Borrelia sp. is a causative pathogen of Lyme disease which has become a worldwide health concern. Non-toxic approaches especially directed toward latent persistent forms of this pathogen are desired. Lipids in the form of volatile and non-volatile oils, and fatty acids with proven anti-borreliae efficacy could become an additional support or an alternative for consideration in treatment approaches.Borrelia burgdorferi s.s. and Borrelia garinii, which are identified as pathogenic factors of Lyme disease in the USA and Europe, using direct microscopic counting and spectrofluorometric measurements.In this study we investigated 47 lipids  of plant and animal origin against typical motile, knob/round-shaped persisters, and biofilm-like aggregates of Borrelia spp. at the same concentration, although with 20–30% eradication mark.Out of all examined lipids, 5 oils  at or below 0.25%, and 3 fatty acids  at or below 0.75 mg/ml, showed bactericidal activity against typical motile spirochetes and knob/round-shaped persisters. Only Bay leaf oil and Cassia oil, including their major constituents, eugenol and cinnamaldehyde, showed to target biofilm-like aggregates of both tested Based on obtained results, volatile oils were more potent than non-volatile oils, and unsaturated fatty acids were more effective than saturated fatty acids. Among all tested oils, Bay leaf oil and Cassia oil, with their major components eugenol and cinnamaldehyde, seem to have the highest anti-borreliae efficacy. Borrelia which is an invasive, host-dependent, semi-aerophilic, and slow-growing pathogen [Borrelia burgdorferi s.s. and Borrelia mayonii (in North America) and Borrelia afzelii and Borrelia garinii (in Eurasia). All of the species can exist in three morphological forms. The vegetative (active) form is the typical corkscrew motile spirochetes. Under stress conditions, including antibiotic exposure in vitro, they have the ability to quickly transform into latent persistent forms such as knob/round-shaped bodies (forms) and/or biofilm-like aggregates [Lyme disease (LD) is a multi-systemic zoonosis transmitted by ticks. According to the CDC, it has become the most common vector-borne disease in Europe and Northern America. In the USA alone the number of diagnosed LD cases reaches 300,000 annually . The caupathogen . These fgregates . Althouggregates –11.Borrelia sp. [The primary and conventional treatment for Lyme patients is based on antibiotics, and the “first choice” of β-lactams and tetracyclines are administered for up to 3–4 weeks. Randomized clinical trials showed that for the acute (early) stage of LD this approach can be nearly 90% effective, as opposed to the late and persistent stages of this disease, whose etiology, although is still not clearly explained, points to lingering persisting infection as one of the reasons , 12–14. elia sp. –27. Howeelia sp. –32. Theielia sp. . Lipids elia sp. –32.Saturated or unsaturated fatty acids are the essential building blocks of other structurally complex lipids, and are important dietary sources of energy for animals. Thus, it is rational to study the biocidal potential of organic oils and fatty acids and validate their specific or general antimicrobial activity.Borrelia burgdorferi s.s. and Borrelia garinii. We evaluated their anti-borreliae effect using a “golden standard” method of direct counting and dark field microscopy, and the high throughput spectrofluorometric screening method to assess viability of the logarithmic phase  and the stationary phase (rich in knob/round-shaped persisters) [Borrelia strains.In this study, we tested 30 volatile and non-volatile organic oils, and 17 fatty acids against all known pleomorphic forms of sisters) . We alsoSalvia triloba) obtained from SunRose Aromatics, LLC (Morrill ME), the Bay leaf oil (Pimenta racemosa) obtained from Penn Herb Company, Ltd. , and 13Z,16Z docosadienoic acid purchased from Cayman Inc. . Their selection was based on the literature review and potential applicability for commercial use as anti-borreliae agents. The stock solution (25–50 mg/ml) of each solid compound was prepared by suspending an individual test compound in DMSO and sterilizing with 0.22 μm syringe filtration. All liquid (stock) compounds were also sterilized with 0.22 μm syringe filtration. No difficulties were observed with dissolving the test lipids in DMSO.The test compounds and antibiotics with the purity between 90 and 98% according to the manufacturer, with the exception of the Greek sage oil -shaped forms, and biofilm-like aggregates. Low passage isolates of the B31 strain of Borrelia burgdorferi and the CIP103362 strain of Borrelia garinii were obtained from the American Type Culture Collection . The B31 strain is an isolate from Ixodes dammini, and the CIP103362 strain is an isolate from Ixodes ricinus. Both Borrelia sp. were prepared for testing as previously reported [2, in 15 ml polypropylene sterile test tubes, respectively. The homogeneous logarithmic culture  of tested Borrelia sp. was obtained by maintaining inoculum in a shaking incubator at 33 °C and 250 rpm for 2–3 days. Stationary culture (enriched in knob/round-shaped cells/persistent forms) of tested Borrelia sp. was generated by maintaining inoculum in an incubator at 33 °C for 7–8 days. Biofilm-like aggregates of tested Borrelia sp. were prepared by incubation of inoculums in four-well chambers  coated with collagen Type I from rat tail for at least 1 week.Two reported . Briefly6–1 × 107 cells/ml  and supplemented with the test compound in concentration ranging from 0.025 to 1.0% for liquid agents, and from 0.025 mg/ml to 1.0 mg/ml for solid agents. Controls were treated with DMSO (0.1–0.4%) or triple combination of antibiotics (daptomycin+cefoperazone+doxycycline)  since this combination of antibiotics showed to be effective against the logarithmic phase and the stationary phase of Borrelia burgdorferi s.s. [2 up to 72 h. Assessment was done using high throughput spectrofluorometric screening and/or direct counting with a bacterial Petroff-Hausser counting chamber and dark field microscopy, supported by SYBR Green I/PI staining and a fluorescence microscope , respectively. The excitation wavelength was set at 485 nm, and the fluorescence intensity at 535 nm (green emission) and 635 nm (red emission). Long-term sub-culture study was performed by transferring 50 μl of the stationary phase of Borrelia sp. culture, previously treated with test lipid and washed with fresh BSK-H medium, to 1 ml of fresh agent-free BSK-H medium for 14 days. The tubes were then incubated at 33 °C with 5% CO2 and bacterial proliferation was assessed using a bacterial Petroff-Hausser counting chamber with a dark field microscope and SYBR Green I/PI staining with a fluorescence microscope  [Testing was assessed using direct counting and spectrofluorimetry as previously described , 34. Brieri s.s. . Next, t7 cells/ml was inoculated into each sterile chamber filled with 1 ml BSK-H medium, and incubated for 1 week at 33 °C with 5% CO2, followed by 72 h of incubation with the test lipids. Control wells received DMSO (0.1–0.4%) or the triple combination of antibiotics (daptomycin+cefoperazone+doxycycline) . Eradication of biofilm-like structures was assessed by using the crystal violet (CV) staining method as previously reported [Quantitative anti-borreliae effect of the test compounds against biofilm-like aggregates was assessed in four-well chambers coated with collagen Type I from rat tail  as described , 6, 26. reported . All expn = 3). The Student’s two-tailed t test was used to determine statistically significant differences set at 0.05 levels. Statistical analysis was performed using GraphPad software.All data are presented as means ± SD (Borrelia burgdorferi s.s. (B31 strain) and Borrelia garinii (CIP103362 strain) are presented as the minimal bactericidal (MBC) concentrations in Tables Borrelia sp. The triple combination of antibiotics (daptomycin+cefoperazon+doxycycline) at the concentration of 0.03 mg/ml  was used as a positive control since this combination at this particular concentration was previously reported to be effective against both typical motile spirochetes and knob/round-shaped persisters of Borrelia burgdorferi [The bactericidal efficacy of selected lipid compounds against typical motile spirochetes (logarithmic phase) and knob/round-shaped persisters (stationary phase) of i s.s. B3 strain a CIP10336 strain agdorferi , 26.Tabl50 value achieved after 24 h  are effective biocides, since no regrowth of viable cells was detected after a 14-day subculture. This was in contrast to 13Z,16Z docosadienoic where ~ 24% regrowth was observed. The 0.03 mg/ml (0.003%) triple antibiotic combination  was effective against typical motile spirochetes and knob/round-shaped persisters, but not biofilm-like aggregates grown on collagen surface, and treatment with the triple antibiotic combination resulted in ~ 11% regrowth of viable cells, as reported previously .Pimenta racemosa) often misled with Laurel leaf (Laurus nobilis), Birch oil (Betula lenta), and Cassia oil (Cinnamomum cassia) misled with Cinnamon oil (Cinnamomum zeylanicum), in contrast to other volatile oils included in this study, e.g. Chamomile oil German (Matricaria chamomilla), Chamomile oil Roman (Chamaemelum nobile), Laurel leaf (Laurus nobilis), Thyme oil (Thymus vulgaris), etc., have not been extensively studied by other research groups against gram-positive and gram-negative bacteria, and there is no record about their anti-borreliae efficacy [, recently reported about the profound efficacy of Clove oil (Syzygium aromaticum), Cinnamon oil (Cinnamomum zeylanicum), and Oregano oil (Origanum vulgare) with its main component carvacrol, against the stationary phase of Borrelia burgdorferi at the MBC90 value established to be at or above 0.1%.Antibacterial efficacy of Bay leaf oil , as well as monounsaturated erucic acid  and petroselinic acid . Interestingly, the unsaturated fatty acids that are classified as omega 6, 9, and 12 were more potent than omega 3. Their molecular target and mode of action still remains unknown, even though there have been several reports that point to the cell membranes as a primary target [, [Based on our study we concluded that unsaturated fatty acids were more potent than saturated fatty acids against y target . Greenwaarget [,  suggestearget [, . In addiBorrelia burgdorferi sensu lato and indicate their being a promising non-synthetic alternative for combating these bacteria. These and earlier reported data by other groups support the hypothesis that organic oils in particular may be valuable and cost-efficient antimicrobials, and could be especially beneficial as topical or oral intervention in the case of other limited resources. Although further in vivo and human studies with attention paid to their safety are warranted, the organic oils could also become important antimicrobial agents improving the outcome of traditional treatments, and/or as an alternative option to conventional approaches.Collectively, this study documents in vitro efficacy of a range of lipid compounds against active and latent forms of


The tumor–node–metastasis (TNM) staging system does not perform well for guiding individualized induction or adjuvant chemotherapy for patients with locoregionally advanced nasopharyngeal carcinoma (NPC). We attempted to externally validate the Pan’s nomogram, developed based on the 8th edition of the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) staging system, for patients with locoregionally advanced disease. In addition, we investigated the reliability of Pan’s nomogram for selection of participants in future clinical trials.This study included 535 patients with locoregionally advanced NPC who were treated between March 2007 and January 2012. The 5-year overall survival (OS) rates were calculated using the Kaplan–Meier method and compared with predicted outcomes. The calibration was tested using calibration plots and the Hosmer–Lemeshow test. Discrimination ability, which was assessed using the concordance index, as compared with other predictors.P < 0.001] and underestimated the 5-year OS of each risk group. The differences between the predicted and observed 5-year OS rates were smallest among low-risk patients  and were largest among high-risk patients (≥ 160 points) . The Hosmer–Lemeshow test suggested that the predicted and observed 5-year OS rates had no ideal relationship (P < 0.001). Pan’s nomogram had better discriminatory ability compared with the levels of Epstein–Barr virus DNA acid (EBV DNA) and the 7th or 8th AJCC/UICC staging system, although not better compared with the combination of EBV DNA and the 8th staging system. Additionally, Pan’s nomogram was marginally inferior to our predictive model, which included the 8th AJCC/UICC N-classification, age, gross primary tumor volume, lactate dehydrogenase, and body mass index.Pan’s nomogram was observed to underestimate the 5-year OS of the entire cohort by 8.65% [95% confidence interval (CI) − 9.70 to − 7.60%, Pan’s nomogram underestimated the 5-year OS of patients with locoregionally advanced NPC at our cancer center, and may not be a precise tool for selecting participants for clinical trials. Nasopharyngeal carcinoma (NPC) arises from the squamous cells of the epithelial lining of the nasopharynx. Radiotherapy is the primary treatment modality because of NPC’s confined anatomical location and high sensitivity to radiation. The non-specificity of nasal and aural symptoms accounts for locoregionally advanced disease in 70% of patients upon initial diagnosis . SubsequP < 0.01) discriminatory power compared to the 8th AJCC/UICC staging system .Pan et al.  have devAlthough Pan’s nomogram may have greater potential than the 8th AJCC/UICC edition to identify patients for inclusion in clinical trials, however, since it was developed from a cohort of patients with stage I–IVa disease, its validity for specifically identifying patients with locoregionally advanced disease remains unknown. Additionally, external validation is important before clinical application to individualized randomized controlled trials of induction or adjuvant chemotherapy. As such, we first assessed Pan’s nomogram discriminatory accuracy and calibration by using a large external cohort of patients with stage III–IVb NPC who underwent intensity-modulated radiotherapy (IMRT) and concurrent chemotherapy alone. Second, we performed a direct comparison of its performance with that of Epstein–Barr virus deoxyribonucleic acid (EBV DNA), the most recent and potential biomarker for NPC , in an a18F]-fluorodeoxyglucose positron emission tomography combined with computed tomography (PET/CT); (3) ages between 20 and 75 years old; (4) treated with IMRT plus concurrent chemotherapy alone; and (5) had pretreatment levels of EBV DNA and hemoglobin. Patients were excluded if they had received anticancer therapy prior to diagnosis at our hospital, were pregnant or lactating, or if they were diagnosed with synchronous/metachronous cancer lesion(s) before or during the treatment or follow-up period.Between March 2007 and January 2012, patients were deemed eligible for this study if they met the following inclusion criteria: (1) newly diagnosed with the World Health Organization type 2 or 3 NPC; (2) restaged to III–IVb  according to pretreatment magnetic resonance imaging (MRI) of the nasopharynx and neck, chest radiography or computed tomography (CT), abdominal sonography or CT, a whole-body bone scan or -fluorodeoxyglucose PET/CT were performed to detect locoregional relapse, distant metastasis, or both. Salvage treatment including reirradiation, surgery or chemotherapy, or both, was delivered to patients with confirmed relapse, distant metastasis, or persistent disease.Patients were followed at least once every 3 months during the first 3 years and every 6 months thereafter. Detailed recordings of history and physical examinations were performed at each follow-up visit. Nasopharyngoscopy with or without biopsy, MRI of the head and neck, chest radiography or CT, abdominal sonography or CT, a whole-body bone scan, or [The 5-year overall survival (OS) rate, defined from the date of treatment to death from any cause, was predicted using Pan’s nomogram for the entire cohort and each of the three different risk groups  as suggested by Pan et al. . The 5-yNext, we assessed the calibration of the model by plotting the observed and predicted 5-year OS outcomes and confirmed the findings using the Hosmer–Lemeshow calibration test ; for whiWe compared the discriminatory accuracy of Pan’s nomogram vs EBV DNA levels, the 7th and 8th editions of the AJCC/UICC staging system, and the best predictive model of our dataset. To develop our best predictive model, prognostic factors such as age , sex 2222, body https://cran.r-project.org/). A two-sided P < 0.05 was considered as statistically significant.Statistical analyses were performed using Stata version 14.1  and R version 3.3.1 , 43 (8.0%), 75 (14.0%), and 74 (13.8%) patients experienced locoregional failure, distant failure, and death, respectively.P < 0.001) in addition to the survival of each risk group. The difference between the predicted and observed 5-year OS rates were smallest among low-risk patients  and largest among high-risk patients . Calibration plots of the predicted vs observed 5-year OS rates and survival curves by stratifying risk are illustrated in Fig. P < 0.001).Table P = 0.005). For the clinical stage determined using the 8th and 7th edition of the AJCC/UICC staging system, the C-index was 0.594 (95% CI 0.536–0.651) and 0.594 (95% CI 0.531–0.656), respectively, which was much lower as compared with that of Pan’s nomogram (both P < 0.001). Further, the advantage conferred by the discrimination ability achieved using Pan’s nomogram sharply decreased when compared with the combination of EBV DNA (categorized) and the clinical stage determined according to the 8th edition of the AJCC/UICC staging system .The C-index for Pan’s nomogram to predict 5-year OS was 0.710 (95% CI 0.649–0.771). When comparing the discrimination ability of Pan’s nomogram with that of other predictors, we observed that for EBV DNA (categorized), the C-index was 0.616 (95% CI 0.551–0.681), which indicated inferiority to Pan’s nomogram (P = 0.097) when compared with that of Pan’s nomogram.Multivariate Cox regression model using backward selection approach ultimately identified the variables, age, BMI, LDH, GTVp, and the 8th AJCC/UICC N-classification as independent prognostic factors Table . AdditioOur findings demonstrated that Pan’s nomogram  underestThe calibration ability of Pan’s nomogram derived from our database differed from the training and validation cohort of Fujian Provincial Cancer Hospital and Pamela Youde Nethersole Eastern Hospital, respectively . This caSecond, the patients in our database received concurrent chemoradiotherapy alone, whereas the patients in the study by Pan et al.  receivedIn contrast, the differences among other characteristics suggest that the prediction of Pan’s nomogram was not precise enough. For example, the LDH levels of patients in our database were significantly lower compared with those of patients included in the study by Pan et al.  Table 11 and thePan’s nomogram discriminated outcomes better compared with other single predictors such as EBV DNA and the 7th and 8th AJCC/UICC staging system. This was expected because Pan’s nomogram combined several prognostic factors with tumor stage. Unfortunately, Pan’s nomogram did not achieve significant superiority over the combination of EBV DNA and the tumor stage based on the 8th edition of the AJCC/UICC staging system. Moreover, it was marginally inferior to the model, which included independent prognostic factors such as the age, BMI, LDH, GTVp, and N-classification based on the 8th AJCC/UICC staging system.Risk prediction programs , 34–37 oThe limitations of this study are as follows. The lack of unified treatment approaches, chemotherapy regimens, and radiation or chemotherapy doses determined by the nature of retrospective design may, to a certain extent, bias the findings of this study. Also, due to the small sample size of patients analyzed, this could have possibly lowered the confidence of validation derived from this study. Lastly, validation by a single institution does not essentially provide a strong evidence and further large cohort, multi-institutional analysis is still required.Pan’s nomogram was observed to significantly underestimate the 5-year OS of patients with locoregionally advanced NPC. It failed to precisely identify high-risk participants for inclusion in randomized controlled trials.


Non-obstructive coronary artery disease (NOCAD) is a common finding on coronary angiography. Our goal was to evaluate the long-term prognosis of NOCAD patients with stable angina (SA).The study cohort consisted of 7478 NOCAD patients with normal EF (≥ 50%), and SA who underwent coronary angiography between 1995 and 2012. We compared NOCAD patients (stenosis< 50%) with 10,906 patients with stable obstructive CAD (≥ 50%). The primary endpoint was all-cause mortality. Secondary endpoints included repeat angiography, progressive CAD, and PCI. A second comparison group consisted of 7344 patients with NOCAD presenting with an ACS. Rates of all-cause mortality of NOCAD ACS patients were compared to NOCAD SA patients.p < 0.001). This was driven by patients with normal coronary arteries , p < 0.001), whereas patients with minimal disease (> 0% and < 50%) were at similar risk as CAD patients , p = 0.06). In NOCAD patients, the strongest predictors of all-cause mortality were age and minimal disease. SA patients with NOCAD had low rates of repeat angiography (7.3%), future CAD (2.3%) and PCI (1.7%). NOCAD ACS patients had a 41% increase in all-cause mortality risk compared to NOCAD SA patients (HR 1.41 (1.25–1.6), p < 0.001).Median follow-up time was 6.5 years. NOCAD patients had a lower risk of all-cause mortality compared to CAD patients (HR CAD vs. NOCAD 1.33 (1.19–1.49); This study underlines the importance of minimal CAD, as it is not a benign disease entity and portends a similar risk as stable obstructive CAD.The online version of this article (10.1186/s12872-018-0908-z) contains supplementary material, which is available to authorized users. Non-obstructive coronary artery disease (NOCAD) is a common finding on diagnostic coronary angiograms with rates of up to 50–60% in patients with stable angina (SA) and of about 30% in certain population with acute coronary syndromes (ACS) –4. SymptFurthermore, it is well known that presentation with an ACS with obstructive CAD portends an increased mortality risk. Previous studies addressed ACS patients with NOCAD, which is also termed myocardial infarction with non-obstructive coronary arteries (MINOCA), but study size was either small or patients were followed only in the short-term –12.Our aim was therefore to evaluate prognosis and its predictors in a large, contemporary population of patients of both sexes with NOCAD. We also sought to investigate whether presentation with an ACS leads to a worse prognosis compared to stable NOCAD patients.Eligible subjects included all adults over the age of 18 years undergoing their first cardiac catheterization between January 1, 1995 to March 31, 2012, registered in the Alberta Provincial Project for Outcome Assessment in Coronary Heart Disease (APPROACH©) database . APPROACData collection included e.g. sociodemographic characteristics, comorbidities and risk factors, disease specific variables , and medications at time of catheterization. Angiography results including coronary anatomy, extent of coronary stenosis, and LV ejection fraction (EF) were also recorded . The degAPPROACH and this protocol were approved in accordance with the Declaration of Helsinki by the Institutional Ethics Review Board of the University of Calgary. Patients signed informed consent to allow data collection, clinical follow-up, and anonymous data reporting.Normal coronaries, minimal disease (i.e. coronary sclerosis) and significant obstructive disease were defined as 0%, > 0 and < 50% and ≥ 50% luminal narrowing in any epicardial coronary artery, respectively. The NOCAD cohort consisted of those with normal coronary arteries or minimal disease. We identified patients with a normal EF (≥50%) who presented with either SA or an ACS.Stable angina (SA) was defined as Canadian Cardiovascular Society (CCS) class 1, 2 or 3 angina with inclusion of clinically stable patients with atypical chest pain. ACS was defined as unstable angina or non–ST-elevation MI or ST-elevation MI in accordance to universal myocardial infarction criteria.Patients with an EF below 50% or prior percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) were excluded, as well as patients with significant valvular disease, or left main disease, or referrals for pre- or post-transplant work-up, evaluation of heart failure, congenital heart disease, or serious arrhythmia. Patients with incomplete data were likewise excluded. The rate of incomplete data was low (3.7%).n = 7478). Comparison Group I contained patients with significant CAD (≥ 50% stenosis) who presented with SA . Comparison Group II included patients with NOCAD presenting with an ACS (n = 7344).The main study group consisted of patients with NOCAD (coronary stenosis < 50%) presenting with SA , repeat angiogram, and future PCI in NOCAD patients. Any patient who had a second angiogram during the follow-up period was counted once as having had a repeat angiogram.Summary statistics for categorical variables presented include counts and percentage, and for continuous variables include mean and standard deviation. The difference for categorical variables between NOCAD and CAD was tested by Chi-square test; and the difference for continuous variables were evaluated by student’s t-test. Cox’s proportional hazard regression model was used to compare all-cause mortality between CAD and NOCAD. The crude hazard ratio (HR) and adjusted HR with their 95% confidence intervals were estimated. In the multivariable-adjusted model, we adjusted the HRs for age, diabetes mellitus (DM), and hypertension. The analyses for Cox’s proportional hazard regression model were stratified by the groups of NOCAD and its subgroups of normal coronaries, and minimal disease (> 0 and < 50%), as well as obstructive CAD. We estimated HRs between NOCAD and CAD patients, stratified by coronary status, and between NOCAD patients presenting with SA or an ACS.Moreover, Cox’s proportional hazard regression models were used to investigate predictors for all-cause mortality among patients with NOCAD. Pre-specified variables for the univariate Cox regression model included age over 55 years, presence of DM, positive stress test, abnormal baseline ECG, hypertension, or previous or current smoking, as well as having undergone more than two angiograms for the same condition.Factors, which showed a significant association with increased mortality in the univariate analysis, were entered in the multivariate regression model using a stepwise method. To explore the effect of sex, we included this factor in our analyses, independently of the significance level in the univariate model. We conducted these analyses for NOCAD patients with SA. All statistical analyses were performed using statistical software SAS .During the study period, there were 141,004 patients in the APPROACH registry with 70.2% men and 29.8% women. For reasons previously described, we excluded 92,126 patients, leaving a population of 48,878 subjects with normal EF. Figure NOCAD patients were significantly younger, were more likely to be female and had significantly lower rates of cardiovascular risk factors compared to CAD patients. A similar picture emerged when patients with normal coronary arteries were compared to patients with minimal disease . SA patients with NOCAD had a lower risk of all-cause mortality than patients with CAD after adjustment for basic risk factors .When NOCAD patients presented with an ACS rather than with SA, they had a 41% increase in mortality risk (NOCAD ACS vs. SA HR 1.41 (1.25–1.6), n = 543 (7.3%)). Simultaneously, progression to obstructive CAD in patients with NOCAD was small (n = 170 (2.3%)), as well as the necessity to perform a PCI (n = 128 (1.7%)). Overall, patients with a NOCAD had significantly lower rates of cardiac procedures than patients with stable one-vessel CAD (p < 0.001).Over a median of 6.5 years of follow up, the percentage of subjects with a repeat catheterization was low , which showed that patients with NOCAD had a similar mortality risk as patients with obstructive 1-vessel CAD . ConcomiOverall, data appears to accumulate that the finding of NOCAD is not benign and should prompt consideration of secondary prevention measures as used in subjects with stable obstructive CAD. Large-scale studies are warranted to determine the benefit of such measures.Some limitations apply to this study. Foremost, this was not a randomized controlled trial but a population-based registry study with all its ensued limitations of potential confounding and unmeasured covariates. However, the strength of the APPROACH registry is that it provides a real-world scenario and it captures all deaths and revascularization procedures within the province.p < 0.05) who present to our region with ACS undergo catheterization. Furthermore, we used multivariable adjustment as strategy to account for baseline differences between patients with NOCAD and CAD. Nevertheless, residual confounding cannot be excluded.Of note is that APPROACH is a procedure-based registry and not a clinical registry. We cannot exclude a diagnosis, angiogram and/or hospitalization referral bias. However, based on information from an APPROACH ACS registry, it is known that 73.9% of women and 85.1% of men


As ageing emerges as the next public health threat in Africa, there is a paucity of information on how prepared its health systems are to provide geriatric friendly care services. In this study, we explored the readiness of Uganda’s public health system to offer geriatric friendly care services in Southern Central Uganda.Four districts with the highest proportion of old persons in Southern Central Uganda were purposively selected, and a cross-section of 18 randomly selected health facilities (HFs) were visited and assessed for availability of critical items deemed important for provision of geriatric friendly services; as derived from World Health Organization’s Age-friendly primary health care centres toolkit. Data was collected using an adapted health facility geriatric assessment tool, entered into Epi-data software and analysed using STATA version 14. Kruskal-Wallis and Dunn’s post hoc tests were conducted to determine any associations between readiness, health facility level, and district.p = 0.015). The difference in readiness between HCIIIs and HCIVs was 2.39 (p = 0.025).The overall readiness index was 16.92 (SD ±4.19) (range 10.8–26.6). This differed across districts; Lwengo 17.91 (SD ±3.15), Rakai 17.63 (SD ±4.55), Bukomansimbi 16.51 (SD ±7.18), Kalungu 13.74 (SD ±2.56) and facility levels; Hospitals 26.62, Health centers four (HCIV) 20.05 and Health centers three (HCIII) 14.80. Low readiness was due to poor scores concerning; leadership (0%), financing (0%), human resources (1.7%) and health management information systems (HMIS) (11.8%) WHO building blocks. Higher-level HFs were statistically significantly friendlier than lower-level HFs (There is a low readiness for public health facilities to provide geriatric friendly care services in Uganda. This is due to gaps in all of the health system building blocks. There is a need for health system reforms in Uganda to adequately cater for service provision for older adults if the 2020 global healthy ageing goal is to be met. In 2017, the global population of older adults (aged 60 years and above) was 962 million. This is expected to rise to 2.1 billion by 2050, with 80% of them living in the developing world; Africa being home to 10.9% of them . In respThere is evidence that Western countries have progressed well regarding the achievement of the objectives above . HoweverUganda’s socio-economic transformation, internal security, and an improvement in health care services like maternal child health, immunisation, surgical care, and management of infectious diseases like HIV/AIDS, malaria, and respiratory tract infections , have seRelatedly, as the burden of infectious diseases declines, just like in other sub-Saharan African countries, there is a notable increase in the burden of non-communicable diseases in Uganda, more so among the older adults –16. AlthLike other developing countries, Uganda’s public health system’s readiness to provide geriatric friendly services is not well documented . ExistinGeriatrics studies in Uganda have mainly focused on: exploring geriatric education needs, knowledge and attitudes of health workers towards geriatric care services , the imReadiness is the ability of a health facility to provide a given health service , and forThe SARA methodology has effectively been used in resource-limited countries to assess: general health facility readiness , progresExcellent leadership and governance are crucial for the institutionalisation of geriatric friendly care services because it is vital in developing policies and guiding documents, providing enabling environments and forging strategic partnerships with all key stakeholders , 38. FurAssessing the readiness of public health systems to provide geriatric friendly care services is vital for countries to come up with policies that enable adequate planning for their ageing population. The objective of our study was to assess the readiness of public primary health care facilities to provide geriatric friendly services in Southern Central Uganda.This cross-sectional study was conducted across public primary health care facilities located in four purposively selected districts of Bukomansimbi, Kalungu, Rakai and Lwengo which according to the 2014 National census data were home to the highest proportion of older adults; 5.9, 5.9, 5.0 and 4.8% respectively in Southern Central Uganda , 40. PreThe study population was public primary health care facilities  located within the four selected districts. HCIIIs are a first level referral HFs located at sub-county level and offer non-specialised preventive, curative, and promotive health care services. In Uganda, they serve an average population of 30,000 people and are headed by a Clinical Officer . HCIVs are second-level referral health facilities located at the county level. On top of offering services offered at HCIIIs, they offer surgical, inpatient, and blood transfusion services. They are led by Medical Officers and are designed to serve 100,000 people. Hospitals are more specialised and are located at the district level. They offer more specialised health care services, are led by a medical director, and serve a population of over 200,000 people , 41. DetWe used the WHO’s workplace violence in the health sector sampling design frame  because Data was collected using a pretested geriatric health facility assessment tool whose variables were adapted from WHO’s 2008 Age-friendly Primary Health Care Centres Toolkit , USAID’sWe determined the readiness index (RI) for each HF based on SARA guidelines  by ascerRI = (a + b + c + d + e + f)/6 . [Where To elicit associations between readiness and level and location of HFs; the Kruskal-Wallis test was run; while Dunn’s post hoc test determined which facility levels or districts differed in readiness.For a HF to be deemed fully ready to provide geriatric friendly care services, it was expected to have an 80–100 RI score. Moderate readiness was a RI of 51–79, while low readiness was scoring 0–50.n = 9) of them were from Rakai district, 22% (n = 4) from Lwengo district, three HFs (17%) from Kalungu district, and two HFs (11%) were selected from Bukomansimbi district. By facility level; 67% (n = 12) were HCIIIs, 28% (n = 5) HCIVs, and one hospital. The overall readiness index (RI) was 16.92 . Of the four districts, Lwengo and Rakai districts had the highest readiness indexes of 17.91(SD 3.15) and 17.63 (SD 4.55) respectively, while Bukomansimbi and Kalungu districts had RIs of 16.51 (SD 7.18) and 13.74 (SD 2.56) respectively. The highest facility-level readiness index was at the hospital with a RI of 26.62, while HCIVs had a RI of 20.05 and HCIIIs a RI of 14.80 (Table p = 0.015) across HF levels, with Dunn’s post hoc test showing that the difference (2.39) was between HC IIIs and HC IVs (p = 0.025).Of the 18 HFs, 50% , HMIS for geriatric care (11.8), and human resource for geriatric care (1.7). The leadership and governance, and financing for geriatric care blocks had a score of zero Fig. .Fig. 2Mn = 18) scored zero concerning this building block. They lacked geriatric care policies, national geriatric care management guidelines, had no geriatric focal personnel, and did not receive geriatric support visits. Relatedly, older adults were not represented on health unit committees and had no community networks to support them  on ageing, with older adults allowed to ask questions. Only 2 HFs had toilets with grab rails Fig. a, and onTable n = 18, 100%) had weighing scales and MUAC tapes, pain killers, anti-hypertensives, eye ointment, and antibiotics. Of the 18 HFs, blood pressure machines were available at 13 (72%), stethoscopes at 15 (83.3%), 11 HFs (61%) had glucometers, and 16 HFs (89%) had anti-malarial drugs. On the other hand, all HFs (n = 18) lacked hearing loss screening equipment, hearing aids, memory loss screening cards, and incontinence bags. Eyeglasses, walking crutches, white canes for the blind and other assistance devices for the blind were found at only one (5.6%) of the 18 HFs. Wheelchairs were only available at four (22%) HFs. For other tracer items, see Table This WHO building block had the highest score of 46.4. All HFs  had out-patient department (OPD) registers that segregated data by age at all HFs , and 17 of them (94.4%) were reporting on geriatric data through DHIS2. On the other hand, all HFs lacked vital tools: the geriatric medical assessment tool, geriatric comprehensive screening tool, geriatric mental state examination tool, memory loss evaluation form, geriatric depression scale, urinary incontinence evaluation form, fall evaluation form, and geriatric daily activity form. No HF was using geriatric data to improve service delivery  across all study districts and HFs, though hospitals (26.62) and HCIVs  were more prepared compared to HCIIIs   across HF levels, where hospitals were more ready (26.62) compared to HCIVs  and HCIII , has been documented in other health systems assessment studies conducted in Uganda. For example, Katende et al., (2015), found hospitals and HCIVs to more equipped with regard to the availability of essential supplies, training, drugs, and diagnostic equipment as compared to hospitals and HCIVs [The significant difference in readiness indexes  can be attributed to the Government of Uganda’s current efforts to improve its health system through making health facilities more accessible and equipped with laboratory sundries, and screening equipment . On the The highest WHO block-level score was with the medical commodities and equipment for geriatric care block (46.4). This is attributable to policies that have led to system improvements at the National Medical Store; Uganda’s autonomous body responsible for procurement and supply of medical equipment and medicines across all public health facilities in Uganda . HoweverThe low HMIS block score (11.8) correlates with existing data that HMIS remains a challenge in Africa  and UganTo our knowledge, this study provides the first published insight on the readiness of public health facilities to offer geriatric friendly care services in Uganda and Africa, thus providing useful information for policy action. Furthermore, our study utilised data from different districts and levels of HFs; making findings more generalizable. During data collection, we used standardised piloted tools and triangulation through observation and verification. Lastly, the tools used for data collection were adapted from published, internationally proven documents like SARA , the HeaOur study was restricted to public primary HFs, leaving out regional, national, private, and private not for profit HFs. This leaves an information gap on geriatric readiness in other health service sectors. Furthermore, the study was limited to districts in Central Uganda which could affect country-level generalisation. However, efforts were made to integrate a diverse group of HFs across various districts and locations to enrich the findings. Lastly, the two hospitals in this study were located within one district. For our study sample, we chose one hospital, which made eliciting associations at that level hard. We recommend country-based surveys that include non-public health facilities, further validation of the geriatric primary health care facility assessment tool, and anthropological studies to augment our findings.These findings indicate that Uganda’s public primary health care system is not adequately ready to provide geriatric friendly care services. This is due to gaps in leadership and governance, financing, human resource, HMIS and equipment and commodities for geriatric care. For Uganda to align with the 2020 global healthy ageing goal, changes in policy, financing, human resource for geriatric care, creating a favourable environment for older adults and HMIS for geriatrics need improvement, more so at lower level community HFs where the majority of older adults seek care.Additional file 1: Table S1. The geriatric readiness score framework.Additional file 2: Table S2. A guide for calculating HF block level scores.


P < 0.001, n = 179), and time-dependent ROC analysis demonstrated that the six-gene signature had good predictive ability for six-year survival for ESCC . We then validated its prediction performance in an independent set by RT-PCR . Furthermore, RNAi-mediated knockdown of genes in the flavoprotein signature led to decreased proliferation and migration of ESCC cells. Taken together, CTTN, GGH, GPD2, PYROXD2, SRC, and SYNJ2BP have an important clinical significance for prognosis of ESCC patients, suggesting they are efficient prognostic markers and potential targets for ESCC therapy.Flavoproteins and their interacting proteins play important roles in mitochondrial electron transport, fatty acid degradation, and redox regulation. However, their clinical significance and function in esophageal squamous cell carcinoma (ESCC) are little known. Here, using survival analysis and machine learning, we mined 179 patient expression profiles with ESCC in GSE53625 from the Gene Expression Omnibus (GEO) database and constructed a signature consisting of two flavoprotein genes (GPD2 and PYROXD2) and four flavoprotein interacting protein genes . Kaplan–Meier analysis revealed the signature was significantly associated with the survival of ESCC patients (mean survival time: 26.77 months in the high-risk group vs. 54.97 months in the low-risk group,  Esophageal cancer has been ranked as the fifth most malignant disease, and it is the fourth leading cause of cancer death in China . There aRiboflavin (vitamin B2) is an essential micronutrient for normal cellular function. Riboflavin is the precursor of both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which play key roles in cell development and growth. Riboflavin participates in intermediary metabolism and is required for many metabolic reactions, such as mitochondrial electron transport, fatty acid degradation, and redox regulation , 6. An iDeficiency of riboflavin can directly lead to the abnormal function of flavoproteins. Flavoproteins are a class of enzymes that catalyze a wide range of redox reactions through a variety of chemical mechanisms. This kind of protein must contain noncovalently bound FAD or FMN as a cofactor. Warburg et al. isolated the first flavoprotein from yeast (flavin phosphoric acid was its prosthetic group), and Banga et al. observed the presence of flavin in muscle tissue. So far, more than 160 kinds of flavoproteins have been isolated and characterized . One of http://www.uniprot.org/) and identified their interacting proteins from the Human Protein Reference Database (http://www.hprd.org/) and BioGRID (https://thebiogrid.org/) database [We selected flavoproteins from the UniProt database (database .https://www.ncbi.nlm.nih.gov/geo/). We used this dataset as a training set [The mRNA expression data and corresponding clinical data of 179 ESCC patients (GSE53625) were obtained from the GEO database  and then used the random survival forests variable hunting (RSFVH) algorithm to filter out genes until 10 genes [N is the number of prognostic flavoproteins and their interacting genes, EXP is the gene's fold change value of expression, and β is the corresponding COX coefficient of the genes.Firstly, we selected survival-related flavoproteins and their interacting genes by univariate Cox proportional hazards analysis  was repeated at least three times [Total RNA from cells and tissues was extracted with TRIzol  according to the manufacturer's protocol. The purity and concentration of RNA were determined by OD260/280 ratio using a NanoDrop spectrophotometer, cDNA was obtained from the total RNA using random hexamers, and real-time PCR was performed by using a SYBR® Premix Ex Taq™ kit . The primer sequences for CTTN (cortactin), GGH (gamma-glutamyl hydrolase), GPD2 (glycerol-3-phosphate dehydrogenase 2), PYROXD2 (pyridine nucleotide-disulphide oxidoreductase domain 2), SRC (non-receptor tyrosine kinase), and SYNJ2BP (synaptojanin 2 binding protein) for real-time RT-PCR are shown in ee times , 24.μg/mL). Cells were incubated at 37°C in 5% CO2.Cell lines used in this study were previously described , 26. In In functional assays, KYSE150 and KYSE510 cells were seeded into plates and cultured for 12–24 h until 70–80% confluence. ESCC cells were transfected with 30 nM or 75 nM siRNA using Lipofectamine 3000 (Invitrogen) according to the manufacturer's instructions. Then, cells were cultured and used for further analysis. Short interfering RNAs for CTTN, GGH, GPD2, PYROXD2, SRC, and SYNJ2BP and a negative control (NC) siRNA were synthesized by GenePharma . The siRNA sequences targeting the flavoprotein signature are described in 2. After washing twice with 4°C precooled PBS, cultures were fixed with ice-cold methanol for 20 min and stained with haematoxylin for 15 min. Colonies were photographed using ChemiDoc Touch (Bio-Rad). Each experiment was performed in triplicate.A colony formation assay was performed as described previously . In brieμL pipette tip. ESCC cells were maintained in RPMI 1640 medium with 2.5% fetal bovine serum. Images were captured at 0 and 36 h using a Leica DMI3000B inverted phase-contrast microscope . The wound closure rate was calculated from 6 images, using Image J  analysis. Each experiment was performed in triplicate.KYSE150 and KYSE510 cells were transfected with siRNAs targeting the flavoprotein signature, and then cells were starved in serum-free medium for 12 h after being transfected for 36 h. Circles 3 mm in diameter were marked on the bottom of each dish to identify the areas for image capture and ensure that measurements were taken at the same locations. A wound was made by scraping the cell monolayer with a 200 4 cells were plated in medium without serum in the upper well of a transwell chamber of a 24-well transwell with 8 μm pores (BD Biosciences), which was placed in a bottom chamber containing medium supplemented with 10% fetal bovine serum. After 48 h, the membranes were fixed and stained with haematoxylin solution and scraped off the cells remaining in the upper chamber. The migration was quantified by counting 10 random fields under a Leica DMI3000B inverted phase-contrast microscope (400) with Image J. Each experiment was performed in triplicate.The transwell assay was performed as described previously . KYSE150http://www.bioconductor.org/). SPSS v13.0 software was used for statistical analysis. Where indicated, statistical analysis was performed by calculating means and SD. Graphs were mainly made by GraphPad Prism 6 . Differences between groups were evaluated with Student's t test. Significance was defined as P < 0.05. Gene set enrichment analysis (GSEA) was performed by the GSEA software [In training datasets, 179 ESCC patients were divided into low-risk (risk score ≤12.86) and high-risk groups (risk score >12.86) by using the median as a cutoff value . In the software .179 ESCC samples  from the GSE53625 dataset were selected according to the dataset screening criteria described in the Materials and Methods section. Several PCGs were identified by probe re-annotation of the Agilent-038314 CBC Homo sapiens lncRNA + mRNA microarray V2.0. We generated a new mRNA expression profile of the GSE53625 profiles by the following steps: First, we keep the probes which were mapped to the genomic coordinates of mRNAs uniquely. Second, we use the arithmetic mean to integrate the values of multiple probes mapping to the same mRNAs. Then, we identified 17434 PCGs in the dataset.P < 0.05), to serve as prognostic genes. Secondly, ten genes most related to the prognostic classification were selected among the flavoprotein family set according to the permutation importance score by using random survival forests variable hunting (RSFVH) algorithm  and four flavoprotein interacting protein genes , and the risk score was obtained as follows: risk score = (−0.77 × expression level of GPD2) + (0.28 × expression level PYROXD2) + (0.33 × expression level of CTTN) + (−0.39 × expression level of GGH) + (0.54 × expression level of SRC) + (0.48 × expression level of SYNJ2BP). AUC of the flavoprotein signature in the prognostic model was 0.76 for survival status  and low-risk group (n = 89). The high-risk group had a significantly shorter OS than the low-risk group . The same flavoprotein model was used to calculate the flavoprotein signature-based risk scores for 121 patients in this dataset. P=0.032, mean survival time: 15.73 months vs. 21.1 months).To confirm the findings described above, we also evaluated the efficiency of the constructed expression-defined flavoprotein prognostic model in independent validation datasets (P < 0.01). Colony formation assay showed that knockdown of the flavoprotein signature genes significantly inhibited the growth of KYSE150 cells and KYSE510 cells . Migration of ESCC cells was examined in each group by wound healing and transwell assays. As shown in Figures P < 0.01, one-way ANOVA). However, six genes in the flavoprotein signature produce different degrees of effect on ESCC cell proliferation and migration. And six genes also play different roles in KYSE150 and KYSE510 cells. After knocking down the PYROXD2 gene in KYSE150 cells, the cell proliferation ability was most significantly reduced , while wound healing and transwell assay showed that knockdown of the PYROXD2 gene in KYSE150 cells had the least effect on cell migration compared to other genes . In KYSE510 cells, the significant effect of inhibiting cell proliferation is knocking down the SYNJ2BP gene . In KYSE150 and KYSE510 cells, the effect of GGH on cell proliferation was minimal compared to other genes . However, after knockdown of GGH, wound healing and transwell assay showed that the GGH gene has a great influence on the mobility of ESCC cells . These results imply that the flavoprotein signature plays important but different roles in the progression of ESCC.To assess the effect of the flavoprotein signature expression in ESCC cells, siRNAs targeting the flavoprotein signature genes were transfected into KYSE510 cells and KYSE150 cells, and the silencing of the flavoprotein signature was determined by qRT-PCR. 10 cells . CompareTo further explore the potential biological function of this signature, we compared the gene expression profiles of ESCC patients classified as high-risk and low-risk by the flavoprotein signature in the training set (GSE53625). The gene sets with significantly different expression (FDR < 0.05) between high-risk and low-risk were selected for gene set enrichment analysis (GSEA). Several clusters of genes functionally related to more than 150 GO terms and 20 KEGG pathways were observed. These data suggest that the flavoprotein signature might affect tumorigenesis and development through interacting with many important biological processes, such as epithelial mesenchymal transition, focal adhesion, oxidative phosphorylation, and long chain fatty acid metabolism .Esophageal cancer is the fifth most common and fourth most lethal malignant tumor in China . CurrentIn this study, we used different statistics and machine learning methods to identify an expression signature, involving flavoproteins and their interacting proteins, that is associated with survival of ESCC patients. The six genes CTTN, GGH, GPD2, PYROXD2, SRC, and SYNJ2BP with the largest AUC were selected as the flavoprotein signature. CTTN and SRC are two of the most studied oncogenes. SRC is a non-receptor protein tyrosine kinase that is activated following engagement of many different classes of cellular receptors . CTTN isFunctionally, we knocked down the flavoprotein signature by transfecting siRNA into ESCC cells. Next, the functionally well-defined CTTN and SRC were used as positive controls to compare the effect of the remaining four genes on proliferation and migration of transfected ESCC cells. Similar to knockdown of CTTN and SRC, ESCC cell growth and motility are significantly reduced following knockdown of GGH, GPD2, PYROXD2, and SYNJ2BP. Among these genes, GPD2 knockdown has the most significant effect on inhibiting the proliferation and migration of ESCC cells. The GPD2 gene encodes the mitochondrial glycerol-3-phosphate dehydrogenase, which is localized to the outer surface of the inner mitochondrial membrane. Mitochondrial glycerol-3-phosphate dehydrogenase, as a component of the glycerophosphate shuttle, functions at the crossroads of glycolysis, oxidative phosphorylation, and fatty acid metabolism. Mitochondrial glycerol-3-phosphate dehydrogenase regulates both the glycerol-3-phosphate and malate-aspartate shuttles, which play important roles in tumor metabolism –58. GSEAIn summary, this is the first study to investigate a signature, comprised of flavoproteins and their interacting proteins, in patients with esophageal squamous cell carcinoma. Furthermore, abnormal expression of the flavoprotein signature promotes proliferation and migration of ESCC cells. These results implicate components of the flavoprotein signature as efficient prognostic markers and potential targets in gene therapy for ESCC.


Background: Alzheimer’s disease (AD) and bipolar disorder (BIP) are complex traits influenced by numerous common genetic variants, most of which remain to be detected. Clinical and epidemiological evidence suggest that AD and BIP are related. However, it is not established if this relation is of genetic origin. Here, we applied statistical methods based on the conditional false discovery rate (FDR) framework to detect genetic overlap between AD and BIP and utilized this overlap to increase the power to identify common genetic variants associated with either or both traits.Methods: We obtained genome wide association studies data from the International Genomics of Alzheimer’s Project part 1  and the Psychiatric Genetic Consortium Bipolar Disorder Working Group . We used conditional QQ-plots to assess overlap in common genetic variants between AD and BIP. We exploited the genetic overlap to re-rank test-statistics for AD and BIP and improve detection of genetic variants using the conditional FDR framework.Results: Conditional QQ-plots demonstrated a polygenic overlap between AD and BIP. Using conditional FDR, we identified one novel genomic locus associated with AD, and nine novel loci associated with BIP. Further, we identified two novel loci jointly associated with AD and BIP implicating the MARK2 gene  and the VAC14 gene .Conclusion: We found polygenic overlap between AD and BIP and identified novel loci for each trait and two jointly associated loci. Further studies should examine if the shared loci implicating the MARK2 and VAC14 genes could explain parts of the shared and distinct features of AD and BIP. About a century ago, Alois Alzheimer and Emil Kraepelin described the historical equivalents of AD and BIP . Still tAlzheimer’s disease is a neurodegenerative disorder  usually Despite several lines of evidence suggesting a relation between AD and BIP, it is not established if the conditions have a shared genetic basis. AD and BIP are in most cases complex traits, i.e., they are influenced by several genetic and environmental factors. Twin studies estimate the heritability of AD and BIP to 60% or higher . Genome With the current sample sizes, however, the power of GWASs can be boosted by leveraging polygenic overlap between complex traits ,b, 2015.p-values for all SNPs) from the IGAP  or 2% , p-value for Hardy–Weinberg equilibrium < 1 × 10-10 in cases or <1 × 10-6 in controls, missingness difference between cases and controls > 2%, or autosomal heterozygosity deviation (|hetF| > 0.2). Individuals with > 2% missing genotypes were also excluded. The IMPUTE2  and for BIP given association with AD as condFDR(BIPjAD) and considered values < 0.01 significant. For further details, we refer to previous studies  to improve power to detect SNPs associated with AD given associations with BIP and -values” . We deno studies ,b, 2015 p-values for both traits are as small or smaller than the observed p-values”  to identify SNPs jointly associated with AD and BIP. conjFDR is defined as “the posterior probability that a SNP is null for either phenotype or both simultaneously, given the -values” . After r-values” ,b, 2015 (ADjBIP) ((BIPjAD) < 0.01   and cond) < 0.01 . We cons) < 0.01 .-8 in the original GWAS(s). Further, we used LDlink (r2> 0.1) with any of the genome-wide significant hits in the original GWAS(s). Finally, we conducted a search on PubMed using the term (“SNP id” OR “gene name”) AND (“Bipolar Disorder”[Mesh] OR “Alzheimer Disease”[Mesh]) to exclude that the variants or implicated genes have been associated with AD or BIP at genome-wide significance in previous GWASs.To determine if a locus was novel, we first checked that the p-value(s) for the implicating variant was > 5 × 10d LDlink  to exclu4 used postmortem brain tissue from over 1,340 samples to provide genome-wide exon-level transcriptome data in 16 cerebral regions (The Human Brain Transcriptome (HBT) project regions . We obtar2> 0.1), and calculated the average empirical cumulative distribution function (ecdf) by using the p-values obtained through 200 iterations. SNPs within the major histocompatibility complex region (defined as chr6:25652429–33368333) and the apolipoprotein E (APOE) gene (chr19:44909039–45912650), and SNPs in LD (r2> 0.1) with these SNPs, were excluded from the analyses due to their complex LD structure (r2< 0.1) intergenic SNPs, which are depleted of true associations, to calculate an inflation factor value  . For detAll GWASs performed and investigated in the present study were approved by the local ethics committees, and informed consent was obtained from all participants. Furthermore, the Norwegian Institutional Review Board for the South-East Norway Region has evaluated the methods used in the current study and found that no additional institutional review board approval was needed because no individual data were used (ref. 2011/1980).vice versa  < 0.01  and MTSS1L . The signal in NDUFS3 was driven by one single SNP and is thus probably a spurious association.We identified 22 SNPs clumped into 19 independent loci at condFDR) < 0.01 . The chr) < 0.01 . Red ann(BIPjAD) < 0.01 , CNTNAP5 , KIAA1109 , SSBP2 , AK091365 , RCOR2 , STARD9 , GRIN2A , THRA , and PRKCA . However; the CNTNAP5 gene has previously been associated with the posterior cortical atrophy variant of AD at genome-wide significance  < 0.01 . As visu) < 0.01 , 17 lociificance  and withificance .Finally, we applied conjFDR to assess for SNPs jointly associated with AD and BIP. We used effect sizes from the original data sources to determine the allelic direction of effects in both traits.(AD&BIP) < 0.05 (MARK2 (rs10792421) was associated with AD and BIP with the same direction of effect on AD and BIP . MARK2 is widely expressed in the developing and adult human brain (VAC14 (rs11649476) was associated with AD and BIP with opposite directions of effect in AD and BIP . VAC14 is also widely expressed in the developing and adult human brain  < 0.05 . A 2 kb an brain . An intran brain . Both SNrg = -0.0222, SE = 0.0519, p = 0.669).We estimated that there is no overall genetic correlation between AD and BIP according to LDSR  had a concordant direction of effect in both traits (MARK2 gene encodes the microtubule affinity regulating kinase 2 (MARK2). The kinase is involved in a diversity of neuronal cellular processes, including neuronal migration, and tau phosphorylation  was related to AD and BIP with opposite directions of effects. The same variant was shared between BIP and intelligence with concordant direction of effects in a recent study using conjunctional FDR P] to phosphatidylinositol 3,5-bisphosphate [PIP2] P2, endosomal dysfunction, and reduced clearance of beta amyloid. Another example of the relationship between the processing of phosphoinositides and APP comes from a study of 2, is negatively associated with a proxy of genetic susceptibility to BIP.The intronic variant within onal FDR . VAC14 eP2] . PIeostasis . A null  neurons . Amyloid neurons . BalklavPRKCA gene encodes the protein kinase C alpha (PKCa). PKCa is described in amyloid plaque of patients with AD  receptor. The NMDA receptor is central for synaptic plasticity and learning . Decreased expression of TRANK1 alters the expression of other genes related to neuronal development and differentiation , clinical features , and treatment response to lithium among patients with AD and BIP. Further, it is unknown if the loci interact with environmental risk factors and other genes implicated in AD and BIP.Our results should be interpreted in light of the following limitations. We can neither exclude that some of the patients with AD have had BIP, nor that some of the patients with BIP will develop AD, which could have confounded our results. However; this could not explain the finding in the conjunctional FDR analyses of one locus implicated in AD and BIP with opposite directions of effect. Due to linkage disequilibrium among SNPs, our findings do not necessarily reflect causal variants, or that the same causal variants are involved in both traits. Although we found indications of modest polygenic overlap using conditional QQ-plots , we onlyMARK2 and VAC14 jointly implicated in AD and BIP are previously described to be involved in neuronal migration, tau phosphorylation, and endosomal homeostasis. Further experimental studies should examine if our findings translate to altered levels of transcription, pathophysiological processes, clinical features, and treatment response to lithium among patients with AD and BIP.We find polygenic overlap between AD and BIP and identify novel loci associated with each trait and jointly with both traits, providing new insights into their genetic architecture. The genes OD, PF, SH, and OA designed the protocol of the study. OA and OS obtained funding. AS, OS, OF, SH, and YW conducted the analyses. OS, AS, AW, OF, YW, SH, and OA interpreted the results. OD, OS, and AS drafted the manuscript. All authors contributed with the further writing of the manuscript and approved the final manuscript.OA has received a speaker’s honorarium from Lundbeck and has a patent application (US 20150356243) pending. AD also applied for this patent application and assigned it to UC San Diego. AD has additional disclosures outside the present work: founder, equity holder, and advisory board member for CorTechs Labs, advisory board member of Human Longevity, recipient of non-financial research support from General Electric Healthcare. The remaining 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.


Infectious diseases in children living in resource-limited settings are often presumptively managed on the basis of clinical signs and symptoms. Malaria is an exception. However, the interpretation of clinical signs and symptoms in relation to bacterial infections is often challenging, which may lead to an over prescription of antibiotics when a malaria infection is excluded. The present study aims to determine the association between clinical signs and symptoms and basic hematology data, with laboratory confirmed bacterial infections.p-value p < 0.1 in the univariate analysis. Only a p-value of < 0.05 was considered as significant for multivariate analysis.A health survey was done by study nurses to collect clinical signs/symptoms in febrile (axillary temperature ≥ 37.5 °C) children under - 5 years of age. In addition, blood, stool and urine specimen were systematically collected from each child to perform bacterial culture and full blood cell counts. To determine the association between a bacterial infection with clinical signs/symptoms, and if possible supported by basic hematology data (hemoglobin and leucocyte rates), a univariate analysis was done. This was followed by a multivariate analysis only on those variables with a p = 0.003), edema (p = 0.010) and convulsion (p = 0.021). Bacterial bloodstream infection was significantly associated with fever> 39.5 °C (p = 0.002), diarrhea (p = 0.019) and edema (p = 0.017). There was no association found between bacterial infections and basic haematological findings. If diarrhea and edema were absent, a good negative predictive value (100%) of a bacterial bloodstream infection was found, but the positive predictive value was low (33.3%) and the confidence interval was very large .In total, 1099 febrile children were included. Bacteria were isolated from clinical specimens  of 127 (11.6%) febrile children. Multivariate logistical regression analysis revealed that a general bacterial infection (irrespective of the site of infection) was significantly associated with the following clinical signs/symptoms: diarrhea (Our study demonstrates that clinical signs and symptoms, combined with basic hematology data only, cannot predict bacterial infections in febrile children under - 5 years of age. The development of practical and easy deployable diagnostic tools to diagnose bacterial infections remains a priority. In resource-limited settings, infectious diseases are mainly presumptively managed on the basis of clinical signs and symptoms , 2. HoweHowever, the use of malaria RDT leaves a significant part of the febrile, non-malaria, patient population undiagnosed. Clinicians that work in areas without laboratory facilities can only manage their patients on the basis of clinical signs and symptoms, which can sometimes be supported by simple tests for hemoglobin and white blood cell count , 6–8. ThHaemophilus influenzae type b was introduced into the extended program of immunization (EPI) in Burkina Faso in January 2006 : 12–32) and 27.8% (306/1099) were children under - 12 months. The main clinical signs and symptoms reported were cough and diarrhea in 43.5% (478/1099) and 37.8% (371/1099) of the cases, respectively , followed by Streptococcus pneumoniae and E. coli in 6.2% (4/65) in both cases, Staphylococcus aureus and Neisseria meningitides in 3.1% (2/65) in both cases, Haemophilus influenzae B and Enterobacter agglomerans in 1.5% (1/65). For bacterial GII, enteropathogenic E. coli was isolated in 50.8% (33/65), Salmonella spp in 44.6% (29/65) and Shigella in 4.6% (3/65). E. coli was the only species isolated from UTI. Three pediatric bottle flagged positive for growth the cultures were due to contamination.The prevalence of the bacterial infections investigated in the present study is reported in Table Criteria for hospital admission were not defined in the present study. The referrals and admissions were done according to the routine practice  In the present study, 17.9% (197/1099) of the recruited febrile children were hospitalized by health professionals. A bacterial infection was found in 3.5% (38/1099) of these children and 14.5% (159/1099) was negative for a bacterial infection. The admission rate was almost two-time higher for children with a confirmed bacterial infection 29.9% (38/127) compared to those that were negative for a bacterial infection 16.4% (159/972) (Table p < 0.1). Children with moderate malnutrition according to z - score calculation (weight/age and height/age) were also prone to have a general bacterial infection. Gender and age were also associated with a general bacterial infection (p < 0.1). Based on the information obtained from the bacterial cultures, it was found that bacterial BSI was significantly associated in the univariate analysis to diarrhea, dehydration, edema, convulsion, pallor conjunctiva, splenomegaly, hepatomegaly and hemoglobin< 8 g/dl (p < 0.1). Age and moderate malnutrition according to z - score calculation (weight/age and height/age) were significantly associated to UTI, after the univariate analysis. Based on stool culture, bacterial GII was associated to gender, age and moderate malnutrition according to z - score calculation (weight/age and height/age) in univariate analysis (p < 0.1) (Table Table 1) Table .Table 2Cp = 0.002), diarrhea (p = 0.003), edema (p = 0.010) and convulsion (p = 0.021) ] and edema . Bacterial GII was not associated with clinical signs and symptoms, and basic laboratory data according to this second criteria. The multivariate analysis was adjusted for age, gender and weight, which are confounding factors.The multivariate logistical regression analysis revealed that a general bacterial infection was significant associated to the following clinical signs and symptoms: high axillary temperature ≥ 39.5 °C , diarrhea and edema were only associated with a bacterial BSI in febrile children under - 5 years of age. However, there is a risk to overlook a real bacterial BSI if only the clinical signs and symptoms combined to basic hematology data are used in the assessment of the child (only 1/65 case of BSI could be diagnosed if presence of edema and diarrhea should be considered). Although clinical signs and symptoms combined with basic hematological data are useful for suspicion of bacterial infections, there is a need to determine the presence of bacteria in clinical specimens related to the site of infection. Furthermore, there is a necessity to develop practical tools to distinguish bacterial infections from other infections in febrile children in order to be able to treat fatal, but treatable, diseases, in particular when a reliable diagnostic test would be available in an early stage of the infection or at the first contact with health professionals . TherefoSalmonella enterica (80.5% of positive blood culture and 50% of stool culture) was the main species isolated from blood and stool culture [Previous study demonstrated that malaria may predispose to non-typhoid salmonella (NTS) bacteremia , 23. In  culture , 24–26. Studies by Mtove et al.  and BrenOur study confirms the limitation of using clinical signs and symptoms, and basic laboratory data to diagnose bacterial infections , 32. In E. coli was the only specie isolated. Maybe the urine samples were contaminated with stool during urine sample collection.Although the multivariate analysis showed an association between UTI and diarrhea, the low prevalence of UTI deserve more attention. Previous studies reported association between UTI with high axillary temperature, sex and age (12–35 months) . HoweverBacterial GII was not associated to any clinical signs and symptoms, and also basic hematology data. This is in line with the general observation that bacterial GII is not associated with fever –42.S. pneumonia and S. aureus is very common in healthy children [A limitation of our study is that it does not present information on bacterial respiratory tract infections. The Pneumonia Etiology Research for Child Health (PERCH) project, a multi-country, case-control study to determine the etiology of and risk factors for (very) severe pneumonia in young children (1–59 months of age), provides a wealth of information on respiratory infection in African children . These schildren  but theiDespite the usefulness of clinical signs and symptoms in combination with basic hematology data in detecting bacterial blood stream infections, our study demonstrated the necessity to confirm the presence of bacterial infection with practical diagnostic tools. Nevertheless, the worldwide concern about the over prescriptions of antibiotics cannot be circumvented if clinical signs and symptoms, combining to basic hematology data remain the only information available in area without laboratory facilities. The development of practical and easy deployable diagnostic tools to diagnose bacterial infections remains a priority.


The prognostic value of body composition in cancer patients has been widely studied during the last decade. The main finding of these studies is that sarcopenia, or skeletal muscle depletion, assessed by CT imaging correlates with a reduced overall survival (OS). By contrast, the prognostic value of fat mass remains ill-defined. This study aims to analyze the influence of body composition including both muscle mass and adipose tissue on OS in a homogeneous population of advanced colorectal cancer (CRC) patients.Among 235 patients with chemorefractory advanced CRC included in the SoMore and RegARd-C trials, body composition was assessed in 217 patients on baseline CT images. The relationship between body composition , body mass index (BMI) and OS were evaluated.Patients with a higher BMI had a better OS . Those with low muscle index and muscle density had an increased mortality . Likewise, low subcutaneous and visceral fat index were associated with an increased risk of dying , as were a high subcutaneous and visceral adipose tissue density . In multivariate analysis, a high visceral fat density was the main predictor of poor survival.Our results confirm the protective role of obesity in CRC patients at an advanced stage, as well as the negative prognostic impact of muscle depletion on survival. More importantly, our data show for the first time that visceral adipose tissue density is an important prognostic factor in metastatic CRC.NCT01290926, 07/02/2011 and NCT01929616, 28/08/2013. Malnutrition has been known for more than 30 years to be associated with reduced overall survival (OS) of cancer patients  and is pCT-based regional analysis of muscle and adipose tissue at the level of the third lumbar vertebra strongly correlates with whole-body fat and muscle mass , making The aim of this study was to assess the association of BMI, skeletal muscle and adipose tissue mass and density with OS in a homogenous group of patients with chemorefractory advanced CRC.18F-fluorodeoxyglucose (FDG)-positron emission tomography (PET) combined with CT. After exclusion of patients without follow up data allowing determination of OS or whose CT was not appropriate for body composition evaluation, 88 patients (91%) of the SoMore study and 129 patients (93%) of the RegARd-C study were included in the final analysis. Overall survival in months was defined as the time from inclusion to the date of death from any cause. Given the similar inclusion criteria and the fact that regorafenib and sorafenib are closely related multikinase inhibitors with limited benefit in our study population [All patients included in the SoMore   and the pulation , 12, the2) was calculated as weight in kilograms divided by the square of height in meters. BMI was then categorized according to the World Health Organization modified by Martin et al.  [2) of skeletal muscle , subcutaneous adipose tissue and visceral adipose tissue was delineated on two adjacent CT slices at the level of the third lumbar vertebra. Skeletal muscle, subcutaneous adipose tissue and visceral adipose tissue were defined by ranges of − 29 to 150, − 190 to − 30 and − 150 to − 50 HU, respectively [2) were normalized for the square of height (m2) as indexes (cm2/m2). Figure r2 ranging from 0.95 to 1.0.BMI (kg/my ≥30.0) . Evaluatectively . Muscle Sarcopenia was defined as a skeletal muscle index < 41 in women, < 43 in men with a BMI < 25, and < 53 in men with a BMI ≥ 25. These cut offs have been validated in a large population of cancer patients . Since tThe Mann-Whitney-Wilcoxon test and the chi-square or Fisher exact test were used to assess differences in continuous and categorical variables, respectively. The association between two continuous variables was assessed with Spearman correlation. To assess the heterogeneity between SoMore or RegARd-C, we used the chi-square test.The primary outcome was OS. To assess whether gender-specific cutoffs or BMI-specific cutoffs for each of the six body composition variables were needed, we fitted a multivariate Cox’s proportional hazards model containing, that variable, gender, BMI (≤25 vs > 25) and the two two-way interactions  and the three-way interaction (variable*gender*BMI25). We performed backward variable selection. If e.g. the interaction variable*gender would be retained, this would indicate that the effect of the variable on OS depends on the gender, and thus stratifying by gender would be of interest when calculating the optimal cutoff.findcut [The optimal cutoff in each of the six body composition parameters was determined by the SASmacro %findcut . The outFor the univariate analysis, Kaplan-Meier curves were used to compare OS of patients below or above the optimal cutoff determined for each body composition parameter. The hazard ratio (HR) and 95% confidence interval (CI) were calculated using Cox’s proportional hazards model, and logrank tests were used to compare survival curves.p < 0.05 level. SAS version 9.4 was used for all statistical analyses.For the multivariate analysis, a stepwise variable selection was performed, considering the study subset (SoMore vs RegARd-c), age, BMI (4 categories), gender, performance status, time interval between diagnosis and inclusion in the respective study (SoMore or RegARd-c), low skeletal muscle index, low muscle density, low subcutaneous adipose tissue index, high subcutaneous adipose tissue density, low visceral adipose tissue index and high visceral adipose tissue density. Results were considered statistically significant at the bilateral This study has been approved by the ethics committee of Institut Jules Bordet and has been performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments.Among the 217 subjects included in the present study, 94 (43%) were women and 123 (57%) were men. Median BMI was 24.9 (14.1–41.0) with 49% of these heavily pre-treated patients being overweight or obese. Sarcopenia was present in 150 (69%) of the study population. Although sarcopenia was less prevalent in obese patients as compared to non-obese, it was still observed in 48% of this subgroup. Table At the time of analysis, 197 (90%) of the patients had died. Median OS (mOS) was 8.2 months (6.8–10.4) in the SoMore study and 7.1 months (3.4 to 13.3) in the RegARd-C study. To make sure that data from these 2 trials could be pooled, a test for heterogeneity comparing SoMore and RegARd-C patients was performed for each body composition variable dichotomized considering the gender-specific median in each dataset (Table p < 0.001) and skeletal muscle index . Men had a lower subcutaneous fat index  but a higher visceral fat index  and lower visceral adipose tissue density . BMI was significantly correlated with skeletal muscle index , subcutaneous fat index , and visceral fat index  but was inversely correlated with muscle density , subcutaneous fat density , and visceral fat density .A wide variation in body composition was found between men and women. Women had a lower BMI ). Neither the presence of sarcopenia nor a low muscle density according to a validated cutoff  were assHowever, a skeletal muscle index below the gender-specific median was associated with increased mortality in both subsets, as shown in Table Patients with a skeletal muscle index and muscle density below these thresholds had an increased mortality, with HR of 2.06 (1.45–2.93) and 1.54 (1.09–2.18), respectively. Low subcutaneous and visceral fat index were also associated with an increased risk of dying, with hazard ratios of 1.63 (1.23–2.17) and 1.48 (1.09–2.02), respectively. Finally, a high subcutaneous and visceral fat density were also correlated with mortality, with a HR of 1.93 (1.44–2.57) and 2.40 (1.79–3.20), respectively. The corresponding survival curves are shown in Fig. A multivariate analysis was then performed, taking into account the population subset (SoMore vs RegARd-C study participants), age, BMI , gender, performance status, time interval between diagnosis and inclusion in the study (SoMore or RegARd-C), low skeletal muscle index, low muscle density, low subcutaneous fat index, high subcutaneous fat density, low visceral fat index, high visceral fat density. Results are shown in Table In this study, we measured skeletal muscle mass as well as subcutaneous and visceral fat mass in a population that is highly homogeneous from an oncologic perspective. Indeed, all patients had been heavily pretreated for a metastatic CRC and received similar although not identical treatments in both study cohorts. More than two thirds of these selected patients were sarcopenic, which is in line with the results of another study assessing the presence of sarcopenia in metastatic CRC patients  but muchpost-hoc nature of our analysis does not allow us to exclude such bias. However, our data were prospectively collected and obesity was found to predict OS in a multivariate analysis taking several other body composition-related factors into account. Moreover, several prognostic models in mixed cancer patient populations show a protective role of overweight and/or obesity [Another important finding of our study is the association of obesity with an increased OS, which is a typical example of the obesity paradox. Indeed, obesity is a known risk factor for colon cancer incidence and mortality in healthy subjects , 18. The obesity , 22. Ano obesity . Indeed, obesity . In anot obesity . Similar obesity . Most ofWhile the prognostic impact of low muscle mass has been shown in numerous studies, the role of adipose tissue mass and density has received much less interest, and research on this topic has yielded conflicting results. For instance, a high visceral fat area was associated with a shorter disease free survival in breast cancer patients treated with neoadjuvant chemotherapy . By contOur results confirm the protective role of obesity in cancer patients at an advanced stage, as well as the negative prognostic impact of low muscle index and muscle density. Finally, and more importantly, this work shows for the first time in metastatic cancer patients that visceral adipose tissue density is an important prognostic factor even when well-known oncologic prognostic variables such as performance status and length of disease are taken into account. Further research is needed to confirm these findings regarding adipose tissue, which may help to better define the prognosis of advanced colorectal cancer patients in the future.


Relapsed Ewing's sarcoma (RES) is an aggressive malignancy with poor survival. Although high-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) given after conventional chemotherapy (CC) has shown survival benefits, it is not generally used in the United States for RES. We performed a systemic review to evaluate the benefits of HDCT for RES.  Literature search involved Medline, Embase, and Cochrane database. We included studies with RES patients treated with HDCT/ASCT.  Twenty-four studies with total of 345 reported RES patients that got HDCT were included in final analysis. Seventeen studies had patients with multiple malignancies including RES, while seven had only RES patients. At 2 and 3–5 years, event-free survival (EFS) in studies with only RES patients ranged 42–47% and 20–61% and overall survival (OS) ranged 50–66% and 33–77%, respectively. In studies with combined patients that reported outcomes of RES separately, the EFS at 1–3 and 4 years was 36–66% and 17–50%, respectively. The OS at 1-2 and 3-4 years was 40–60% and 50–70%.  Most studies using HDCT/ASCT as consolidation regimen showed improved survival benefits compared to CC. Randomized controlled studies are needed to determine true clinical benefits of HDCT followed by ASCT in patients with RES. Patients with localized primary Ewing's sarcoma (ES) have 60–70% 5-year overall survival (OS) with multimodality treatment . In patiComprehensive literature search was conducted using Medline (PubMed and Ovid SP), Embase, and Cochrane Database of Systematic Review (CDSR) up to April 2017. The search strategies included various combinations of text words and controlled vocabulary when available. We used the following keywords while building search; Ewing sarcoma, relapsed/refractory, chemotherapy, high-dose chemotherapy, transplant, tandem transplant, treatment. There was no language or year limits placed on the search. Three reviewers  independently applied the inclusion and exclusion criteria to the articles that were identified by the search strategy and extracted data using the standardized data extraction form. In addition, more articles were added from bibliographical data of searched articles found through search engines. Full articles of potentially useful articles were reviewed before confirming the inclusion criteria.p values, which are expressed as reported in published record.We included all clinical studies that had patients with relapsed Ewing's sarcoma who were treated with HDCT followed by ASCT using either bone marrow or peripheral blood stem cell rescue regardless of treatment settings. We excluded studies that had patients with only primary local or metastatic Ewing's sarcoma or other soft tissue sarcomas and without relapsed disease. As our article mainly aims to compare long-term outcomes of patients that got HDCT to those that did not, we also excluded ten case reports and case series that did not mention long-term outcomes of relapsed ES in their studies. Following data were extracted from the individual study: demographics, pathology, disease stage, treatment, response, and survival outcome data. When not explicitly stated, outcomes were calculated based on the information included within the published record. Outcome data were presented as mean or median, and all values are expressed to 1 decimal place unless the original article did not provide this degree of accuracy. The exceptions are The primary outcome measures included complete remission (CR), partial remission (PR), progressive disease (PD), stable disease (SD), no response (NR), overall survival (OS), and event-free survival (EFS).The systematic search identified a total of 1005 records. These records were screened for relevance based on their titles and abstracts. Of these studies, 64 were deemed potentially eligible and retrieved for full text review. After detailed review, a total of 43 studies were further excluded for the following reasons: duplicate study data, not focused on relapsed sarcoma, or not receiving ASCT. A total of 21 articles met our inclusion criteria. In addition, 3 published abstracts were also included .n=205)  . Fourteen=205) . Four stn=205) . In addin=205) .n=205). McTiernan et al. [n=24) in comparison to just CC (n=31) was noted by Palmerini et al. [n=7) with relapsed ES that showed response (CR/PR) with CC and subsequently received HDCT/ASCT  that showed response to CC but did not receive HDCT (p=0.018 for both EFS and OS). Rasper et al. [n=20). The two- and five-year EFS for patients who were treated with HDCT were 44–47% and 20–24%, respectively, depending on the regimen of HDCT that was used. The corresponding EFS at 2 and 5 years for patients that got only CC was 10% and 6% (p=0.01). The two- and five-year OS rates for patients who got HDCT were 53–66% and 40–42%, respectively, compared to 22% and 10% in patients with only CC (p=0.01).Barker et al.  studied To summarize, for most studies consisting of only relapsed patients that received HDCT/ASCT after CC, EFS rates at two- and five-year ranged 42–47% and 20–61%, respectively. The OS rates at two years and at three to five years ranged 50–66% and 33–77%, respectively, depending on the study. Patients in Bacci et al. [p=0.0001) (There were 9 studies (8 retrospective and 1 prospective), in which 105 relapsed ES patients received HDCT/ASCT after CC. In most studies, only patients that showed response (CR/PR) to CC proceeded to get HDCT. The response status after CC was not clearly outlined in 2 studies , 28. Fro=0.0001) . Burdach=0.0001) . Seo et =0.0001) . The OS =0.0001) . SimilarEkert et al.  and Parep=0.08), respectively. The one and half and three-year OS for patients who received HDCT was 70% and 59%, respectively, whereas it was 44 and 34% for patients who got only CC (p=0.06) (n=63) included in the study (primary metastatic EFT and relapsed EFT) at 5 years was 27% (Five studies (2 prospective and 3 retrospective) reported combined outcomes of all patients in their study. At least 35 relapsed ES patients received HDCT. In most studies, patients who proceeded to get HDCT/ASCT showed initial response (CR/PR) with CC. The response status of patients was not clearly outlined in one study . Al-Fari(p=0.06) . Pape et(p=0.06) . In anot(p=0.06) . Ladenst was 27% . Kavan e was 27% .To summarize, for most studies that only reported combined outcomes for patients with relapsed/primary metastatic ES along with other malignancies (that got HDCT/ASCT following CC), EFS at 1.5–3 and 5 years was 39–60% and 27%, respectively. OS at 1–1.5 and 3–3.5 years was 58–70% and 31–59%, respectively. In studies that reported outcomes for relapsed ES patients separately (that received HDCT/ASCT after CC), EFS at 1–3 and 4 years was 36–66% and 17–50%, respectively. The OS at 1–2 and 3–4 years was 40–60% and 50–70%, respectively, depending on the study. Patients in Graham-Pole et al.  did excen=42) that were published by Thiel et al. [We also found three abstracts . The patients in this study were only followed for 10 weeks; hence, long-term survival is not known . AnotherThe usage of HDCT followed by ASCT rescue is based on observation that outcome in many malignancies that are chemosensitive is dependent on the dosage of chemotherapy used. Steep dose-response is noticed for both toxic and therapeutic effects. Preclinical studies documented linear log correlation between dose and tumor cytotoxicity. An increase in dose by three to tenfold, particularly for alkylating agents, can result in a multiple log increase in tumor cell death. Myelosuppression is one of the major side effects at this dose . ASCT heWe compared outcomes of HDCT with ASCT given as consolidation regimen after CC to results from historical studies which used only CC. In most studies, patients with relapsed ES that received HDCT/ASCT showed initial chemosensitivity (CR/PR) to CC. The OS was 20–60% at 1-2 years and 20–25% at 5 years when only CC was used in historical studies , 8, 11. Five studies that compared outcomes of patients with relapsed ES who received HDCT and ASCT after CC with those who received only CC showed improved survival with HDCT/ASCT , 16–18. Among all positive OS survival outcomes, HDCT regimens used in eight studies (Barker et al. , Ferrari1The superior outcomes seen in the studies could also have been impacted by many factors, such as selection bias in favor of younger, fit patients who were offered HDCT. Most studies included in our analysis were retrospective in nature, and patients who did not respond to CC did not proceed to receive HDCT, thus adding a further selection bias for chemosensitive patients, who are more likely to be offered HDCT/ASCT, which may affect the positive impact observed with this intervention. Limitation of our review also includes significant heterogeneity in included study designs, type of CC and HDCT used, local versus metastatic relapse, different inclusion criteria, follow-up duration, and study end points in various trials included in our analysis. These factors made it difficult to perform meta-analysis of the available data. In addition, patients included in studies had combined data on relapsed and primary metastatic disease. Despite these limitations, results of this review suggest that HDCT has a positive impact on survival.In conclusion, HDCT and ASCT given as a consolidation treatment after CC appear to be beneficial in improving survival in patients with relapsed Ewing's sarcoma. It appears to be more prominent in chemosensitive diseases. HDCT given as induction treatment did not appear to be beneficial in improving survival, but it was used in only two studies. The heterogeneity in the included studies suggests that prospective randomized controlled studies are needed to definitively show the benefit for single and two cycles of HDCT and ASCT in relapsed Ewing's sarcoma.