRP) between the ECMO-dependent, the ECMO-independent and the healthy control group. The platelet count (Plt) at day 0 was significantly lower in the ECMO-dependent compared to the ECMO-independent group . In the dim/CD34+, EPC-CD45dim/CD34+/CD133+, EPC-CD45dim/CD34+/CD133+/CD31+) were significantly higher in the ECMO-dependent group compared to the ECMO-independent group and to the healthy control group  in the ECMO-dependent group, whereas subpopulation counts of MSC did not change significantly . Furthermore, VEGF serum levels were associated with MSC CD34−/CD29+/CD73+/CD90+ counts in the ECMO-dependent group .Correlation analysis revealed a significant association between decreased VEGF serum levels and EPC CD45In this study, we examined the influence of ECMO on the mobilization of EPC and MSC in newborns with CDH. Contrary to our hypothesis, our results indicate, that ECMO support in newborns with CDH is associated with decreased numbers of circulating EPC and MSC. Furthermore, ECMO support in newborns with CDH is associated with decreased VEGF and increased Ang2 serum levels.The mobilization of EPC and MSC is differentially regulated in several diseases with marked endothelial dysfunction or systemic inflammation such as sepsis and ARDS , 20, 21.Furthermore, our results show that—besides EPC- also mean MSC counts were elevated in CDH patients compared to healthy controls at day 0, when excluding the influence by ECMO. As ECMO seems to be associated with suppressed EPC and MSC mobilization, this could negatively—or at least not beneficially- impact vessel development in the severely impaired pulmonary vessel network in CDH patients undergoing ECMO. This hypothesis is supported by the current literature, which suggests that low numbers of EPC in newborns are associated with endothelial and vascular dysfunction and thereby with the development of bronchopulmonary dysplasia , 30. As +/CD45dim numbers during ECMO support. A previous study demonstrated elevated VEGF-A serum levels in newborns with CDH compared to normative data available in the literature  and could not be detected in ECMO-independent newborns. In general, VEGF is strongly involved in the mobilization of EPC, promotes EPC- and MSC-dependent angiogenesis and improves MSC viability –35. Our terature . Contrarterature , but itsterature –40. The terature .To interpret the results of our study within a clinical context and with respect to the design, we have to discuss the limitations of our study. Although our study has a prospective design, it is—however—an observational and not an interventional study, which has an impact on gross data interpretation. We were not in control of certain confounders and can mainly observe associations and not deduce cause and effect. Thus, the conclusions drawn from our results on the impact of ECMO on EPC, MSC, VEGF, and Ang2 remain mainly associative. Also, the need for blood products or volume support during ECMO, which we did not assess systematically, might have confounded serum levels and cell counts in patient samples. Further, we could demonstrate a slight decrease of leucocyte numbers directly after initiation of ECMO support, which might indicate that there has been a dilutional effect on early blood samples by priming of the ECMO circuit. A further limitation of our study is that we did not investigate the biological behavior of both the isolated EPC and MSC in mechanistic experiments. Thus, we can only speculate about the potential clinical impact of our findings at this time.In conclusion, our study results indicate, that ECMO might be associated with an impairment of endogenous mobilization of both EPC and MSC subpopulations, as well as VEGF serum levels in infants with CDH. These results raise the question whether an increase of EPC, MSC or VEGF could beneficially influence the clinical course of CDH patients undergoing ECMO, which needs to be addressed in future studies. Furthermore, future research questions in that regard should also address the necessary amount of stem cells to transplant and the optimal timepoint for stem cell transplantation in the course of ECMO-therapy in CDH. An increase of either EPC or MSC could be accomplished by stimulation of the endogenous mobilization, or by exogeneous transplantation. A potential source of stem cells for therapeutic usage in prenatally diagnosed CDH could be the umbilical cord . CurrentAll datasets generated for this study are included in the article/The studies involving human participants were reviewed and approved by Ethics committee of the Medical Faculty Mannheim of the University of Heidelberg. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.NR, CP, US, and TS contributed to the concept and design, acquisition, interpretation of data, and drafting of the article. TV, CW, BT, and GB contributed to the interpretation of data and revised the article for important intellectual content. All authors approved the final version of the article.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.


We investigated whether changes in adiponectin levels over time predict incident metabolic syndrome (MetS) in a population-based prospective study. In total, 1110 subjects were categorized into four groups according to their sex-specific median baseline adiponectin levels and the change in adiponectin levels at follow-up: low baseline adiponectin and decreased adiponectin during follow-up (LB&DF), low baseline adiponectin and increased adiponectin during follow-up (LB&IF), high baseline adiponectin and decreased adiponectin during follow-up (HB&DF), and high baseline adiponectin and increased adiponectin during follow-up (HB&IF). During the median 2.4-year follow-up period, 180 (16.2%) subjects developed MetS. Compared to the LB&DF group, the fully adjusted hazard ratio  for incident MS was the lowest in the HB&IF group ), followed by the HB&DF group ) and LB&IF group ). This phenomenon was more prominent in men than in women. Among the individual MetS components, increased adiponectin levels during follow-up were significantly associated with lower risks of incident low high density lipoprotein (HDL) cholesterol and incident high blood pressure. This finding suggests that a change in adiponectin level, as well as the baseline adiponectin level, might have a clinical role in the development of MetS among men. Metabolic syndrome (MetS) refers to a cluster of cardiovascular disease risk factors whose underlying pathophysiology is related to insulin resistance; these factors include central obesity, dyslipidemia, impaired glucose tolerance, and hypertension . MetS isMetS is closely linked to obesity and adipose tissue dysfunction. Dysfunctional adipose tissue secretes signaling molecules and hormones, which are collectively termed adipokines. Adiponectin, one of the adipokines exclusively secreted from adipose tissue, has protective properties against obesity-related diseases . AlthougAlthough previous studies have reported that higher baseline adiponectin levels are protective against incident MetS , little n = 334); (2) low adiponectin at baseline and increased adiponectin during follow-up (n = 221); (3) high adiponectin at baseline and decreased adiponectin during follow-up (n = 401); and (4) high adiponectin at baseline and increased adiponectin during follow-up (n = 154). At each visit, informed written consent was obtained from all participants. The study protocol was approved by the Ethics Committee of the Korean Center for Disease Control and the Institutional Review Boards of the Yonsei University Wonju College of Medicine (YWMR-15-9-075).The present study used data from the Korean Genome and Epidemiology Study on Atherosclerosis Risk of Rural Areas in the Korean General Population (KoGES-ARIRANG), a population-based prospective cohort study to assess the prevalence, incidence, and risk factors for chronic degenerative disorders such as hypertension, diabetes, and cardiovascular disease . KoGES-AAt the baseline and follow-up examinations, study participants completed a standardized medical history and lifestyle questionnaire and underwent a comprehensive health examination according to standard procedures. Body weight and height were measured while participants were wearing light indoor clothing without shoes. Blood pressure was measured from the right arm after the participant had rested for at least 5 min in a quiet room using a standard mercury sphygmomanometer . Two measurements were made with at least 5 min intervals in between, and the mean of the two measurements was used in the analyses. Smoking status and exercise level were determined based on self-reports. Current smokers were defined as participants who had smoked ≥100 cigarettes in their lifetime and who reported “currently smoking”’ in the questionnaire. Subjects who answered “yes” to the question “Do you perform physical exercise regularly enough to make you sweat?” were assigned to the regular exercise group. A venous blood sample was drawn from study participants after fasting for ≥12 h or overnight. Serum aliquots were centrifuged then stored at −80℃ until thawed for adiponectin analysis within one week after centrifugation. The serum concentrations of adiponectin were measured using radioimmunoassays (RIAs)  with intra-assay and inter-assay coefficients of variation ranging between 2.9% and 6.6%. Fasting glucose was determined using a glucose oxidase-based assay. The serum concentrations of total cholesterol, high density lipoprotein (HDL) cholesterol, and triglycerides (TG) were measured using enzymatic methods . High sensitivity C-reactive protein (hs-CRP) was measured using the Denka Seiken  assay, which has been validated against the Dade Behring method. The homeostasis model assessment for insulin resistance (HOMA-IR) levels were calculated using the following formula: fasting plasma insulin (μ IU/mL) × fasting plasma glucose (mg/dL)/405.The study endpoint was incident MetS at the follow-up visit. The definition of MetS in this study was based on a modification of the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) criteria . MetS wap-value of <0.05 was considered statistically significant, and SAS software, version 9.2  was used to conduct all analyses. Since the women had higher levels of adiponectin compared to the men, we performed all analyses separately for men and women. The distributions of serum TG, adiponectin, hs-CRP, and HOMA-IR scores were markedly skewed; therefore, they were log-transformed for the analyses where normality was required. We divided the study population according to the median baseline serum adiponectin level with sex-specific cut-offs of 9.31 mg/mL for men and 13.62 mg/mL for women. Additionally, we divided participants into two categories according to the change in adiponectin level at the follow-up. Differences among the groups were analyzed using analysis of variance (ANOVA) with the Bonferroni post hoc analysis method for continuous variables and the chi-squared test for categorical variables. Cox proportional hazards analyses were conducted to estimate the hazard ratio (HR) and 95% confidence interval (CI) for the association between adiponectin levels and the development of MetS. A p < 0.001). However, there were no significant differences in follow-up adiponectin levels between the LB&IF group and HB&DF group. The proportions of regular exercise were not different among four groups.The baseline characteristics of the study participants according to the baseline and change in adiponectin levels are presented in p = 0.10).p for trend <0.001). The risk of incident high TG was the lowest in the HB&IF group followed by the HB&DF group and the LB&DF group, in that order (p for trend = 0.002). The risk of incident high blood pressure was the lowest in the HB&IF group followed by the LB&IF group and the HB&DF group, in that order (p for trend = 0.002). When the analysis was stratified by sex (p for trend = 0.002).d by sex , the resIn this longitudinal cohort study in a healthy Korean population, we demonstrated that an increase in adiponectin level over the 2.4-year follow-up period was significantly associated with a lower risk of developing MetS. We also found that the lower risk of incident MetS in subjects with increased adiponectin levels during follow-up was significantly independent of baseline adiponectin levels. Moreover, we observed that the association between changes in adiponectin levels and the risk of developing MS was more prominent in men than in women. To the best of our knowledge, this is the first population-based study of the relationship between changes in adiponectin level and MetS that considers the sex-specific effect of circulating adiponectin. Adiponectin has beneficial effects on glucose homeostasis, chronic low-grade inflammation, oxidative stress, and atherosclerotic processes, so this molecule has usually been considered a salutary adipokine . ParadoxThe other principal finding of our study is the sex-specific association between circulating adiponectin levels and incident MetS with the salutary effect of increased adiponectin levels during follow-up being observable only among men. Contradictory findings have previously been reported regarding a sexually dimorphic association of adiponectin with metabolic disease ,18. To iOur study has several limitations. First, we measured only the total adiponectin level. Adiponectin is secreted from adipocytes into the bloodstream in three oligomeric complexes: trimers, hexamers, and high molecular weight (HMW) multimers comprising at least 18 monomers. Generally, HMW adiponectin is responsible for the majority of the insulin sensitizing effects . HoweverIn summary, this longitudinal study performed in a healthy, rural, Korean population revealed that changes in circulating adiponectin level over 2.4 years of follow-up, as well as adiponectin levels at the baseline, were independently associated with a lower risk of MetS. When the analysis was stratified by sex, these associations were only observed in men, whereas no associations regarding developing MetS or majority of its components were detected in women. These results suggest that high or increased serum adiponectin levels might play a protective role against metabolic disease especially in men. Additional long-term prospective studies in other populations, including subjects with various other health conditions, are needed to support our conclusion that increased adiponectin levels, as well as high baseline adiponectin levels, independently lower the risk of incident MetS.


India had the largest number of under-5 deaths of all countries in 2015, with substantial subnational disparities. We estimated national and subnational all-cause and cause-specific mortality among children younger than 5 years annually in 2000–15 in India to understand progress made and to consider implications for achieving the Sustainable Development Goal (SDG) child survival targets.We used a multicause model to estimate cause-specific mortality proportions in neonates and children aged 1–59 months at the state level, with causes of death grouped into pneumonia, diarrhoea, meningitis, injury, measles, congenital abnormalities, preterm birth complications, intrapartum-related events, and other causes. AIDS and malaria were estimated separately. The model was based on verbal autopsy studies representing more than 100 000 neonatal deaths globally and 16 962 deaths among children aged 1–59 months at the subnational level in India. By applying these proportions to all-cause deaths by state, we estimated cause-specific numbers of deaths and mortality rates at the state, regional, and national levels.In 2015, there were 25·121 million livebirths in India and 1·201 million under-5 deaths . 0·696 million (57·9%) of these deaths occurred in neonates. There were disparities in child mortality across states (from 9·7 deaths [Goa] to 73·1 deaths [Assam] per 1000 livebirths) and regions (from 29·7 deaths [the south] to 63·8 deaths [the northeast] per 1000 livebirths). Overall, the leading causes of under-5 deaths were preterm birth complications , pneumonia , and intrapartum-related events , with cause-of-death distributions varying across states and regions. In states with very high under-5 mortality, infectious-disease-related causes (pneumonia and diarrhoea) were among the three leading causes, whereas the three leading causes were all non-communicable in states with very low mortality. Most states had a slower decline in neonatal mortality than in mortality among children aged 1–59 months. Ten major states must accelerate progress to achieve the SDG under-5 mortality target, while 17 are not on track to meet the neonatal mortality target.Efforts to reduce vaccine-preventable deaths and to reduce geographical disparities should continue to maintain progress achieved in 2000–15. Enhanced policies and programmes are needed to accelerate mortality reduction in high-burden states and among neonates to achieve the SDG child survival targets in India by 2030.Bill & Melinda Gates Foundation. An estimated 1·201 million deaths occurred in children younger than 5 years in India in 2015—the largest number of such deaths among all countries. The number reduced from 2·516 million in 2000,5Information on national, regional, and state-level under-5 mortality rates and causes of deaths is of great value to guide health spending on child survival programmes. This is particularly true for India, with its decentralised health policy and decision making,Evidence before this studyWe have been routinely reviewing and publishing studies on child mortality by cause estimates at global and national level since 2003. This study shared the same search strategies and screening criteria as our last publication for the 2000–15 period, with search terms covering child, mortality or death, and leading causes of deaths. We identified child cause-of-death studies published until Feb 12, 2015, in the following databases: PubMed, Embase, ISIS Web of Knowledge, Medline BIOSIS, Popline, WHOLIS, and IndMed. We have previously reported estimates of all-cause mortality at national and district levels for India for the years 2001 and 2012, as well as cause-specific child mortality estimates based on empirical data from the Million Deaths Study for major states and selected major causes for children younger than 5 years. The Global Burden of Disease Study has published mortality by cause among all age groups, including deaths among children younger than 5 years, in India. However, there are few data currently available for children younger than 5 years, especially at the subnational level, for the years 2000–15. Thus, this study aimed to fill this gap and provide more detailed modelled estimates by state, age, and cause.Added value of this studyWe present all-cause and cause-specific mortality estimates at state level annually for 2000–15 based on a comprehensive set of high-quality national and subnational verbal autopsy studies and a multicause model. We provide transparent time trends for neonates and children aged 1–59 months at national and subnational levels, reflect on achievements against Millennium Development Goal (MDG) 4, and highlight areas where greater efforts are required if states are to achieve the Sustainable Development Goal (SDG) 3 child survival targets by 2030.Implications of all the available evidenceInformation on child mortality by cause at the state, regional, and national levels shows that achievements with regard to child survival have been made in India in the MDG era, informing policy making and resource allocation in the SDG era. For example, efforts in integrated newborn care packages could further improve neonatal health outcomes. High and consistent commitment from local governments is crucial to reduce subnational disparities. Further work is required to improve the resolution of all-cause and cause-specific estimates to support decentralised decision making. Continued investment in high-quality and timely collection, dissemination, and use of child cause-of-death data at the subnational level in India is essential. Although some states are on track or have already achieved the SDG child mortality targets, ten of the 25 major states need to accelerate progress during 2015–30 to meet the under-5 mortality target, and 17 need to accelerate progress to meet the neonatal mortality target by 2030.We largely followed our previously published methods to estimate livebirths, under-5 all-cause mortality rates, and number of deaths per state (including union territories).We derived state-level neonatal mortality rates using similar methods and data sources to those used for crude birth rate, with missing values imputed on the basis of the relative relationship of infant mortality rate among states. State-level under-5 mortality rates were obtained with one of two approaches. For 20 large states, we used the under-5 mortality rate from the sample registration system,State-level cause-specific mortality proportions for neonates and children aged 1–59 months were estimated separately with multinomial logistic regression models using data from verbal autopsy studies. The analytical framework has been published elsewhere.24The model input data were different for the two age groups. For neonates, input data from all countries with high mortality where verbal autopsy studies were available were used.For children who died at between 1 month and 59 months of age, we developed a subnational verbal-autopsy-based multicause model, using only subnational verbal autopsy studies from India to best account for the cause-of-death profile in this unique setting . Verbal 26We excluded deaths due to AIDS and malaria in the multinomial model because they were difficult to estimate stably because of the low disease burden. Instead, AIDS and malaria were estimated separately. AIDS deaths per state were estimated by the Indian Council of Medical Research using Spectrum, an analytical software that has been routinely used by UNAIDS. Malaria deaths per state were estimated by the WHO Malaria Programme on the basis of reported malaria cases and case fatality rates.We grouped the remaining causes of death into the following categories, based on International Classification of Diseases 10th revision:Haemophilus influenzae type b (Hib) vaccine on deaths due to pneumonia and meningitis, we included a post-hoc adjustment, details of which have been published previouslyAfter the verbal-autopsy-based multicause model had been parameterised, we used state covariates to predict state-level cause-specific mortality proportions. Details of how the state prediction covariate database was prepared and the final covariates retained are provided in the We applied the state-level cause-specific mortality proportions by age to state-specific and age-specific all-cause deaths to derive age-specific and cause-specific numbers of deaths annually from 2000 to 2015 for each of the 35 states. An overview of the estimation process is shown in the We report deaths by cause for each of the 23 major states, with the 12 smaller states grouped into northeastern states  and other states  on the basis of geographical location . Small sWe report estimates nationally and by state, region , and EAGA status . We alsoWe compared our estimates with other estimates for the same period based on the 2000–15 Million Death Study (MDS)Our study conformed to the Guidelines for Accurate and Transparent Health Estimates Reporting (GATHER) statement .30 AnalyThe funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to all the data in the study, and the corresponding author had final responsibility for the decision to submit for publication.In 2015, an estimated 25·121 million children were born in India, and 1·201 million children died before the age of 5 years, equating to an under-5 mortality rate of around 47·81 per 1000 livebirths . 0·696 mStates with the highest burden (numbers) of under-5 deaths were mostly those clustered in the central and east regions, with half of all under-5 deaths occurring in three states: Uttar Pradesh, Bihar, and Madhya Pradesh . A similNationally, the leading causes of under-5 deaths in 2015 were preterm birth complications , pneumonia , and intrapartum-related events . Among neonates, the leading causes were preterm birth complications , intrapartum-related events , and neonatal sepsis or meningitis  had a lower proportion of neonatal deaths (56·0% vs 63·2%), but higher proportions of preterm birth complications (28·1% vs 25·7%) and diarrhoea (10·6% vs 6·3%) as causes of death  to the lowest (south region), increased from 1·4 to 2·1 in 2000–15. This increase appeared to accelerate after 2005 . The norReductions in under-5 mortality rate also varied greatly across states, from 62·6% in Tamil Nadu to 26·2% in Assam. Of the five states with the highest under-5 mortality rate in 2000, four  reduced their under-5 mortality rate by nearly half (46·6–49·3%). The exception was Assam, which had the smallest reduction in under-5 mortality rate during 2000–15, and had the highest under-5 mortality rate in 2015. When the reduction was annualised, ten of the 25 major states had annual rate of reduction in under-5 mortality rate greater than 4·4%. Most states (80%) had much slower declines in neonatal mortality rate than those among children aged 1–59 months. Uttar Pradesh, for example, had one of the highest annual rates of reduction among children aged 1–59 months (6·1%), but one of the lowest annual rates of reduction in neonatal mortality rate 2·9%; .Cause-specific mortality proportions changed gradually at the national level during 2000–15 . The thrAmong children aged 1–59 months, the three leading causes of death in 2000 were all related to infectious diseases: pneumonia, diarrhoea , and measles. During 2000–15, the measles-specific mortality rate declined rapidly and, by 2015, injuries had overtaken as the third leading cause of death in this age group . Annual Disparities in annual rates of reduction in 2000–15 were observed across causes and across states . GeneralAlthough a few states have already achieved and some others are on track to achieve the SDG child survival targets, the remaining states, and India as a whole, will need to accelerate progress substantially. To achieve the SDG under-5 mortality rate target by 2030, ten of the 25 major states need to accelerate progress in terms of their annual rate of reduction during 2015–30 , includiIn 2015, India had the largest number of under-5 deaths globally, and almost 60% of these deaths occurred in the neonatal period. Compared with estimates from the National Family Health Survey 4, our estimated under-5 mortality rate and neonatal mortality rate were similar in the 2010–15 period, but showed a faster decline since 2000, both nationally and in major states.Geographical and socioeconomic disparities in child survival exist throughout India. Regional disparities worsened during 2000–15, especially after 2005. Pneumonia and diarrhoea were among the top three leading causes in the northeast region, which had the highest under-5 mortality rate. Preterm birth complications were particularly important in the EAGA states. Disparities were also present across age groups and causes. In the majority of the states, reductions in under-5 mortality rate were driven by reductions in deaths among children aged 1–59 months, whereas most states had a much slower decline in neonatal mortality rate. The relative importance of preterm birth complications increased over the 2000–15 period in states with large numbers of under-5 deaths. Diarrhoea had a faster decline than many other causes across almost all states. However, the decline was slower in states with high versus those with low under-5 mortality rates.National efforts have been made to improve child survival in 2000–15 in India. The National Rural Health Mission was implemented in 2005 to increase maternal and child health services in rural areas, strategically focused in EAGA states and the northeast region, which might have contributed to the improvement in child survival and mitigated regional disparities.Progress in India is crucial to improving child survival globally. The leading causes of preterm birth complications, pneumonia, intrapartum-related events, neonatal sepsis or meningitis, diarrhoea, and injuries should receive more attention from child survival policy makers and programmes. In this study, we considered the effect of the Hib vaccine on pneumonia and meningitis mortality. The newly introduced pneumococcal conjugate vaccinePreterm birth complications were the leading cause of under-5 deaths in India in 2000–15, and the rate of mortality from this cause saw little change during this period, especially in EAGA states. Cost-effective interventions, such as kangaroo care, thermal control, breastfeeding support, and basic care for infections48Diarrhoea remains an important cause of death, particularly in states with high under-5 mortality rates and low socioeconomic status. However, improvement of diarrhoea survival has never been more promising.The differences in numbers of cause-specific deaths between our study (MCEE), MDS, and GBD were largely due to 17% fewer all-cause under-5 deaths and 8% fewer livebirths estimated by the UN Inter-agency Group for Child Mortality Estimation than were estimated by GBD .1, 19, 2Each of the approaches used to estimate state-level mortality rates in this study and in MDS had strengths and limitations. MCEE estimates were based on standardised procedures, making the estimates internationally comparable, but they might be less capable of reflecting short-term changes. By contrast, MDS estimates were based on annual data, which show more fluctuations for smaller causes (data not shown). Such fluctuations could reflect both real changes and data quality issues. Although representative and timely data from a high-quality vital registration system would be the ideal source of data, we believe in the value of both estimation approaches, which serve different audiences and purposes. We intended to compare our approach with the GBD state-level estimates, but were unable to do so because GBD estimates are only available through the visualisation tool, but are not yet downloadable. Improved transparency would promote understanding, comparison, and enhancement of global health estimates.The present study had some limitations. First, many states in the northeast region are small states with little data available from the sample registration system. To fill these data gaps, for example on neonatal mortality rate, we chose to borrow information from nearby states . In Assam, the proportion of under-5 deaths that were neonatal was 37·3%. States in the northeast for which information was borrowed from Assam had a large share of the regional under-5 deaths, and the relatively low proportion of neonatal deaths in Assam might have led to an artificially low proportion of neonatal deaths in the entire northeast region. This borrowing of data would also affect cause-specific estimates because cause-specific mortality fractions were applied to all-cause deaths among neonates and older children separately. Consequently, although preterm birth complications were the leading cause among neonates, they were not the leading cause among all children under 5 years of age in the northeast region. This limitation might also explain why, despite a high under-5 mortality rate, preterm birth complications were not the leading cause in the northeast region but were in the other regions.A second limitation was that, although the modelling strategies used for neonates and older children were similar, the two age groups relied on different input data: neonatal data were taken from verbal autopsy studies from all high-mortality countries, whereas data for older children relied on Indian subnational verbal autopsy studies. Such a difference was justified because data availability varies by age group. The number of studies reporting Indian subnational causes of death among neonates was too small to derive stable estimates. Additionally, many of the India subnational studies had missing data for important causes such as pneumonia, thereby further reducing the reliability of estimates based only on Indian studies. When comparing the input data from India versus other high-mortality countries, we found no substantial differences in the cause-of-death distributions among neonates . Thus, wFinally, our uncertainty calculations did not consider all sources of errors. For example, although we adopted the exponential growth assumption when interpolating for annual population estimates, the likely errors  were not explicitly propagated in our uncertainty estimates. Similarly, borrowing estimates  from another state might have incurred errors, and such errors were also not explicitly accounted for in our uncertainty estimation. To do so, we would have had to make assumptions about the distribution of such errors, which itself cannot be empirically tested.In summary, our estimates are based on the most comprehensive set of high-quality, subnational, verbal autopsy studies to date. This study provides reliable, albeit conservative, time trends on all-cause and cause-specific under-5 mortality at the national and subnational levels in India with transparency. Future work is needed to further increase the resolution of estimates on all-cause estimates and particularly cause-specific estimates . The demand for this information is driven by decentralised decision making and child survival programme efforts, such as the scaling-up of pneumococcal conjugate vaccine and the continuation of Mission Indradhanush at district level. Among older children, as vaccine coverage data were used as model inputs and in post-hoc adjustment, these estimates should not be used to infer causality between vaccine and mortality decline.The evidence base for child mortality and causes of death can be improved through a combination of more and better data collection. Standard verbal autopsy instruments and cause-ascertaining methods should be implemented. Innovative data collection, such as minimally invasive tissue samplingstudy data on the MCEE website see https://www.jhsph.edu/research/centers-and-institutes/institute-for-international-programs/current-projects/maternal-child-epidemiology-estimation/maternal-newborn-and-child-cause-of-death/index.htmlFor access to Spectrum see https://www.avenirhealth.org/software-spectrum.phpFor more on Additional details of the input data, estimation methodology , and estimates are publicly available through the MCEE website.


High level of reactive oxygen species (ROS) has been detected in almost all cancers, which make it become one of the best-characterized phenotypes in cancers. Though ROS plays an important role in tumors, the degree of oxidative stress can be better evaluated by assessing stable metabolites of oxidative reactions because of its high instability. 8-hydroxy-2′-deoxyguanosine (8-OHdG), a product of oxidative damage to 2′-deoxyguanosine, is known as a useful marker for assessing oxidative DNA damage and has been a feature of carcinogenesis in several researches. But the exact prognostic value of 8-OHdG expression in patients with cancer is still unclear.A comprehensive search was performed in PubMed, Web of Science, EMBASE. Eligible studies were included based on defined exclusion and inclusion criteria to perform a meta-analysis. STATA 14.0 was used to estimate pooled hazard ratios (HRs) with 95% confidence interval (95% CI), the heterogeneity among studies and publication bias to judge the prognostic value.A total of 2121 patients from 21 eligible studies were included in the meta-analysis. A significant association was found between elevated 8-OHdG expression and poor OS  in cancer patients ; In the subgroup analysis, race of sample, cancer types, detection method of 8-OHdG, sample classification, detection location of 8-OHdG and paper quality (score more or less than 7) did not alter the association between 8-OHdG expression and cancer prognosis. Furthermore, 8-OHdG expression was an independent prognostic marker for overall survival in patients with cancer  using Cox multivariate analyses.This meta-analysis found that highly expressed 8-OHdG in tumor tissues may be a predictor of prognosis in most solid tumors. However, especially in breast cancer, low 8-OHdG expression is associated with poor prognosis, which is partly because of the increased antioxidant mechanisms in breast cancer tissues. This study demonstrates for the first time that 8-OHdG expression is associated with the prognosis of cancer patients. In the future, whether the expression level of 8-OHdG can be used as a biomarker for the prognosis of all human cancers requires more research. Tumor cells constantly suffer various endogenous and environmental attacks, which make high level of reactive oxygen species (ROS) be detected in almost all cancers and become one of the best-characterized phenotypes –3. The rThough ROS plays an important role in tumors, the degree of oxidative stress can be better evaluated by assessing stable metabolites of oxidative reactions because of its high instability. ROS can cause oxidative damage to double-stranded DNA directly, or to free bases in the cellular and mitochondrial deoxynucleoside triphosphate (dNTP) pool . Among aThere is an increasing body of evidence indicating that 8-OHdG is a useful marker for assessing oxidative DNA damage and has been a feature of carcinogenesis in several researches , 18. HigThis analysis was conducted following the meta-analyses and systematic reviews guidelines for prognosis-related tumor marker researches , 42. An Studies included in the present meta-analysis were independently reviewed by two investigators (XQ and DS) and should meet the following criteria: 1) The prognostic data of 8-OHdG in any type of human solid tumors needed to be presented; (2) All cancer patients were diagnosed according to the gold standard for diagnosis, based on histopathological examinations; (3) 8-OHdG levels in tumors, blood samples or urine were estimated in each study; (4) The patients were divided into two groups according to the levels of 8-OHdG; (5) Sufficient data should be provided to obtain hazard ratios (HR) for survival rates and their 95% confidence intervals (95%CI). Studies were excluded from the present meta-analysis if one of the following criteria was met: (1) Case reports, reviews, meta-analysis, letters, editorials, comments, expert opinions or any other reviews that didn’t contain raw data; (2) Full text could not be obtained; (3) Researches on non-English writing; (4) Repetitive publications; (5) No survival data or data insufficient to be extracted and analyzed; (6) Survival data was acquired based on animal studies and no follow-up of patients. Detailed inclusion and exclusion criteria of each study are presented in Additional file  The progData was extracted independently by the two researchers (XQ and DS), and final consensus was reached through discussion. Data were retrieved from each study including: author; year of publication; country of the population enrolled; ethnicity; tumor stage; sample size; study design; follow-up data; survival data; survival analysis methodology; expression levels, location and laboratory methods of 8-OHdG; cut-off values; HR values and their 95% confidence intervals. Quality assessment of cohort studies in this meta-analysis was performed using the Newcastle-Ottawa scale (NOS) as recommended by the Cochrane Non-Randomized Studies Methods Working Group. Studies with score ≥ 7 were considered high quality according to the NOS. Detailed NOS scores of all included studies were shown in Table 2 based Cochran Q test and Higgins I2 statistic. I2 values > 50% indicated heterogeneity among studies. If there existed heterogeneity, a random-effect model, subgroup analysis and meta regression by factors contributing to heterogeneity would be carried out. Influence analyses was performed to examine the effect of each study on the overall pooled results. The presence of publication bias was evaluated by using funnel plots, Begg’s test and Egger’s test. P values < 0.05 were considered statistically significant.The meta-analysis was performed as previously described . In the n = 915), Web of Science (n = 1319) and EMBASE (n = 1303). After removing duplicates, 1665 articles were left. Furthermore, 1607 of the remaining articles were excluded according to the titles and abstracts. Finally, a total of 21 relevant articles were included in this meta-analysis after a more careful full-text reading. The detailed screening process is shown in Fig. Based on our searching strategy, a total of 3537 articles were identified from PubMed n = 95, Web ofAmong the 21 studies, a total of 2121 patients were included, with mean sample size of 101 patients (range 30 to 252). The period of these studies ranged from 2003 to 2017. The regions represented in the studies include various countries around Europe, Asia and America, of which the race contains both Caucasoid and Mongoloid. Eight different types of cancer were evaluated. Most studies analyzed the expression level of 8-OHdG by IHC or ELISA, while there was one study unitizing liquid chromatography electrochemistry. Overall survival (OS), cancer-specific survival (CSS), recurrence-free survival (RFS), disease-free survival (DFS) and progression-free survival (PFS) were estimated as survival outcomes in the studies. RFS, DFS and PFS were merged into the event-free survival (EFS) group for analysis. Cox multivariable analyses were performed in 17 studies. Further detailed characteristics of each study are presented in Table 2 = 0.2298; χ2 = 53.52, df = 16, p < 0.0001; I2 = 70.1%). Since obvious heterogeneity was observed, subgroups analysis was performed by factors of the race of sample, cancer types, detection method of 8-OHdG, detection location of 8-OHdG, sample classification and research quality  based on different 8-OHdG expression levels was reported in 8 types of solid tumors from 15 of the 21 included studies with a total of 1596 patients. Elevated 8-OHdG was significantly associated with poor OS in these patients    while the Egger’s test showed there was significant publication bias (p = 0.007)  with reduced heterogeneity (I2 = 78.7%)  in 489 patients. A close relationship was observed between elevated 8-OHdG expression and EFS  , corresponding to hepatocellular carcinoma, melanoma, renal cell carcinoma and breast cancer, including a total of 495 patients. After summarizing the results, we found there was no significant association between 8-OHdG expression and CSS . , and heterogeneity was still observed among studies (Taup = 0.276) and Egger’s test (p = 0.031) showed opposite conclusion.  with 8-oxo-dGTP hydrolyzing activity, OGG1 (8-oxoguanine DNA glycosylas) with 8-OHdG DNA glycosylase activity and MUTYH (MutY homolog) with adenine DNA glycosylase activity, all play roles in minimizing 8-oxoG accumulation in cellular DNAs . Thus, tIn our meta-analysis, we examined 21 independent studies enrolling a total of 2121 cancer patients. After systematic review of these studies, we discovered that 8-OHdG was highly expressed in various types of tumors except a few specific tumors such as breast cancer. By combining the survival data obtained from these studies, we found that high 8-OHdG expression was a biomarker for poor prognosis for overall survival in most solid cancer patients.Because obvious heterogeneity was observed among studies, we performed s subgroup analysis, meta regression analysis and influence analysis to examine the source of heterogeneity and the stability of the pooled result. In subgroup analysis, we still found that high 8-OHdG expression was associated with poor overall survival in most subgroups. The factors such as race of sample, cancer types, detection method of 8-OHdG, detection location of 8-OhdG, sample classification and research quality would not influence the pooled result. Meta regression analysis found that the subgroup of breast cancer would be the major source of heterogeneity. After excluding the corresponding study, we could still find significant association between elevated 8-OHdG expression and poor OS in cancer patients with reduced heterogeneity. In addition, influence analysis was performed and confirmed the stability of our pooled result. Furthermore, through summarizing the data from studies using Cox multivariate analysis, we found that 8-OHdG could be an independent prognostic risk factor for overall survival. Besides, by collecting the survival data of cancer recurrence or progression, we found that elevated 8-OHdG expression was associated with event-free survival of cancer patients. However, the number of these studies was relatively limited, which made the conclusion not so convincing as above. It should be noted that there were three studies reporting the association between 8-OHdG expression and prognosis of breast cancer patients. One was analyzed with overall survival data and the other two were cancer specific survival data. All of the three studies reported that negative or weak 8-OHdG expression was associated with poor survival of breast cancer patients. These results were contrasted with the other studies and the pooled result.There are several potential mechanisms behind the different association of 8-OHdG levels and tumor prognosis in breast cancer. To deal with the threats posed by high ROS production, tumor cells evolve lots of antioxidant mechanisms, which would prevent ROS from interacting with DNA or directly eliminate 8-OHdG, thus decreasing the expression level of 8-OHdG in tumor tissues. For example, transcription factor NF-E2-related factor 2 (Nrf2), the main inductor of multiple antioxidant enzymes, has been revealed to be highly expressed in various cancer cells , 46–49. In our study, a few limitations should be pointed out. First, the cut-off values of high and low 8-OHdG expression were different among studies. Most were set to be the median, while some of them were set by different standards. Second, as for the race of included patients, there were only Caucasoid and Mongoloid, the representativeness of our results could be limited. Third, several HRs could not be directly obtained from the publications. Data extracted and calculated through survival curves might not be precise enough. Fourth, the association between 8-OHdG expression and clinicopathological characteristics could not be analyzed due to the insufficient data. Therefore, larger-scale, multicenter, and high-quality studies are highly necessary to further confirm our findings. Fifth, although we have confirmed that all the antibodies used in involved studies were mouse original and commercial antibodies, it’s definite that different clones may target different parts of the interest protein, which may possibly be a source of heterogeneity. Furthermore, it is necessary to discuss those different samples with various detecting laboratory methods to evaluate 8-OHdG. Because there hasn’t been a golden standard technique for detecting 8-OHdG, different samples (shown in Table This meta-analysis found that highly expressed 8-OHdG in tumor tissues may be a predictor of prognosis in most solid tumors. However, especially in breast cancer, low 8-OHdG expression is associated with poor prognosis, which is partly because of the increased antioxidant mechanisms in breast cancer tissues. This study demonstrates for the first time that 8-OHdG expression is associated with the prognosis of cancer patients. In the future, whether the expression level of 8-OHdG can be used as a biomarker for the prognosis of all human cancers requires more research.Additional file 1: Table S1. Inclusion and exclusion criteria.


Drosophila melanogaster influence the mRNA transcriptome of early embryos. We used mRNA-seq to evaluate differential expression in Drosophila embryos lacking endosymbionts (control) to those harbouring the male-killing Spiroplasma poulsonii strain MSRO-Br, the CI-inducing Wolbachia strain wMel, or Spiroplasma poulsonii strain Hyd1; a strain that lacks a reproductive phenotype and is naturally associated with Drosophila hydei. We found no consistent evidence of influence of symbiont on mRNA composition of early embryos, suggesting that the reproductive manipulation mechanism does not involve alteration of maternally-loaded transcripts. In addition, we capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of depurination of rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs) encoded by Spiroplasma poulsonii. We found small but statistically significant signals of depurination of Drosophila rRNA in the Spiroplasma treatments (both strains), but not in the symbiont-free control or Wolbachia treatment, consistent with the action of RIPs. The depurination signal was slightly stronger in the treatment with the male-killing strain. This result supports a recent report that RIP-induced damage contributes to male embryo death.Maternally-transmitted endosymbiotic bacteria are ubiquitous in insects. Among other influential phenotypes, many heritable symbionts of arthropods are notorious for manipulating host reproduction through one of four reproductive syndromes, which are generally exerted during early developmental stages of the host: male feminization; parthenogenesis induction; male killing; and cytoplasmic incompatibility (CI). Major advances have been achieved in understanding mechanisms and identifying symbiont factors involved in reproductive manipulation, particularly male killing and cytoplasmic incompatibility. Nonetheless, whether cytoplasmically-transmitted bacteria influence the maternally-loaded components of the egg or early embryo has not been examined. In the present study, we investigated whether heritable endosymbionts that cause different reproductive phenotypes in  With few exceptions3, inheritance is achieved via the mother, generating an asymmetry in the interests of the symbiont regarding the host’s sex, whereby males are generally dead-ends for the symbiont. Consistent with this inequality, numerous heritable bacteria manipulate host reproduction in favour of symbiont-bearing females. Four such reproductive phenotypes have been described4. Feminization occurs when genetic males develop and function as females. Parthenogenesis-induction occurs in haplo-diploid systems, where unfertilized eggs, which would otherwise develop as males, develop instead into reproductively functional females. In male-killing (or son-killing), the symbiont eliminates infected males to the presumed advantage of surviving infected female siblings5. In cytoplasmic incompatibility (CI)6, matings between symbiont-infected males and uninfected females result in death of offspring at the embryonic stage. The CI mechanism involves symbiont-mediated damage to the male sperm that is rescued in the presence of a compatible symbiont strain in the egg4.Heritable associations between arthropods and endosymbiotic bacteria are widespread and influential to their hosts7. As such, they may manipulate the female host’s reproductive system or usurp her cellular machinery in order to invade developing oocytes10. Infection-induced changes during oogenesis may thus have effects on the composition of an egg . Drosophila melanogaster eggs contain maternal RNAs that are exclusively expressed during early development . Zygotic transcription is silent during this period and therefore maternal mRNAs play a crucial role in early embryonic development12. The egg, in a sense, is a point of convergence between the existing host, symbiont, and new host, and consequently could undergo symbiont-induced changes that could lay the foundation for the occupation of the symbiont within the new host. Furthermore, it is possible that infection by a reproductive parasite could cause changes in maternally-derived components that are necessary to induce a reproductive phenotype. As hosts of two independent lineages of maternally transmitted bacteria, Wolbachia and Spiroplasma13, members of the genus Drosophila, have emerged as a model system for heritable symbiosis e.g.23.Most maternally inherited symbionts are transmitted to a new host through the egg cytoplasmSpiroplasma (class Mollicutes) is generally associated with arthropods and plants, and can reside intra- and extra-cellularly24. The nature of Spiroplasma-host associations ranges from pathogenic to mutualistic, although the fitness consequences of the majority of Spiroplasma strains remain unknown. Most strains of Spiroplasma known to date appear to transmit horizontally from the environment or via a vector . A few strains of Spiroplasma, however, are maternally-transmitted by their arthropod hosts. Among these, ~20 species in the genus Drosophila are reported to harbour Spiroplasma26. A subset of maternally-transmitted Spiroplasma of Drosophila and other arthropods are male killers. All of the Drosophila-associated Spiroplasma male-killing strains that have been genetically characterized to date fall within the poulsonii clade; one of the four Spiroplasma clades that independently invaded the genus Drosophila26. The poulsonii clade also contains non-male-killing strains such as “Hyd1” and “sNeo”; naturally-occurring defensive mutualists of Drosophila hydei and Drosophila neotestacea, respectively27, as well as several strains that lost the ability to kill males in the lab29.The wall-less bacterial genus Spiroplasma exerts death of Drosophila male embryos is not fully understood, but several aspects have been elucidated35. First, a functional dosage compensation complex  is required, as mutants of components of this complex that are infected with male-killing Spiroplasma, fail to express male-killing32. Secondly, the DCC, which normally acetylates X chromatin in males, mis-localizes to other regions of the nucleus immediately prior to the killing stage35. This phenomenon is accompanied by inappropriate histone acetylation, and genome-wide transcription misregulation35. Thirdly, abnormal massive apoptosis and neural disorganization occurs34. The male X chromosome exhibits signs of DNA damage (chromatin bridges and segregation defects), and bridge breakage triggers sex-specific abnormal apoptosis via p53-dependent pathways30. Recently Harumoto and Lemaitre15 identified spaid, a Spiroplasma-encoded gene that appears to be responsible for male killing. Overexpression of spaid in D. melanogaster embryos causes death of males, but not females, and induces massive apoptosis and neural defects, reminiscent of the Spiroplasma-induced male-killing phenomenon in pattern, but in a somewhat delayed fashion . Spaid contains an OTU (ovarian tumor) deubiquitinase domain and ankyrin repeats (ANK). Harumoto and Lemaitre15 propose that the OTU domain promotes nuclear localization of Spaid , while the ankyrin repeats interact with DCC complex itself or with its associated histone modifications. How this leads to DNA damage and segregation defects of the male X chromosome, as well as to other phenotypes associated with male-killing described above, and which are the host cellular targets of Spaid remain unknown.The mechanism by which Spiroplasma. Ribosome inactivating proteins (RIPs) are plant- (e.g. ricin and saporin) and bacteria-encoded (e.g. Shiga toxin) enzymes that cleave an adenine base (hereafter “depurinate”) from a specific position within a motif of the 28S rRNA that is universally conserved across eukaryotes. This motif is known as the sarcin-ricin loop (SRL). Depurination of this site renders the ribosome incapable of protein synthesis36. Several confirmed and predicted RIPs are encoded in the genomes of Spiroplasma strains that associate with Drosophila, including the male-killing strain native to D. melanogaster (MSRO) and the closely-related non-male-killing strains sNeo38 and Hyd139. These poulsonii-clade strains are also known for their defensive abilities against certain parasitic wasps and nematodes of Drosophila41. Evidence of SRL depurination consistent with the action of RIP has been detected in nematodes and wasps exposed to Drosophila harbouring Spiroplasma37, and has led to the hypothesis that RIPs play a major role in the Spiroplasma-mediated defence against wasps and nematodes. Depurination of Drosophila ribosomes in Spiroplasma-infected treatments had been detected in larvae, but was mostly restricted to “cell-free hemolymph”, suggesting only ribosomes outside the cell were targets37. Furthermore, despite detecting significant levels of depurinated Drosophila ribosomes in Spiroplasma-infected flies, this phenomenon is generally not accompanied by a significant decrease in intact  ribosomes37. This, along with the observation that the detected levels of depurination were not associated with larva-to-adult fly mortality, was interpreted as RIP activity having a negligible direct effect on fly fitness37. A more recent study, however, revealed that Spiroplasma-mediated depurination of Drosophila ribosomes varies widely by life stage. It is strongest in embryos and old adults38, but also not accompanied by a detectable decrease in intact ribosomes. In addition, significant ribosome depurination (but not significant depletion of intact ribosomes) occurs under heterologous expression of two Spiroplasma RIP genes in D. melanogaster, confirming their RIP activity. Their expression was also associated with embryo mortality , and with a reduction in fly lifespan and in adult hemocyte number38.In addition to Spaid, a different type of toxin has been reported in Spiroplasma influences the composition of maternally loaded transcripts of host eggs , particularly in a manner that may facilitate male-killing. To do so we compared the effects of three heritable endosymbionts  on the composition of mRNA transcripts of early embryos of Drosophila melanogaster. Furthermore, we used data from this study and published RNA-seq data to analyse whether there is a difference in signals of rRNA depurination of host embryo ribosomes in the presence of male-killing and non-male-killing symbionts.In this study we examined whether Wolbachia and two Spiroplasma strains, and a symbiont-free control  that naturally harbour the Wolbachia strain wMel were used to generate the Wolbachia treatment (W + S−). Positive infection for wMel was confirmed based on PCR with Wolbachia-specific primers targeting the wsp gene42. The same stock was reared in tetracycline food  for two generations, followed by three generations of antibiotic-free food to generate a Wolbachia-free (W−) stock. The W− flies served as the symbiont-free control. The Spiroplasma infection treatments (W−S+) were generated by artificially infecting Wolbachia-free (W−) flies with the strain MSRO-BR (Red 42) native to D. melanogaster43 or Hyd1 (TEN-104-106) native to D. hydei13. Fifteen Wolbachia-free (W−) females (15 lines) were infected per Spiroplasma strain. These artificially infected lines were maintained for 3–5 generations before being used for the experiment. Spiroplasma-infected (W−S+) lines were selected every generation to ensure positive infection status, based on PCR with Spiroplasma-specific 16S ribosomal DNA primers43. MSRO treatment lines were backcrossed to Wolbachia-free (W−) CS males every generation, as male-killing by this strain is nearly perfect. A minimum of four infected lines was combined, per replicate, at the start of the experiment, to create a total of three biological replicates for each Spiroplasma treatment. The biological replicates for the Wolbachia treatment and control were maintained as three different populations for four generations prior to the start of the experiment.We used three infection treatments, one rol Fig. . LaboratWolbachia-free (W−) CS males during the collection period, and allowed to lay eggs on cornmeal food plates. The initial batch of eggs was discarded to improve the chances of collecting fertilized eggs from the same stage44. Thereafter, egg laying was monitored and cornmeal plates were changed approximately every 45 min, so as to collect embryos that were on average ~60–75 min old. Eggs were collected from each replicate with a small brush 6–8 times over a 2-day period for each treatment and the control. The eggs were placed in sterile RNase-free 1.7 ml microtubes, and immediately put on dry ice during the collection period, after which they were transferred to −80 °C for storage.Approximately 40–50 three-day-old virgin females, from each replicate of each treatment, were allowed to mate in cages with Three to four biological replicates per condition  were used for the extractions  Genome Analyzer II platform  with HiSat2 (v.2.0.5)46 to obtain bam files. The files containing the mapped read information were then analysed with HTseq (0.6.1)47 to obtain read counts for both genes and exon regions specified in a corresponding gff file for the dmel-all-r6.18 genome sequence. The resulting count files were then manually formatted into a count matrix suitable for differential expression analysis . Command lines, as well as input and output files needed to replicate our analyses, from the different pipelines used are available in Supporting Data Files see Fig. . The pro48 and Limma + Voom v. limma_3.34.549 and DeSeq2 v.1.18.150. Command line and relevant input files for these analyses are provided in Supporting Data Files p-value  of 0.05 or less were considered to be potentially differentially expressed (DE).Differential expression analyses were performed with three different pipelines edgeR v.3.20.5et al.51. The parameters  used to run the simulations are provided in Table To identify possible limitations of our experimental design in the detection of DE genes, we performed a statistical power analysis with the method of Hart 52. When a reverse transcriptase encounters an abasic site, it preferentially adds an adenine in the nascent complementary (c)DNA strand53. This property, which results in an incorrect base at the RIP-depurinated site in the cDNA and all subsequent PCR amplification steps, can be used to detect evidence of RIP activity in any procedure that relies on reverse transcription (e.g. RNA-seq or reverse-transcription qPCR). To examine whether a signal of depurination consistent with RIP activity was detectable in Spiroplasma-infected flies, we used the RNA-seq data generated in the present study, as well as RNA-seq data  from two published studies that also compared patterns of gene expression from Spiroplasma-infected and Spiroplasma-free D. melanogaster embryos: NCBI Acc. No. PRJDB446930; and PRJNA31837335. The Spiroplasma strain examined by35 and30 is substrain UG (for Uganda) of MSRO. Bowtie2 v.2.1.054 and Samtools 0.1.1955 were used to map and extract, respectively, the reads from the three studies to the 28S rRNA gene of D. melanogaster (Acc. No. NR_133562.1 positions 2591–3970). We verified that the reads that mapped to NR_133562.1:2591–3970 (hereafter “28S reads”) did not belong to other parts of the Drosophila genome or to the genomes of Spiroplasma or Wolbachia, as follows. We used HISAT2 v.2.0.2-beta46 (for Drosophila) and Bowtie2 v. 3.4.254 (for the prokaryotic genomes) to attempt mapping the “28S reads” to the following reference genomes: (1) the D. melanogaster genome (BDGP6); (2) MSRO-UG (NZ_PETG00000000.1); (3) a draft genome of Spiroplasma Hyd1 (unpublished data); and (4) Wolbachia wMel (NC_002978). As expected, the “28S reads” only mapped to (1), and specifically to regions whose annotation terms included “28S rRNA”. To visualize and count the shift from A to T (or other bases) in the source RNA pool, the “28S reads” were mapped again to the NR_133562.1:2591–3970 in Geneious v.11.1.2 . The number of reads containing each of the four bases or a gap at the target site was counted by selecting the position at all the reads to be counted, and recording the counts reported by Geneious under the “Nucleotide Statistics” option . The proportion of reads with an A at the target site  was calculated and compared among treatments and replicates. We used a mixed model in JMP Pro 13  to examine the effect of symbiont treatment (fixed) on the proportion of adenines (arcsine square root transformed) at the target position . Source Study was treated as a random effect.RIP toxins remove a specific adenine present in the sarcin-ricin loop (SRL) of the 28 S rRNA leaving an abasic site D. melanogaster. To achieve this, we used mRNA-seq to evaluate differential expression (DE) in Drosophila embryos lacking endosymbionts (control) to those harbouring one of the following heritable endosymbionts: the male-killing Spiroplasma poulsonii strain MSRO-Br; the CI-inducing Wolbachia strain wMel; and the non-male-killing Spiroplasma poulsonii strain Hyd1 (native to D. hydei). A power analysis was performed to determine the limitations of our experimental design. A secondary goal of this study capitalized on several available mRNA-seq datasets derived from Spiroplasma-infected Drosophila melanogaster embryos, to search for signals of damage  to Drosophila rRNA, consistent with the activity of Ribosome Inactivating Proteins (RIPs), which are encoded in the genomes of several Spiroplasma strains. This assay examined the proportion of adenines at the RIP target position (indicative of intact rRNA) in Spiroplasma-infected vs. Spiroplasma-free treatments.The present study aimed at determining whether heritable symbionts alter the composition of maternally-loaded mRNAs in Drosophila genome is shown in Table The number of reads that was obtained, passed QC, and mapped to the Spiroplasma) and wMel (CI-inducing Wolbachia) treatments (Table Spiroplasma) treatment, half of the pipelines did not detect DE genes, whereas the other half detected a few DE genes  were found in common among these pipelines. For all of these, the Hyd1 treatment exhibited lower expression, and lower variation among replicates, than the other treatments  tests were performed by comparing each symbiont treatment to the symbiont-free control. None of the eight pipelines used detected differentially expressed (DE) genes in the MSRO , whereas source study did not . Both MSRO (UG and BR) treatments had a significantly (p ≤ 0.001) smaller proportion of As (mean = 96.78%) than the control (mean = 99.79%) and wMel (mean = 100%) treatments. Hyd1 exhibited intermediate proportions of As  by treatment replicate, are shown Fig. 54 with default settings to map the reads from each symbiont-infected replicate to the closest symbiont reference genome available. The number of reads per replicate mapped to the symbiont genome ranged from 5 (for a Hyd1 replicate) to 8619 Spiroplasma strain (MSRO-Br), the CI-inducing Wolbachia strain, and the symbiont-free control. These results suggest that these symbionts do not influence the composition of initial maternally-loaded transcripts or their degradation up to the examined stage, unless they do so to a degree below what was detectable by our experimental design . Furthermore, it is possible, that such cytoplasmically transmitted symbionts could influence the composition of maternally-loaded proteins, or regulate the protein complement of the early embryo in a transcriptionally-independent manner . To our knowledge, these features have not been compared in Spiroplasma-infected vs. uninfected embryos.The present study aimed to test whether infection by a heritable bacterium strain that kills males during the embryonic stage influences the composition of mRNA transcripts present in the early embryo (ca. 60–75 min post-oviposition), and thus, before the onset of zygotic transcription. Based on several analysis pipelines and assumptions, we did not detect any differential expression in genes among embryos harbouring the male-killing Spiroplasma on maternally-loaded mRNA composition are consistent with what is known about the male-killing mechanism. A Spiroplasma-encoded protein (Spaid) appears to interact with the DCC, which assembles onto the X chromosome of wild-type males but not females, and is accompanied by DNA damage and abnormal apoptosis15. This model does not require “priming” of the egg during oogenesis by the symbiont. Nonetheless, the delayed expression of male death in males flies over-expressing wild type Spaid , compared to those harbouring male-killing Spiroplasma, requires further investigation and may indicate that other Spiroplasma-encoded factors are relevant to the male-killing phenotype (e.g. RIP)38.Our results showing no effect of the male-killing Spiroplasma strain native to D. hydei), yielded equivocal results, with four pipelines revealing no differentially expressed genes, and the other four pipelines revealing an intersection of five DE genes . All of these five genes had a lower expression level in the Hyd1 treatment compared to the other three treatments  is expected to have very low expression (FlyBase RPKM = 1) at the stage we examined. It is expressed later (Stages 13–16 ≈ embryo 14–16 h) in several tissue types: foregut, epidermis, trachea, and hindgut, with possible roles in cuticle development and tubular formation 59. Osiris 6  and Osiris 7  are also expected to have very low expression  at the stage examined; with the highest expression at later embryonic stages . Both genes are physically close to each other and belong to the Osiris gene cluster . The OSI family of genes shares a highly conserved protein domain is present, and retains high synteny, in all insects examined to date, and appears to have evolved via gene duplications at the base of the Insecta61. In D. melanogaster, these genes are within the dosage-sensitive Triploid-lethal (Tpl) locus; individuals with one or three copies of Tpl die as late embryos or early first instar larvae62. Although Osiris genes are generally considered of unknown function, based on expression patterns or knock-down experiments, they have been associated with cuticle formation, metamorphosis, digestion, resistance/tolerance to toxins and viruses, wing formation, and phenotypic plasticity . FBgn0038339 (CG6118) contains a BTB/POZ domain, and appears to be involved in regulation of transcription by RNA polymerase II. FBgn0037099 (CG7173) contains a Kazal domain . Both of these genes are expected to have very low expression  at the stage we examined, with peak expression levels occurring in the 14–16 h embryo.Available information on the function of the five genes at which the Hyd1 treatment had significantly lower expression provides little insight into the possible causes or consequences. Based on FlyBase, D. melanogaster is outside the fundamental niche of Hyd1, as also suggested by its lower vertical transmission efficiency in this host (80%64), and by the difficulty of maintaining this artificial association in the lab . Reports of additional phenotypic effects of Hyd1 in D. melanogaster have been anecdotal or contradictory. Concerning adult longevity, Kageyama, et al.65 reported substantially shorter adult life spans of both males and females, whereas Silva, et al.66 detected little to no adult mortality. The study of Silva, et al.66 is expected to more closely represent the conditions and specific D. melanogaster  and Hyd1  genetic backgrounds of the present transcriptomics study. Concerning fly fecundity, both Kageyama, et al.65 and Hutchence, et al.64 report anecdotal evidence of lower female fecundity in Hyd1-infected D. melanogaster than their uninfected counterparts; Silva, et al.66 did not examine this trait. Consistent with the detrimental effect of Hyd1 on fecundity of D. melanogaster, the microarray-based study of Hutchence, et al.64 (focused on adults), revealed downregulation of a cluster of genes involved in egg production and fertilization. Nonetheless, in general, Hutchence, et al.64 found that D. melanogaster adults infected with Hyd1 exhibited less perturbation of gene expression than those infected by MSRO-Br and Spiroplasma poulsonii strain NSRO64 . The apparently different patterns of gene expression perturbation by Hyd1 between our study and that of Hutchence, et al.64 could be due to the different fly stages examined (embryos vs. adults), different experimental tools (RNA-seq vs. microarrays), or other factors .Assuming that the significant results for the five genes are repeatable, they imply more perturbation of gene expression by Hyd1 than by the other symbiont strains; a finding that could reflect that wMel, a CI-inducing strain, on D. melanogaster early embryo mRNA composition, is not unexpected given what is known about the CI mechanism. Essentially, wMel encodes two contiguous protein coding genes (cifA and cifB)18. Each of these proteins is capable of causing CI when expressed in the male germline, whereas only cifA (when expressed in embryos) is able to rescue CI19. Our results suggest that wMel does not “prime” the egg for rescue by manipulating maternally-loaded transcripts or their degradation.The lack of an effect of 67. Furthermore, the freezing to which these samples were subjected between collection and library preparation might have decreased the detectability of depurination53. In addition, it is not known whether the polyA-RNA enrichment protocol used prior to library preparation, which is aimed at depleting rRNA in the sample, could bias representation of depurinated vs. intact rRNA. Only one study has compared inferences of ribosome depurination from mRNAseq vs. qPCR assays in the same system. Based on mRNAseq, Hamilton et al.17 detected ~3.8% depurination  in the nematode Howardula infesting adult Drosophila neotestacea harbouring the non-male-killing Spiroplasma sNeo strain. Comparatively, using the qPCR approach, the abundance of depurinated template representing RIP-induced depurination increased ~20-fold, whereas the levels of intact nematode rRNA were reduced ~six-fold in the presence of Spiroplasma17. Notwithstanding the potential biases, the consistent finding of no depurination in Spiroplasma-free treatments vs. depurination in Spiroplasma-present treatments across three independent studies, lends credibility to the mRNAseq-based approach we employed for inference of depurination.Our study capitalized on the presence of (non-target) rRNA reads in RNAseq datasets prepared by poly-A-tail enrichment, to estimate levels of depurination at the RIP target position of the sarcin-ricin loop (SLR) in the 28 SrRNA of eukaryotes. We acknowledge that the levels of depurination estimated from mRNAseq data may be downwardly biased because rRNA depurinated by RIPs is highly prone to hydrolysis of the sugar-phosphate backbone at the lesion siteSpiroplasma treatments vs. the Wolbachia treatment and the control is consistent with the action of a Spiroplasma-encoded RIP, and supports recent findings that: (1) MSRO-UG infection causes ribosome depurination in D. melanogaster embryos; (2) that heterologous expression of a Spiroplasma RIP1 and RIP2 genes (separately) also causes ribosome depurination in D. melanogaster embryos (measured by a qPCR approach); and (3) that the degree of ribosome depurination  in the different Spiroplasma treatments or RIP transgene constructs is positively correlated with embryo mortality38. Importantly, despite detecting significantly higher levels of depurinated template with the qPCR approach, none of the qPCR assays revealed significantly lower levels of intact template38. This suggests that detectable depletion of intact template in the qPCR approach is not a pre-requisite for detecting a phenotypic effect of depurination.The significantly lower proportion of adenines at the RIP target site for the 66, and with the identical content of RIP-encoding genes (unpublished data). Interestingly, the male-killer MSRO generally exhibited a higher signal of depurination than the non-male-killer Hyd1  in the presence of sNeo17. Therefore, the three members of the poulsonii clade examined to date  encode RIPs capable of depurination of Drosophila ribosomes, but the male-killing strain exhibits the highest levels depurination, a phenomenon that appears to contribute to the male-killing mechanism38. The general patterns, however, indicate that Spiroplasma RIPs are particularly efficient at depurinating the ribosomes of natural enemies of Drosophila , leading to the hypothesis that RIP-induced depurination plays an important role in defence mechanism37. The higher depurination levels of fly ribosomes detected in embryos (and ovaries) compared to other fly stages could be due to greater exposure of ribosomes to RIP prior to cellularization, after which Spiroplasma becomes effectively extra-cellular38. The relatively high levels of depurination in old adults appears to be the result of higher Spiroplasma densities at that stage38. Additional roles have been attributed to RIPs, which could contribute to the male-killing phenotype. For example, several RIPs are reported to cause DNA damage69. Thus, one or more Spiroplasma-encoded RIP might directly contribute to the DNA damage reported during the process of male-killing30.Our results suggest that MSRO substrain Brazil (MSRO-BR) causes comparable levels of depurination to the Uganda substrain, which is consistent with its strong male-killing effectDrosophila, influence composition maternally-loaded mRNAs. The results revealed that mRNA composition does not differ significantly among the embryos harbouring the reproductive parasites Spiroplasma MSRO and Wolbachia wMel and those lacking endosymbionts. Only the symbiont Spiroplasma Hyd1, which does not manipulate reproduction, appeared to alter expression levels of a handful of genes (5–12), but not all analytical approaches supported this finding. Our power analyses indicated that our experimental design should be able to detect most of the genes exhibiting ≥ 3-log-fold change in expression among treatments. Collectively, our results suggest that these cytoplasmically-transmitted bacteria do not alter the composition of mRNAs of the early embryo, and are thus unlikely to use this mechanism to exert their reproductive phenotypes. Capitalizing on several transcriptomics datasets, this study also detected signals of Spiroplasma-induced damage to ribosomes in early Drosophila embryos, with greater damage caused by the male-killer (MSRO) than the non-male-killer (Hyd1), consistent with recent results implicating this mechanism in male killing.This study employed a transcriptomics approach to examine whether cytoplasmically-transmitted bacteria, including reproductive manipulators that strongly impact survival of the embryonic stage of Description of Supplementary FilesSupporting Table S1Supporting Table S2Supporting Table S3Supporting Data File S1Supporting Data File S2Supporting Data File S3