To evaluate whether early and acquired daily fluid overload (FO), as well as fluctuations in fluid accumulation, were associated with adverse outcomes in critically ill children with severe sepsis.This study enrolled 202 children in a pediatric intensive care unit (PICU) with severe sepsis. Early fluid overload was defined as ≥5% fluid accumulation occurring in the first 24 hours of PICU admission. The maximum daily fluid accumulation ≥5% occurring during the next 6 days in patients with at least 48 hours of PICU stay was defined as PICU-acquired daily fluid overload. The fluctuation in fluid accumulation was calculated as the difference between the maximum and the minimum daily fluid accumulation obtained during the first 7 days after admission.Of the 202 patients, 61 30.2%) died during PICU stay. Among all patients, 41 (20.3%) experienced early fluid overload, including 9 with a FO ≥10%. Among patients with at least 48 hours of PICU stay n = 154), 36 (23.4%) developed PICU-acquired daily fluid overload, including 2 with a FO ≥10%. Both early fluid overload  and PICU-acquired daily fluid overload  were independent risk factors associated with mortality after adjusting for age, illness severity, etc. However, fluctuations in fluid accumulation were not associated with mortality after adjustment. Length of PICU stay increased with greater fluctuations in fluid accumulation in all patients with at least 48 hours of PICU stay (FO <5%, 5%-10% vs. ≥10%: 4 0.2% died vs. 10  days; P 4, 36 (23Both early and acquired daily fluid overload were independently associated with PICU mortality in children with severe sepsis. Sepsis remains a leading cause of death among children ,2. FluidFluid administered to critically ill children during the resuscitation phase has the potential to accumulate, which might lead to subsequent fluid overload associated with adverse outcomes –14. AlthIn adult patients with severe sepsis and septic shock, persistent fluid overload is common and associated with increased hospital mortality –24. In cDeclaration of Helsinki. Informed consent was not required because all data were collected retrospectively. Patient records were anonymized and de-identified prior to analysis.All children with severe sepsis who were admitted to the 20-bed medical/surgical PICU in a single standing children’s hospital in an urban setting from January 2011 to March 2015 were eligible for inclusion in the study. The selection of the time period was arbitrary. The exclusion criteria were age of less than 1 month or more than 18 years, unexpected discharge from the PICU or transfer to another hospital, and incomplete data on fluid balance. The Institutional Review Board of the Children’s Hospital of Soochow University approved the study, and all clinical investigations were conducted according to the principles expressed in the Clinical and laboratory data were collected on the day of admission and included age, gender, and admission diagnosis. Clinical status as defined by illness severity; therapeutic interventions and medication management, including antibiotics, inotropes/vasopressors, steroids, mechanical ventilation, diuretics, and renal replacement therapy (hemofiltration); and comorbidities were recorded daily until hospital discharge or death. The inotropic score obtained during each patient’s PICU stay was adapted from Wernovsky et al.  and calcThe pediatric index of mortality 2 (PIM2) score, which was calculated based on physiological parameters collected in the first hour after PICU admission, according to methods described in the original study, was used as a measure to assess illness severity and to predict probability of death .The presence, severity, and fluctuation of fluid overload were evaluated during a period defined as the entire length of a PICU stay if it was <7 days or as the initial 7 days of a PICU stay if it was ≥7 days. Daily fluid accumulation was calculated for each patient, according to the following formula: Percentage of fluid accumulation = [fluid intake (L)—fluid output (L)] / PICU admission weight (Kg) × 100% –31. DailA percentage fluid accumulation ≥5% was defined as fluid overload (FO%), which has been used in similar pediatric studies ,32 as weIn addition, the degree of fluctuation in fluid accumulation was stratified into 3 categories: <5%, 5%-10%, and ≥10%. We explored outcomes among patients with fluctuations in fluid accumulation ≥10% above baseline. Net fluid accumulation, an expression of the cumulative FO% from the day of PICU admission to the given study day was also calculated using a formula listed in the Supporting Information .Protocolized fluid therapy was not utilized. Fluid administration and nutritional support route were determined by the treating physicians. Patients with acute kidney injury (AKI) or fluid accumulation ≥10% were considered to need renal replacement therapy (RRT).Sepsis was defined as the presence (probable or documented) of infection together with systemic manifestations of infection. The diagnoses of severe sepsis and septic shock were based on the International Guidelines from the Surviving Sepsis Campaign . The diaPICU mortality was designed as the primary outcome and defined as all-cause mortality occurring during the PICU stay, including deaths resulting from withdrawal of therapy. The secondary clinical outcomes were the incidences of AKI, ALI, shock, DIC, and MODS; the need for and duration of mechanical ventilation; and the length of the PICU stay.Statistical software  was used for data analyses. Assumptions of normality and homogeneity of variance were first checked. The Mann–Whitney U test was used to determine differences in continuous variables between two groups, and the Kruskal-Wallis H test was used to analyze differences among groups. The chi-square test or Fisher’s exact test were used to compare differences in categorical variables between groups. Spearman’s analysis was used to examine correlations. Univariate regression analysis was performed to calculate odds ratio (OR) with 95% confidence interval (CI) to assess the association of fluid overload with clinical outcomes. Multivariable logistic regression models were performed to identify independent variables associated with outcomes. Model fit was assessed by the Hosmer-Lemeshow goodness-of-fit test. A non-significant value (P >0.05) for the Hosmer-Lemeshow goodness-of-fit test suggested the absence of a biased fit. Variance inflation factor and tolerance values were calculated to determine multicollinearity. For PICU-acquired daily fluid overload analysis, continuous variables entered into the multivariable analysis were log-transformed to fit model assumptions. The sensitivity and specificity were calculated at the optimal cut-off values using Sigma Plot 10.0 software. Analysis of the area under-the-receiver-operating-characteristic curve (AUC) was used to assess the predictive strength, and the nonparametric method of Delong was performed to compare differences between AUCs.In total, 222 children were admitted to the PICU with severe sepsis during the study period. Among these, 20 were excluded: 1 patient was older than 18 years, 7 were unexpectedly discharged due to economic reasons, 3 were transferred to another hospital, and 9 had incomplete data on fluid balance. The remaining 202 patients made up the study cohort. The leading cause of PICU admission was respiratory diseases (40.1%), followed by gastrointestinal diseases (21.8%) and neurologic diseases (19.3%). There was no significant difference in the admission diagnoses between the survivors and the non-survivors (P = 0.703).Severe sepsis was diagnosed in 200 (99.0%) patients on the first day after admission and in 2 (1.0%) patients on the second day after admission. Among all the patients, gram-positive bacteria were isolated from 60 (29.7%) patients, including from the blood in 44 patients, from the cerebrospinal fluid in 7 patients, from the sputum in 6 patients, from the bone marrow in 1 patient, from pleural effusion in 1 patient, and from urine culture in 1 patient. Gram-negative bacteria were isolated from 34 (16.8%) patients, including from the blood in 27 patients, from the sputum in 4 patients, from the cerebrospinal fluid in 2 patients, and from pleural effusion in 1 patient. Mycoplasmas were isolated from 8 (4.0%) patients, including from the blood in 3 patients and from sputum cultures in 5 patients. One hundred (49.5%) of the patients were diagnosed with culture-negative sepsis. In addition, among all the patients, viral DNA was isolated from the blood of 10 (5.0%) patients, and anti-virus IgM antibodies were detected in 5 (2.5%) patients. There were no patients with positive fungal culture.The PICU mortality rate of the whole cohort was 30.2% (61/202). The median length of PICU stay was 4 (IQR 2–8) days. Among all patients, 68.3% were infants. There was a weak correlation between age and PIM2  or mortality . AKI was diagnosed in 36 (17.8%) patients, including 2 (5.6%) classified as stage 1 on the first day after PICU admission; 8 (22.2%) classified as AKIN stage 2, 7 on the first and 1 on the fifth day after PICU admission; and 26 (72.2%) classified as stage 3, 17 on the first, 6 on the second, 1 on the third, and 2 on the fourth day after PICU admission. Among all the patients, 23 developed both AKI and fluid overload during the first 7 days after PICU admission: 8 patients were diagnosed with AKI prior to the occurrence of fluid overload, 3 patients occurred fluid overload prior to AKI diagnosis, and 12 patients were diagnosed with AKI and fluid overload on the same day.All patients received antibiotics on the first day after admission, and 24 (11.9%) received a change in antibiotic administered based on their culture results during their PICU stay. Among the 65 patients who received inotropes during their PICU stay, 58 (89.2%) received on the first day and 7 (10.8%) received inotropes on the second day after admission. There was no significant difference when comparing the time to inotropes between the survivors and the non-survivors (P = 0.695). A comparison of the demographic and clinical characteristics between the patients with severe sepsis who did and did not survive is displayed in Early fluid overload was analyzed in all patients (n = 202). The median fluid accumulation in the first 24 hours of admission was 1.32% (IQR -0.24%-4.07%). Among all the patients, 41 (20.3%) experienced early fluid overload (FO ≥5%) in the first 24 hours of PICU admission, including 9 (4.5%) with FO ≥10%. Early fluid overload was significantly positively correlated with fluid intake  and negatively correlated with fluid output .PICU-acquired daily fluid overload and the fluctuation in fluid accumulation were analyzed in patients who had at least 48 hours of PICU stay (n = 154). Forty-eight (23.8%) patients were excluded, including 28 (46%) non-survivors and 20 (14%) survivors (P <0.001). The median maximum daily fluid accumulation in patients with at least 48 hours of PICU stay during the 2 to 7 day period following admission was 3.48% (IQR 1.73%-4.83%). Among these patients, 36 (23.4%) developed PICU-acquired daily fluid overload (FO ≥5%), including 2 (1.0%) with FO ≥10%. PICU-acquired daily fluid overload was negatively correlated with fluid output , but it was not correlated with fluid intake .To evaluate whether the largest change in fluid balance during the first 7 days after PICU admission was associated with adverse outcomes, the fluctuation in fluid accumulation was defined. The median fluctuation in fluid accumulation during the first 7 days of PICU stay was 4.96% (IQR 3.17%-7.87%) in patients with at least 48 hours of PICU stay (n = 154). Among these patients, 77 (50.0%) had fluctuations in fluid accumulation <5%, 59 (38.3%) had fluctuations between 5% and 10%, and 18 (11.7%) had fluctuations ≥10%.The early fluid accumulation, PICU-acquired daily fluid accumulation, and fluctuation in fluid accumulation between patients with severe sepsis who did and did not survive are compared in Early fluid overload  and fluid intake occurring in the first 24 hours of PICU admission , but not fluid output , were significantly associated with PICU mortality. The association of early fluid overload , but not fluid intake , with PICU mortality remained significant after adjustment for age and illness severity as assessed by PIM2 score.Both PICU-acquired daily fluid overload  and fluid intake occurring during the same day , but not fluid output , were significantly associated with PICU mortality. The association of PICU-acquired daily fluid overload , but not fluid intake , with mortality remained significant after adjustment for age and PIM2 score.The fluctuation in fluid accumulation, which was defined as the difference between the maximum and the minimum daily fluid accumulation obtained during the first 7 days after PICU admission, was associated with mortality . However, the association did not remain significant after controlling for age and PIM2 score .Comparisons of mortality rate between patients with and without early fluid overload, between patients with and without PICU-acquired daily fluid overload, and among patients with fluctuations in fluid accumulation<5%, 5%-10%, and ≥10% are displayed in To identify whether fluid overload was independently associated with increased risk of death in patients with severe sepsis, variables with p <0.05 in the comparison of For early fluid overload analysis (n = 202), the final model retained age , MODS , mechanical ventilation , change in antibiotics , and early fluid overload  as significant factors associated with mortality, as shown in For the analysis of PICU-acquired daily fluid overload (n = 154), backward logistic regression analysis showed that PICU-acquired daily fluid overload was an independent risk factor associated with PICU mortality , as displayed in The performances of fluid overload and confounding variables in predicting PICU mortality are assessed in To identify whether fluid overload, coded as a continuous variable, was independently associated with secondary outcomes in patients with severe sepsis, variables that were related to the outcome in univariate analysis were entered into the multivariable binary logistic regression analysis as confounding factors. Early fluid overload (n = 202) was significantly associated with AKI after adjustment for PIM2 score, ALI, DIC, MODS, shock, and the use of mechanical ventilation, inotropes, and furosemide , with shock after adjustment for PIM2 score, AKI, ALI, DIC, MODS, mechanical ventilation, inotropes, and furosemide , with DIC after adjustment for age, PIM2 score, AKI, ALI, MODS, shock, mechanical ventilation, and inotrope , and with the use of mechanical ventilation after adjustment for age, PIM2 score, AKI, ALI, DIC, MODS, shock, inotropes, and furosemide . In contrast, early fluid overload was not associated with ALI and MODS after multivariable adjustment. Early fluid overload was also not associated with the duration of mechanical ventilation or the PICU length of stay even in univariate analysis (data not shown).The association of PICU-acquired daily fluid overload and AKI remained significant after adjustment for PIM2 score, ALI, DIC, MODS, shock, and the use of mechanical ventilation, inotropes, and furosemide . The fluctuation in fluid accumulation of ≥10% during the first 7 days of PICU stay remained significantly associated with shock  and MODS , but not with AKI, ALI, DIC, and the use of mechanical ventilation after adjustment for the confounding factors.In addition, the PICU length of stay increased with greater fluctuations in fluid accumulation in all patients with at least a 48-hour PICU stay  with an early FO ≥5% died during their PICU stay.This study has some limitations. First, we were not able to include fluid intake and output prior to PICU admission due to practical constraints. This might have resulted in an underestimation of fluid overload, as many patients would likely have received large volumes of fluid resuscitation prior to PICU admission . Second,Our study indicates that both early and PICU-acquired daily fluid overload were independently associated with PICU mortality in children with severe sepsis. Further randomized clinical studies are needed to determine whether interventions to limit the development of fluid overload in critically ill children with severe sepsis are associated with improved outcomes.S1 FigA: Comparison between survivors and non-survivors who died within the first 48 hours of PICU admission. B: Comparison between survivors and non-survivors who died during their PICU stay. Boxes represent medians and interquartile ranges and whiskers represent minimums and maximums. P-values refer to comparison of net fluid accumulation between survivors and non-survivors.(PPTX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Epithelial cancer constitutes a major clinical challenge and molecular mechanisms underlying the process of tumour initiation are not well understood. Here we demonstrate that hair follicle bulge stem cells (SCs) give rise to well-differentiated sebaceous tumours and show that SCs are not only crucial in tumour initiation, but are also involved in tumour plasticity and heterogeneity. Our findings reveal that SC-specific expression of mutant Lef1, which mimics mutations found in human sebaceous tumours, drives sebaceous tumour formation. Mechanistically, we demonstrate that mutant Lef1 abolishes p53 activity in SCs. Intriguingly, mutant Lef1 induces DNA damage and interferes with SC-specific gatekeeper functions normally protecting against accumulations of DNA lesions and cell loss. Thus, normal control of SC proliferation is disrupted by mutant Lef1, thereby allowing uncontrolled propagation of tumour-initiating SCs. Collectively, these findings identify underlying molecular and cellular mechanisms of tumour-initiating events in tissue SCs providing a potential target for future therapeutic strategies. The presence of multiple stem and progenitor cells in the skin has a major impact on the formation of different epidermal tumours. Here the authors identify bulge stem cells as the cells of origin of sebaceous tumours through genetic lineage tracing and molecular studies in a mouse model. Epithelial skin tumours are generally heterogeneous cell populations embedded in a unique microenvironment13+/Itga6high)7891011121318The skin epithelium generates various types of tumours. Multiple stem and progenitor cell populations ensure maintenance of the interfollicular epidermis (IFE) and its appendages, including hair follicles (HFs) and sebaceous glands (SGs) under homoeostatic conditions and therefore could contribute differently to this tumour heterogeneity5Recently, multiple epidermal cell populations have been tested for their potential to initiate tumour growth in mouse models for different types of skin cancer including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). These studies have identified different HF progenitor pools as cellular source for SCC and potentially BCC formation220212223242526in vivo mouse model to induce well-differentiated sebaceous adenomas in a spatially and temporally controlled manner2930Previously, we have established a robust in vivo. One approach to address this issue of cell-of-origin is lineage tracing of individually labelled cells in the process of tumour formationHere we use this inducible tumour model to explore whether multipotent bulge SCs are involved in tumour initiation and examine the potential role of lineage-restricted stem and progenitor cells in propagation and progression of epithelial tumours +) (K14ΔNLef1 mice) with a single sub-threshold dose of the carcinogen DMBA anthracene) induced the formation of sebaceous tumours in high frequency in a spatially and temporally controlled manner  mice (K15_YFP/K14ΔNLef1), which allow fate mapping of individual bulge SC descendants35T2 mice results in Cre activation specifically in SCs of the HF bulge region and bulge SCs are also marked in deformed HF of K14ΔNLef1 mice but not in control (oil-treated) mice of the same genotype genotype 35. Imporgenotype . As expeef1 mice . Subsequks later . Four werol mice . This warol mice . Furtherrol mice . YFP+ ceulge SCs . Contrib tumours  suggestiWe and others have previously demonstrated that HF bulge cells contribute to the renewal of the SGs during skin homoeostasis9de novo tumours outside the normal SC niche (tumour-propagating cells). Therefore, we isolated primary tumour cells and performed transplantation assays into grafting chambers placed onto the back of nude mice  possess a growth advantage following transplantation in a new physiological environment. Surprisingly, CD34+/Itga6high and CD34−/Itga6high tumour cells display similar tumour growth rates and sizes +/Itga6high tumour cells  under control of the bulge-specific K15 promoter were analysed 7935. Imp79293031354142Surprisingly, major differences were observed in the morphology of sebaceous tumours developing in K14ΔNLef1 and K15ΔNLef1 mice. Whereas benign sebaceous adenomas were observed in K14ΔNLef1 mice , K15ΔNLeThe repertoire of sebaceous tumour types seen in SC-driven epidermal tumours was reflected by an impaired differentiation profile detected in the tumour tissues . Marker +/Itga6high SCs from K14ΔNLef1 mice  and squamous differentiation  incorporation within tumour tissues. As expected, proliferating cells were confined to the undifferentiated basal keratinocytes at the periphery of tumour lobules in sebaceous adenoma of K14ΔNLef1 mice . In cont44To determine the proliferative potential of tumour cells, clonogenicity assays were performed with primary tumour cells isolated from K14ΔNLef1 and K15ΔNLef1 mice. Keratinocytes isolated from SC-driven tumours formed more large colonies (holoclones) and less small meroclones and abortive paraclones+ tumour cells was seen in SCC control tumours as analysed by immunofluorescence stainings  irradiation experiments to examine whether normal p53 response is defective in HF bulge SCs expressing mutant Lef1. Generally, nuclear p53 accumulation is detected following UV irradiation of skin46pidermis .Our data imply that normal p53 signalling is disturbed in SCs and tumours of K15ΔNLef1 mice. Thus, the p53 defect is an early event potentially contributing to SC-driven tumorigenesis as expression of mutant Lef1 results in a defective p53 response in HF bulge SCs.EKO mice . p53EKO mice were generated by crossing p53fl/fl to K14Cre animals48EKO and p53EKO control mice, we analysed transcription levels of p53 mRNA and nuclear accumulation of p53 protein following UV radiation. As expected, p53 protein was detected in numerous epidermal cells of wild-type and p53fl/fl mice following UV treatment  in epidermis of p53EKO mice, whereas normal p53 transcript levels were seen in the epidermis of wild-type and K15ΔNLef1 mice  by indirect immunofluorescent stainings. Typically, bulge SCs are quiescent in telogen, the resting phase of the hair cycleEKO mice . RemarkaEKO mice  and K15ΔEKO mice  demonstrEKO mice . In additermates  further Bcl-2 normally detected upon UV treatment of skin in bulge SCs of wild-type mice, was completely abolished in SCs of K15ΔNLef1 animals in vivo. It was previously shown that bulge SCs are characterized by accelerated DNA repair activity and concomitant attenuation of p53 activationIn conclusion, our study identifies a novel mechanism for SC-driven tumour initiation in vivo model for sebaceous skin tumours was used as one example for well-accessible solid tumours with a known differentiation profile and localization of marker molecules. Given that diverse pools of stem and progenitor cells have been characterized within certain regions of the HF in mammalian skinIn this study, we identify novel, divergent functions of epidermal SCs in the process of tumour initiation and the control of the tumour phenotype and tumour cell identity. An inducible Lineage tracing experiments of bulge SCs within an inducible skin tumour model for sebaceous adenomas show clonal expansion of individual labelled SCs and contribution of labelled cells to tumour formation in high frequency. Thus, bulge SCs serve as cellular source for well-differentiated epidermal tumours in their natural niche. Based on our observation that sebaceous tumours are not monoclonal derived, other un-labelled bulge SCs or cells outside the bulge compartment could potentially contribute to tumour growth. Thus, our study sheds light on the important fact that the process of tumour initiation impacts on tumour heterogeneity often seen in epidermal cancer.+ tumour cells, is enriched and therefore, could drive tumour growth outside the normal SC niche. Alternatively, transplantation could select for tumour cells that dedifferentiate as it has elegantly been shown for intestinal tumorigenesis. In this experimental setting, tumour initiation involves a bidirectional conversion of differentiated cells into SC-like populations therefore causing cellular plasticityBy transplantation experiments, we identified tumour-propagating cells in well-differentiated epidermal tumours. In this experimental setup, bulge SC signature does not result in a tumour growth advantage. Our experiments imply that although bulge SCs are a cell-of-origin for differentiated epidermal tumours in their normal SC environment, tumour cell propagation and tumour initiation are not enhanced following transplantation of tumour cells with bulge SC signature. In addition, expression of bulge SC marker molecules, including NFATc1, K15 and CD34 were decreased in sebaceous tumours. In contrast, a pool of more lineage-restricted SCs, for example, Lrig1Based on the observation that mice expressing mutant Lef1 in different epidermal compartments generate a diverse repertoire of sebaceous tumours, we conclude that the cell population that carries the genetic mutations and thus drives tumour formation has a major impact on the tumour phenotype and grade of malignancy. SC-driven tumours exhibit a heterogeneous and aggressive phenotype. The differentiation defects observed in K15ΔNLef1 mice might be a consequence of an erroneous response towards instructive signals and could promote aggressive growth of the affected tumour cells. This observation suggests that cellular plasticity in lineage commitment and loss of cell identity result in dedifferentiation of tumour cells and could be part of the malignant progression of SC-driven tumours. In addition, expression levels of mutant Lef1 were different in SCs isolated from K14ΔNLef1 and K15ΔNLef1 mice indicating a specific and sensitive response of bulge SCs towards abnormal Lef1 activity. The results suggest that the expression level of any oncogene by SCs significantly impacts on the process of tumour initiation.Human sebaceous lesions are often associated with hereditary colorectal carcinomas and the Torre-Muir syndrome that involve a loss of DNA mismatch repair proteins MLH1 and MSH2 ref. . InteresT2, R26REYFP, p53fl/fl, K14Cre, K14ΔNLef1 and K15ΔNLef1 mice have been described before31353648K15CreER(G)T2 mice were crossed with R26REYFP mice. Cre reporter lines were always kept in a homozygous state to maximize visualization of positive recombinants. To perform lineage tracing experiments in the process of tumour formation, K15CreER(G)T2/R26REYFP (K15_YFP) were crossed into the K14ΔNLef1 background (K15_YFP/K14ΔNLef1). K15ΔNLef1 mice were crossed to K14Cre and p53fl/fl mice to generate K15ΔNLef1/p53EKO mice. All mouse strains were maintained on a C57Bl/6 background. All in vivo experiments were carried out according to guidelines and an animal license given by the State Office North-Rhine Westfalia.K15CreER(G)n=6), K15ΔNLef1 (n=17), wild-type (n=10) and K15_YFP/K14ΔNLef1 mice (n=6) were treated once with a sub-threshold dose of DMBA To initiate tumorigenesis, 7-week-old K14ΔNLef1 (−2) using a Waldmann TP-4 lamp. Unexposed and exposed skin were harvested either 4 or 24 h after irradiation.Mice were treated with one single dose of ultraviolet-B . For YFP detection, tumours were fixed with 4% paraformaldehyde before incubation with 30% sucrose over night at 4 °C.−1 bodyweight BrdU (Sigma-Aldrich). Tumour tissue was harvested 1.5 h post injection, fixed in 4% formaldehyde and embedded in paraffin. BrdU incorporation was detected by immunofluorescent staining.Mice were injected with 100 mg kgEpidermal whole mounts of tail skin were isolated as described before55Epidermal whole mounts and sections were stained according to the published protocols35−1 Thermolysin (Sigma) in DMEM/HAM’s F12 low-calcium medium (Biochrom). The suspension was filtered and washed with DMEM/HAM’s F12 low-calcium medium. Afterwards, cells were suspended either in DMEM/HAM’s F12 low-calcium medium supplemented with 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 1.8 × 10−4 M adenine, 2 mM L-glutamine, 0.5 μg ml−1 hydrocortisone, 10 ng ml−1 EGF, 10−5 M cholera enterotoxin, 5 μg ml−1 insulin and 10% FCS Gold for subsequent engraftment experiments or in 5% FCS/PBS for FACS experiments. For FACS staining, cell suspensions were incubated in 5% FCS/PBS for 30 min at 4 °C with the following antibodies: phycoerythrin-conjugated α6-integrin , AlexaFluor647-coupled CD34 . To analyse apoptosis (FITC)-conjugated Annexin V  was added. Cell viability was assessed by 7AAD (BD Bioscience) labelling. Subsequent analysis was carried out using a FACSCantoII Cytometer (BD Bioscience) equipped with BD FACSDiva Software. Cell sorting (α6-integrinhigh/CD34+ versus α6-integrinhigh/CD34−) on a FACSAriaIII cell sorter (BD Bioscience) was performed to specifically analyse the bulge SC compartment in engraftment studies and colony-forming assay.Skin and tumour samples were harvested, minced with a scalpel and incubated for 45 min at 37 °C in 0.25 mg ml3–2 × 106) mixed with 2 × 106 primary dermal fibroblasts from newborn mice. In some experiments, primary keratinocytes were grafted together with fibroblasts. A volume of 50 μl cell suspension was injected into the implanted silicon chamber. After 1 week, chambers were removed and 2 weeks following transplantation, tumour growth was monitored twice a week.Grafting studies were performed on the back skin of anaesthetized Balb/c-nude mouse (Charles River) as described before 37n=3) were plated onto collagen G-coated six-well plates . Cells were co-cultured with mitomycin-treated J23T3 feeder cells (Swiss Albino) in defined low-calcium keratinocyte DMEM/HAM’sF12 medium40n=3 tumours per genotype) were separated into CD34+/Itga6high and CD34−/Itga6high fractions by FACS. Subsequently, 3,000 and 6,000 sorted cells were seeded. Clonal growth was assessed for 2–4 weeks of cultivation. Cells were fixed with 4% paraformaldehyde and stained with rhodanil (1% rhodamine/2% nileblue) Triplicates of 2,500 and 5,000 freshly isolated tumour cells derived from K14ΔNLef1 or K15ΔNLef1 mice 5′- GGCCAGGCTGAGCAGGGTCTTC -3′ (reverse)Bid5′- AACCGCGACCATGGAAAGACC -3′ (forward)5′- ATGCAGGAGCCGGCGTAAACT -3′ (reverse)Noxa5′- CGCTTGCTTTTGGTTCCCTGAG -3′ (forward)5′- CAAACGACTGCCCCCATACAAT -3′ (reverse)Myc-Tag5′- GATCAGCGAGGAGGAC -3′ (forward)5′- CTAAGTCGCCCTCCTCTTC -3′ (reverse)Puma5′- CGGCGGAGACAAGAAGAG -3′ (forward)5′- GAGGAGTCCCATGAAGAGATTG -3′ (reverse)p215′- CTGAGCGGCCTGAAGATT -3′ (forward)5′- GCTAAGGCCGAAGATGGGGAAGA -3′ (reverse)p535′- TCATCTTTTGTCCCTTCTC -3′ (forward)5′- TTATTGAGGGGAGGAGAG -3′ (reverse)Standard derivations are presented for technical replicates. Three independent experiments were performed and analysed.high/CD34+ cells were used in neutral comet assayFAC-sorted α6-integrint-test, except for analysing differences in tumour frequency. Statistical analysis of differences in tumour frequency was carried out using two-way analysis of variance with repeated measures. Quantification methods are indicated in the respective figure legends. All P-values below 0.05 were considered significant. Statistical analyses were done using Microsoft Excel and GraphPad Prism.Statistical significance was calculated using Student’s The study was designed by C.N. and M.P. Tumour experiments and analysis were performed by C.N., M.P. and A.K. Lineage tracing of SCs was done by M.P. Immunostainings were performed by K.R., M.P. and H.B. Clonogenicity assay was performed by M.P and P.S. and transplantations by H.B., A.K. and M.P. FACS analysis was done by H.B., M.P., K.R. and P.S., M.P. performed qRT–PCR studies. K.R. did western blotting, ultraviolet treatment and comet experiments. C.N. and M.P. wrote the manuscript. All authors discussed the results and commented on the manuscript.How to cite this article: Petersson, M. et al. Interfering with stem cell-specific gatekeeper functions controls tumour initiation and malignant progression of skin tumours. Nat. Commun. 6:5874 doi: 10.1038/ncomms6874 (2015).Supplementary Figures 1-7


Tumour location within the breast varies with the highest frequency in the upper outer quadrant (UOQ) and lowest frequency in the lower inner quadrant (LIQ). Whether tumour location is prognostic is unclear. To determine whether tumour location is prognostic, associations between tumour site and clinicopathological characteristics were evaluated.n = 122) or tumours spanning multiple quadrants (n = 381) were excluded from further analysis. Clinicopathological characteristics were analysed using chi-square tests for univariate analysis with multivariate analysis performed using principal components analysis (PCA) and multiple logistic regression. Significance was defined as P < 0.05.All patients enrolled in the Clinical Breast Care Project whose tumour site—UOQ, upper inner quadrant (UIQ), central, LIQ, lower outer quadrant (LOQ)—was determined by a single, dedicated breast pathologist were included in this study. Patients with multicentric disease (P = 0.003) and size (P < 0.001), metastatic lymph nodes (P < 0.001), and mortality (P = 0.011). After multivariate analysis, only tumour size and lymph node status remained significantly associated with survival.Of the 980 patients with defined tumour location, 30 had bilateral disease. Tumour location in the UOQ (51.5%) was significantly higher than in the UIQ (15.6%), LOQ (14.2%), central (10.6%), or LIQ (8.1%). Tumours in the central quadrant were significantly more likely to have higher tumour stage (Evaluation of tumour location as a prognostic factor revealed that although tumours in the central region are associated with less favourable outcome, these associations are not independent of location but rather driven by larger tumour size. Tumours in the central region are more difficult to detect mammographically, resulting in larger tumour size at diagnosis and thus less favourable prognosis. Together, these data demonstrate that tumour location is not an independent prognostic factor. Although mortality rates from breast cancer have decreased since 1990, it is estimated that >39,500 women in the United States died from breast cancer in 2013 –7. DespiTumour location within the breast has been proposed as an independent prognostic factor. For example, the frequency of axillary lymph node metastasis was significantly lower in the upper inner quadrant UIQ, 20.6%) compared to all other quadrants (33.2%) . In cont.6% compaReasons for discordant results are varied. For example, the manner in which tumour locations are grouped can affect survival results: when quadrants were evaluated individually, patients with tumours in the LIQ had a twofold increase in mortality; however, using the same data set, when tumour sites were combined into inner versus outer or lower versus upper, survival did not differ . Other sFor inclusion in the CBCP, all patients met the following criteria: (1) adult over the age of 18 years, (2) mentally competent and willing to provide informed consent, and (3) presenting to the breast centres with evidence of possible breast disease. Tissues were collected with approval from the Walter Reed National Military Medical Centre (WRNMMC) Human Use Committee and Institutional Review Board. All enrolled subjects voluntarily agreed to participate and were provided with layered consent forms that included permission to gather tissue and that described the primary research uses of the samples.http://seer.cancer.gov/archive/manuals/2010/AppendixC/breast/coding_guidelines.pdf). Pathological characterisation of all specimens was performed by a single, dedicated breast pathologist as previously described [Cancer Staging Manual seventh edition [in situ hybridisation using the PathVysion® HER-2 DNA Probe kit .The CBCP database was queried to identify all patients with known tumour location diagnosed 2001–2013. Tumour location was classified using the SEER Coding guidelines . After initial univariate analysis revealed highly associated variables, a multivariate approach was used to determine the extent to which each could be considered an independent prognostic factor. In the initial multivariate analysis, seven possible predictor variables were included: lymph node status, PR status, ER status, HER2 status, location, tumour grade, and size. Principal components analysis (PCA) was performed in order to reduce the dimensionality of a subsequent regression model and to create uncorrelated variables. Multiple logistic regression analysis was performed to assess the influence of these components on patient survival. The informative components were then decomposed to determine the true independent predictors of patient survival resulting from the regression. Further multiple logistic regression analysis was performed on lymph node status, tumour size, and tumour grade in order to determine how strongly other characteristics were associated with these variables.Univariate analysis was performed using chi-square analysis .Because our variables were categorical, one category for each was established as the reference point to which the other categories were compared in regression modelling. The reference categories corresponded to a negative status within the lymph node, PR, ER, and HER2 variables, the central region for tumour location, tumour grade 1, and tumour size T1. The odds ratios established through regression for the complementary categories represent the relative odds that a change from the reference category to the alternative category would cause a change in the dependent variable when all other variables are held constant. Statistical significance was defined as As of July 2014, 1,483 patients were enrolled in the CBCP. Of these, 122 were diagnosed with multicentric disease, and 381 had tumour locations spanning multiple quadrants , leaving 980 eligible patients for this study. The average age at diagnosis was 58.7. The majority of patients were Caucasian (80%), and the majority of tumours were early stage (88%), ER+HER2− (72%), T1 (66%) with negative lymph nodes (70%). Tumour grade was split into well-differentiated (29%), moderately differentiated (38%) and poorly differentiated (33%). Twenty-nine patients have died of disease . Median Tumour location was higher in the UOQ (51.5%) compared to the UIQ (15.6%), lower outer quadrant , central (10.6%), or LIQ (8.1%). To evaluate whether breast cancer risk factors are associated with tumour location, age at diagnosis, ethnicity, body mass index (BMI), and breastfeeding were evaluated by quadrant ; none ofP = 0.026) and 5.1 (P = 0.019), respectively. Tumour size was significantly associated with positive lymph node status with T2 and T3 tumours having expected odds ratios of 12.3 (P < 0.001) and 24.7 (P < 0.001); high-grade tumours were also associated with positive lymph node status with an expected odds ratio of 1.6 (P = 0.045). Tumour location was only associated with tumour size, with all quadrants having smaller tumour size compared to the central region (expected odds ratios range (0.30–0.41).Given the linear relationship between tumour size and lymph node involvement , lymph nP = 0.0754).Determination of whether tumour location can be used prognostically is important in optimising treatment. Tumour location is highest in the UOQ (50–58%) across multiple populations, including Chinese, Danish, the United Kingdom and women treated within the United States Department of Defence healthcare system , 14, 30.The less favourable prognosis seen in patients with tumours in the central region can be attributed to increased tumour size and positive lymph node status. Tumour size is thought to reflect the chronological age of the tumour, with smaller tumours being resected earlier than larger tumours . Tumour Previous studies have demonstrated that tumours within the central region are harder to detect than at other sites , 48 and n = 13,984) or Danish Breast Cancer Cooperative Group . In both studies, UOQ was associated with more favourable prognosis, with one study reporting a hazard ratio of 0.820 and the other a 20% improved survival in patients whose tumours were in the UOQ [P = 0.0754) when compared to the central region; this survival advantage may have reached the level of significance with a larger sample size. In addition, the median follow-up for these patients was 5.5 years; thus, differences in late recurrence (>5 years after diagnosis) by tumour location may not have been detected. Finally, treatment data for each patient were not available. While some studies suggest that less favourable prognosis by tumour location has been associated with under-staging and suboptimal treatment of tumours in the LIQ when lymph node status of the internal mammary chain is not evaluated [Although this study included data from 980 patients, two other studies evaluated much larger data sets including tumour registry data from the US Department of Defence  is an independent prognostic factor for both lymph node metastases and poor outcome. Together, these data suggest that tumour location is not prognostic.The author(s) declare that they have no conflict of interest.Seth Rummel collected the data and performed preliminary analyses; Nick Constantino performed multivariate analysis, Matthew T Hueman and Craig D Shriver provided clinical input and reviewed the manuscript, Rachel E Ellsworth conceptualised the study and drafted the manuscript.


Critical Care by Glassford et al. [We read with interest the recently published article about the physiological changes after fluid bolus therapy (FBT) in d et al. . We are The authors claim that alternative interventions to FBT may include a diagnostic low-volume FBT, classic fluid challenge, low-volume FBT and low dose vasopressor therapy, or cardiac output-guided therapy. So, FBT could be interpreted as a treatment and a diagnostic method for hypovolemia. We thought this interpretation would cause misunderstanding about FBT. FBT is commonly used to assess fluid responsiveness in hemodynamic management, which is also called ‘fluid challenge’ . FBT essFBT resulted in a positive outcome in only about 50 % of cases in the ICU . In othe Liu and He ask if FBT is a medical therapy or diagnostic modality; the answer is, of course, context sensitive. In the context of the immediate management of the septic, critically ill patient, the administration of, often large, volumes of fluid in the form of FBT are an international therapeutic standard of care , 5. FBT We wonder if the confusion arises from one of the fundamental problems with research into fluid administration, fluid resuscitation and fluid responsiveness - the lack of fixed terminology. Moreover, fluid responsiveness is a complex topic, and not what we sought to investigate. We note that, in general, fluid responsiveness is identified retrospectively, and therefore tautologically. Indeed, from the evidence we were able to identify, there is very limited evidence for persistent physiological improvement even in patients identified as ‘fluid responsive’ .We feel the question is not one of individual patient fluid responsiveness, but a larger one regarding the need to demonstrate the independent efficacy of FBT in improving patient outcomes. Such evidence is lacking in critically ill patients, whether fluid responsive or not.


AbstractChlamysfarreri  ♀ and Argopectenirradiansirradians  ♂, two of the dominant cultivated scallop species in China. Genomic in situ hybridization (GISH) was used to examine the chromosome constitution and variation in hybrids at early embryonic stage. The number of chromosomes in 66.38% of the metaphases was 2n = 35 and the karyotype was 2n = 3 m + 5 sm + 16 st + 11 t. After GISH, two parental genomes were clearly distinguished in hybrids, most of which comprised 19 chromosomes derived from their female parent (Chlamysfarreri) and 16 chromosomes from their male parent (Argopectenirradiansirradians). Some chromosome elimination and fragmentation was also observed in the hybrids.Interspecific crossing was artificially carried out between  Chlamysfarreri  ♀ × Patinopectenyessoensis  ♂, whose offspring has a high production trait as well as strong disease resistance ability  and Argopectenpurpuratus , and the hybrids exhibited a great increase in production traits as well as some interesting new characteristics  and Chlamysfarreri by Argopectenpurpuratus and Argopectenirradiansirradians  were obtained from Changfei Scallop Hatchery in Shandong Province, China. Artificial hybridization was carried out in the lab. The main procedures are as followed. Mature parents were induced to spawn by exposure to air for 30 min followed with a temperature shock in 20 °C seawater. Because Argopectenirradiansirradians is hermaphroditic, sperm was filtered by a 25 µm mesh screen in order to avoid introducing eggs of Argopectenirradiansirradians. After collection of the gametes, eggs from Chlamysfarreri were mixed with sperms from Argopectenirradiansirradians to produce hybrids. Hybrid larvae were reared at 20 °C and sampled at the swimming trochophore stage (approximate 20 h after fertilization) and used for chromosome preparation.Sexually mature scallop PageBreakFollowing colchicine (0.01%) treatment for 2 h at room temperature, the larvae were exposed to 0.075 M KCl for about 30 min. After fixation in Carnoy’s fixative  for 3 times (each 15 min), samples were stored at -20 °C. The fixed larvae were dissociated into fine pieces by pippetting in 50% acetic acid. The cell suspension was dropped on hot-wet glass slides and air-dried. For FISH analysis, the chromosome preparations were air-dried and preserved in a moist chamber at -20 °C until use.Total genomic DNA was extracted from adductor muscle using traditional phenol/chloroform method described by in situ hybridization and probe detection were performed as described by Genomic PageBreakDigital images were recorded using a CCD camera (COHU) and analyzed with software of Lucia - FISH Image System. The karyotype was determined from more than 10 good metaphase plates and classified according to the criteria defined by Chlamysfarreri is 2n = 38 = 6m + 10sm + 22st .In most metaphases, hybridization signals were not uniform along chromosomes. Some strong signals are located on telomeric region of long arms and centromeric regions in eri Fig. , and on ans Fig. . These ufarreri . In ArgoA probes . The hetA probes , musselsA probes  and oystA probes . In addiA probes , plants A probes , insectsA probes  and mammA probes . In ArgoISH Fig. . The morISH Fig. . The strISH Fig. . So we sPageBreakPageBreakChlamysfarreri   . We spec


Halyomorpha halys (Stål) (BMSB) is an exotic invasive insect originating in East Asia, currently causing significant damage to fruits, vegetables and other crops throughout most of the Mid-Atlantic states of the U.S. It also is a nuisance pest, entering homes in the fall in search of suitable overwintering sites. Two formulations of BotaniGard® with a strain of Beauveria bassiana (GHA) as the active ingredient were tested against second instar BMSB. Both the wettable powder and the emulsifiable suspension formulations were efficacious at 1 × 107 conidia mL−1, causing 67%–80% mortality 9 days post treatment and 95%–100% after 12 days. The wettable powder formulation was slightly more efficacious.The brown marmorated stink bug,  Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) (BMSB) is an exotic invasive insect in the U.S. that has spread extensively from Pennsylvania, its first site of an accidental infestation [The brown marmorated stink bug, estation . It has estation ,3. Outsiestation . Originaestation ,6.The efficacy of several chemical insecticides and their residual effects on adult BMSB have been assessed in fruit orchard field trials. Several of the products tested showed a high level of efficacy when BMSB were exposed to treated branches immediately after the pesticide application but, in general a residual effect 3 and 7 days post treatment was not observed .Ophiocordyceps nutans, was shown to be effective against BMSB in Japan [Beauveria bassiana and Metarhizium anisopliae caused significant mortality to the adults [B. bassiana isolate, GHA, the active ingredient in the commercial product BotaniGard® . Therefore, protection of the crop from damage by BMSB could be enhanced by targeting the early instars, thereby stopping further reproduction and preventing future generations. The purpose of the research reported herein was to determine the efficacy of two formulations of BotaniGard® against second instar BMSB.In recent years, extensive research on biological control of BMSB has been conducted in the U.S. A wide variety of natural enemies have been identified in the native range of BMSB, and high rates of parasitism 63%–85%) in Asia were also reported [% in Asiain Japan . The pote adults . One of Daucus carota var. sativus L.), green string beans (Phaseolus vulgaris L.) and sunflower seeds (Helianthus annuus L.). Water was provided for the insects by placing cotton balls moistened with tap water in the cage three times per week.Test insects (second instars) were obtained from a continuous lab culture of BMSB maintained at the University of Vermont Entomology Research Laboratory. Adult BMSB originally obtained from field-collected individuals in vegetables and fruits in New Jersey were reared in clear plastic cube cages (29.85 cm on each side) having three side panels with 24 × 24 cm plastic mesh screening (1.1 mm × 0.1 mm opening) and one side with a 16 cm diameter opening with a nylon sleeve to access the insects . To insure BMSB escape was prevented, each BugDorm was held within a larger cage (92 cm L × 61 cm W × 82 cm H) made with a PVC frame covered on all sides with nylon thrips-proof screening. The room in which the cages were held was lighted with four Sylvania Daylight Deluxe F40/DX T12  fluorescent bulbs with a 16 h light (L):8 h dark (D) photoperiod. Mean temperature in the cage was 23 °C ; RH was 30% . They were cultured on carrots (6 and 1 × 107 conidia mL−1) of Beauveria bassiana (GHA strain) formulations were tested: BotaniGard® 22WP (wettable powder), and BotaniGard® ES (emulsifiable suspension), both obtained from BioWorks, Inc., Victor, NY, USA. In addition, two control treatments were tested: distilled water and untreated. Both formulations contained 11.3% fungal conidia as active ingredients and 88.7% inert material (2 × 1010 conidia g−1 or mL−1). The formulations were diluted to deliver the desired test concentrations. These concentrations were selected because in previous BMSB tests, they produced mortalities between 20% and 80% and were most appropriate for the evaluation of efficacy.Two concentrations . Data on the percentage of mortality among BMSB nymphs for the entire experimental period, combining results from all of the experiments, were analyzed using a general linear model (GLM), followed by Tukey’s honestly significant difference (HSD) for multiple comparisons. The analysis was conducted using SPSS ver. 17.1 at the 0.05 (α) level of significance [Prior to analysis, data were tested for normality and found to be normally distributed . Mortality of BMSB nymphs among the two control treatments (distilled water and untreated) ranged from 0 to 4.2% throughout the experimental period and were not significantly different (p = 0.633) (p < 0.001).When comparisons were made among the four separate experiments, differences in mortality levels were not significant, which allowed the combination of the full data set (= 0.633) . Mortali7) than the lower concentration (5 × 106) . At the higher spore concentration, the wettable powder formulation exhibited greater efficacy than the emulsifiable suspension . In contrast, for the low concentration, the two formulations showed similar levels of efficacy . Mortality of the BMSB nymphs increased gradually over the 12 days after treatment with the wettable powder formulation reaching a maximum of around 78% for the low concentration and 100% for the higher concentration. For the emulsifiable suspension treatments, approximately 67% mortality was obtained for the low concentration compared to 95% for the higher concentration.For both formulation types, BMSB mortality was significantly greater for the high concentration treatment  against BMSB adults [7, 80%–86% mortality was obtained for adults and immatures [6), at 9 days after treatment, 12% mortality was obtained in the trial with adults, compared with 48% for the immatures. At 12 days post-treatment, 60% mortality occurred for adults, and 88% for immatures [B. bassiana GHA strain. In addition, the immature stage is more sensitive to infection than the adults, therefore, growers may want to specifically target that stage to maximize reduction of the pest population. However, although these results show promise, trials are still needed to fully assess the efficacy of these fungal formulations under different environmental field conditions. In general, many comparisons of results from lab-based and field trials on the lethality of various chemical pesticides to BMSB have shown that mortality is considerably less in the field than in the laboratory [These bioassay results confirm that significant mortality of BMSB nymphs can occur following a spray treatment of the GHA strain of B adults . At 9 dammatures . One hunmmatures . This su


We aimed to clarify that the size of the lung adenocarcinoma evaluated using mediastinal window on computed tomography is an important and useful modality for predicting invasiveness, lymph node metastasis and prognosis in small adenocarcinoma.We evaluated 176 patients with small lung adenocarcinomas  who underwent standard surgical resection. Tumours were examined using computed tomography with thin section conditions (1.25 mm thick on high-resolution computed tomography) with tumour dimensions evaluated under two settings: lung window and mediastinal window. We also determined the patient age, gender, preoperative nodal status, tumour size, tumour disappearance ratio, preoperative serum carcinoembryonic antigen levels and pathological status . Recurrence-free survival was used for prognosis.Lung window, mediastinal window, tumour disappearance ratio and preoperative nodal status were significant predictive factors for recurrence-free survival in univariate analyses. Areas under the receiver operator curves for recurrence were 0.76, 0.73 and 0.65 for mediastinal window, tumour disappearance ratio and lung window, respectively. Lung window, mediastinal window, tumour disappearance ratio, preoperative serum carcinoembryonic antigen levels and preoperative nodal status were significant predictive factors for lymph node metastasis in univariate analyses; areas under the receiver operator curves were 0.61, 0.76, 0.72 and 0.66, for lung window, mediastinal window, tumour disappearance ratio and preoperative serum carcinoembryonic antigen levels, respectively. Lung window, mediastinal window, tumour disappearance ratio, preoperative serum carcinoembryonic antigen levels and preoperative nodal status were significant factors for lymphatic vessel, vascular vessel or pleural invasion in univariate analyses; areas under the receiver operator curves were 0.60, 0.81, 0.81 and 0.65 for lung window, mediastinal window, tumour disappearance ratio and preoperative serum carcinoembryonic antigen levels, respectively.According to the univariate analyses including a logistic regression and ROCs performed for variables with p-values of <0.05 on univariate analyses, our results suggest that measuring tumour size using mediastinal window on high-resolution computed tomography is a simple and useful preoperative prognosis modality in small adenocarcinoma. We previously reported that the size of lung adenocarcinoma, evaluated using mediastinal window (MD) settings on computed tomography (CT), is a more important predictive prognosis factor than the total tumour size, evaluated using lung window (LD) settings This investigation aimed to clarify the importance of the tumour size evaluated by MD settings as a preoperative prognostic predictive factor for anatomical pulmonary resection in patients with small adenocarcinomas (1–3 cm). Furthermore, we would clarify that the preoperative evaluation of tumour diameter by CT with MD settings would enable the prediction of prognosis, lymph node metastasis and tumour invasiveness for patients with clinically early-stage tumours.This was a retrospective study conducted between October 2003 and December 2008 in patients with small lung adenocarcinomas (diameters of ≤3 cm) that underwent standard surgical resections  at the Cancer Institute Hospital.Tumour dimension was evaluated under two different CT imaging conditions: LD  and MD . The CT  images were evaluated for the maximum tumour dimension.Tumour disappearance ratio (TDR) was defined as 1− MD/LD.“For all patients, preoperative staging was assessed using chest CT, CT or ultrasonography for abdominal metastasis, brain CT or magnetic resonance imaging for the brain metastasis and bone scanning for bone metastasis.”Clinical mediastinal and hilar lymph node status was deemed positive if the chest CT findings revealed a lymph node short axis of >1.0 cm. The status of mediastinal, hilar or interlobar nodes was assessed according to the classification for lung cancer in the TNM Classification of Malignant Tumours, Seventh Edition We excluded tumours comprising 100% GGO from this study because most of them were believed, on microscopic examination, to be non-invasive or precancerous lesions. The GGO component was defined as hazy and amorphous with increased lung attenuation, but without obscuration of the underlying vascular markings and bronchial walls. In addition, we excluded a subgroup with BAC or mucinous BAC on microscopic examination that were defined as adenocarcinoma in situ Patient records were examined for age, gender, preoperative nodal status and tumour size, as evaluated using both MD and LD. Preoperative serum carcinoembryonic antigen (CEA) levels, TDR and pathological status were evaluated using elastic stain, and included lymphatic vessel (ly), vascular vessel (v) and pleural (pl) invasion.Because individual patients were not identified, our institutional review board  approved this study without the requirement to obtain patient consent. The patient records/information was anonymized and de-identified prior to analysis.P-values of <0.05 on univariate analysis using the logistic regression test or the Cox proportional hazards model. All analyses were performed using the JMP 10 software  and results with P-values of <0.05 were considered statistically significant.Disease-free survival was assessed. Survival duration was defined as the interval between surgery and either tumour relapse or the most recent follow-up. The Kaplan–Meier method was used to calculate the recurrence-free survival rates. Univariate analyses included a log-rank test, chi-square test and logistic regression. Receiver operating characteristic analyses (ROC) were performed for variables with In total, 176 patients were enrolled. This subgroup that excluded BAC and small tumours (<1 cm) comprised 99 females and 77 males, with ages ranging from 34 to 78 (median  = 61) years. The follow-up periods ranged from 24–84 (median  = 49) months.P<0.001) (As shown in P<0.001) .As shown in As shown in Tumour diameter is a major prognostic factor for lung cancer. The most common method for determining tumour size before surgery is by CT using lung window settings It has been reported that tumour size was not associated with either indicators of proliferation or tumour invasiveness Furthermore, it has been reported that the larger or more desmoplastic fibrous scars is asosiated with more aggressive tumour invasion and poorer prognoses In the present study, we confirmed that tumour dimensions determined using MD settings provided additional useful prognostic data that could not be evaluated using LD settings P<0.01). When the mediastinal size was>20 mm, one-fourth of the patients in this cohort showed serum CEA levels beyond cut off value at 5 ng/ml.Tumour markers are used clinically to assist in the diagnosis of patients with non-small-cell lung carcinoma and to monitor progression, recurrence and/or efficiency of treatment. Among these markers, CEA is considered to be the most prevalent marker for the diagnosis and monitoring of lung adenocarcinoma. Serum CEA levels are an important prognostic factor for early-stage adenocarcinoma, such as clinical stage IA tumours TDR is an established and important prognostic factor. Suzuki et al. reported that radiological non-invasive (neither ly nor v) peripheral lung adenocarcinoma could be defined as an adenocarcinoma of ≤2.0 cm with ≤0.25 consolidation The solid type adenocarcinoma on CT was highly associated with non-lepidic predominant adenocarcinoma, such as acinar predominant, papillary predominant and solid predominant with mucin production th general rule for clinical and pathological record of lung cancer, the recommended LD are a window level of −500 to −700 and a window width of 1000–2000 HU, whereas we used −500 HU and 1500 HU, respectively. The recommended MD are a window level of 30–60 H and a window width of 350–600 HU, while we used 60 HU and 350 HU, respectively.In this study, the patient population was limited to those with tumour diameters of 1–3 cm. Patients with tumour diameters of <1 cm were excluded because of the small number of cases and the major bias to perform wedge resection. Another problem was the LD and MD imaging conditions for CT. According to 7The differences in these imaging conditions for CT may have affected the MD, LD and TDR results. Furthermore, the solid component size defined as a lesion without GGO evaluated by LD is a promising method of the solid area representing an invasive lesion (scar) of the adenocarcinoma,Therefore, further investigations on the optimal CT imaging conditions for assessing the aggressiveness of small adenocarcinomas should be performed in multiple centres with larger sample sizes. The size of the solid lesion (excluding the GGO component) evaluated using LD is also a useful CT-based prognostic factor. Further examination is necessary to clarify the relative utility of both tumour size measured using MD and extracted solid size measured by LD.In conclusion, our results suggest that measuring tumour size with MD on high-resolution CT is a simple and useful preoperative modality for predicting invasiveness, lymph node metastasis and prognosis. An increased tumour size with MD correlated with pathological malignant potential reflecting tumour aggressiveness and degree of invasion.


Mathematica graphs. The present model is applicable to study the behavior of intestinal flow . It is also relevant to simulations of biomimetic pumps conveying hazardous materials, polymers, and so forth.A mathematical study on creeping flow of non-Newtonian fluids (power law model) through a nonuniform peristaltic channel, in which amplitude is varying across axial displacement, is presented, with slip effects included. The governing equations are simplified by employing the long wavelength and low Reynolds number approximations. The expressions for axial velocity, stream function, pressure gradient, and pressure difference are obtained. Computational and numerical results for velocity profile, pressure gradient, and trapping under the effects of slip parameter, fluid behavior index, angle between the walls, and wave number are discussed with the help of  In biolocomotion, earthworms also use peristalsis as an external motion achieving very efficient “geonautical” mobility, aided by the secretion of lubricating mucus. This also serves to subject the soil to continuous biological “pistons” forcing air through burrowed tunnels, promoting aeration and soil mixing, and encouraging mineralization of nutrients and their uptake by vegetation [The transportation of physiological fluids due to continuous wavelike muscle contraction and relaxation of physiological vessels such as the oesophagus, stomach, intestines, ureter and blood vessels , and other hollow tubes is known asistalsis . Peristaistalsis , swallowgetation . Roller getation . Ostwald-DeWaele power law model which successfully simulates viscosity, shear thickening, and shear thinning effects. Representative studies deploying this model in peristaltic fluid dynamics include [The behavior of most of the physiological fluids is known to be non-Newtonian. A simple yet versatile rheological model is the include –9 wherei include –18 have  include , couple  include , William include , Eringen include , fractio include , Oldroyd include , second- include , micropo include , and Her include , gastric include , and abn include .Kwang et al.  studied −1) can be represented by a power law fluid. Also, it is found that physiological organs are generally nonuniform ducts [ peristaltic transport of power law fluid in a nonuniform channel under a slip boundary condition. The effects of slip parameter, fluid behavior index, angle between the walls, and wave number on pumping characteristics and trapping phenomenon are investigated numerically and depicted graphically.It has been pointed out by Charm and Kurland , 28 thatrm ducts , 30. Remh, a, b, λ, x, α are transverse vibration of the wall, half width of the channel, amplitude, wavelength, axial displacement, and angle between walls of channels, respectively. The sinusoidal nature of peristaltic waves is established in numerous clinical studies and we refer readers to the standard monograph Keener and Sneyd [We consider the peristaltic flow of power law fluid in a nonuniform channel under a hydrodynamic slip boundary condition see . Let thend Sneyd .τxx, τxy, τyy are the shear stress components and ρ, u, v, y, p are the fluid density, axial velocity, transverse velocity, transverse coordinate, and pressure, respectively.The governing equations of the motion of power law fluids . Using the above nondimensional variables and taking into account long wavelength and low Reynolds number approximation, after dropping the primes, the governing equations for flow of a power law fluid reduce toβ(=L/a) is the dimensionless slip parameter and L is the dimensional slip parameter.We introduce the following dimensionless parameters:y and using condition , rheological fluid power index (n), angle between the walls (α), and wave number (δ) on the velocity profile, pressure gradient, and the trapping phenomenon.We systematically study the effects of slip parameter ( transverse (y-)axis. ϕ = 0.5, ∂p/∂x = 1, x = 1, n = 1, α = π/4, δ = 1. Evidently the curves for velocity profile are displaced downwards when the magnitude of β increases from 0 to 0.3. The curve for β = 0 represents the velocity profile for a uniform no-slip channel. The slip boundary condition defined in (u) is evidently boosted. The fluid moves faster at the boundary with greater slip. This adds momentum to the near wall flow which is transferred to the core region also and generates a consistent acceleration in the flow. In the absence of the momentum slip effect (β = 0) the magnitude is suppressed. The implication is that, with a slip effect, the axial flow distribution receives a nontrivial modification (acceleration) which is generally ignored in the majority of peristaltic flow models, and this can influence the efficiency of the peristaltic pumping. It may further be noted that with heat and species diffusion present  thermal jump (slip) and solutal slip (mass slip) at the deformable boundaries can also be incorporated and this is being considered by the authors for future investigations.Figures fined in  is a Navβ = 0.1, α = π/4, δ = 1, ϕ = 0.5, ∂p/∂x = 1, x = 1. It is found that the curve is displaced in an upward direction with increasing the value of n. The curve for n < 1 (n = 0.8) represents the velocity profile for pseudoplastic and for n > 1 (n = 1.2) represents the dilatant fluid and for n = 1 represents Newtonian fluid. α = π/3, π/4, π/6) at fixed values β = 0.1, n = 1, δ = 1, ϕ = 0.5, ∂p/∂x = 1, x = 1. The curves of velocity profile move downwards with large inclination between the peristaltic walls. δ = 1, 2, 3) at fixed value of ϕ = 0.5, ∂p/∂x = 1, x = 1, β = 0.1, n = 1, α = π/4. The magnitude of axial velocity increases with increasing the wave number.β), rheological fluid behavior index (n), channel inclination angle (α), and wave number (δ). Inspection of these figures confirms the sinusoidal nature of the axial flow in the direction of propagation of the peristaltic waves. In all these graphs the channel is diverging (α > 0). The axial velocity is generally enhanced in magnitude with greater wall slip effect (n > 0) biofluids clearly propel slower than pseudoplastic (n < 0) biofluids. x > 0 whereas they are decreased for x < 0.Figures p effect , and the nonlinear and is opposite to the geometry of nonuniform peristaltic channel across the longitudinal axial line. It is apparent that pressure gradient is maximized at the point of contraction and minimized at the point of relaxation. Pressure enhances with distance between the walls. The effect of slip parameter on pressure gradient at fixed values of other physical parameters ϕ = 0.5, q = 1, n = 1, α = π/4, δ = 1 is shown in  increases with increasing the magnitude of β. The impact of fluid behavior index (n) on pressure gradient at fixed values ϕ = 0.5, q = 1, β = 0.1, α = π/4, δ = 1 is illustrated in α = π/3, π/4, π/6) at fixed values ϕ = 0.5, q = 1, β = 0.1, n = 1, δ = 1. It is found that the pressure gradient is maximum with small inclination and minimum with large inclination. ϕ = 0.5, q = 1, β = 0.1, n = 1, α = π/4. The pressure gradient once again is found to be enhanced with increasing the wave number.Figures β), fluid behavior index (n), angle between the walls (α), and wave number (δ) is that the trapped bolus increases in size as β, n, and α increase. However, the size of the trapped bolus decreases in size as δ increases. Evidently slip exerts a nontrivial influence on pressure gradient, velocity, and bolus magnitude and growth.Trapping is an interesting phenomenon in peristaltic motion in which an internally circulating bolus of fluid is formed by closed streamlines and this trapped bolus is pushed ahead along with the peristaltic wave. The effects slip parameter, power law index, angle between the walls, and wave number are illustrated with the help of contour plots Figures  of strea(i)β and α and the converse behavior with n and δ.Axial velocity across the transverse displacement is parabolic in nature and shifted in a downward direction increasing (ii)Pressure gradient across the axial length increases with slip parameter, fluid behavior index, and wave number and decreases with increasing inclination between walls.(iii)β, n, and α increase and decreases with δ.The size of trapped bolus increases with The present study has ignored curvature effects of the physiological vessel which are important in clinical applications and also biomimetic pumps employed in chemical engineering. These introduce a Coriolis effect and can lead to secondary vortex effects. They have been studied by other authors [In this study the two-dimensional peristaltic flow of a power law physiological fluid with the effect of slip condition through a nonuniform channel has been investigated. On the basis of computational and numerical results, the main findings of the present study are as follows: authors  and will


Detection of specific viral antibody or nucleic acid produced by infection or immunization, using oral fluid samples, offers increased potential for wider population uptake compared to blood sampling. This methodology is well established for the control of HIV and measles infections, but can also be applied to the control of other vaccine preventable infections, and this review describes the application of oral fluid assays in support of mumps, rubella and varicella national immunization programs. In England and Wales individuals with suspected mumps or rubella, based on clinical presentation, can have an oral fluid swab sample taken for case confirmation. Universal varicella immunization of children has led to a drastic reduction of chickenpox in those countries where it is used; however, in England and Wales such a policy has not been instigated. Consequently, in England and Wales most children have had chickenpox by age 10 years; however, small, but significant, numbers of adults remain susceptible. Targeted varicella zoster virus (VZV) immunization of susceptible adolescents offers the potential to reduce the pool of susceptible adults and oral fluid determination of VZV immunity in adolescents is a potential means of identifying susceptible individuals in need of VZV vaccination. The main application of oral fluid testing is in those circumstances where blood sampling is deemed not necessary, or is undesirable, and when the documented sensitivity and specificity of the oral fluid assay methodology to be used is considered sufficient for the purpose intended. Mumps is a highly infectious, generally benign disease defined by acute onset of unilateral or bilateral tender, self-limited swelling of the parotid or other salivary glands, lasting two or more days and without other apparent cause . A numbeParamyxoviridae, subfamily Paramyxovirinae, genus Rubulavirus. It is a non-segmented, negative strand RNA virus comprising a helical nucleocapsid surrounded by a lipid envelope. The complete genome of mumps virus has been sequenced [The causative agent of mumps, the mumps virus, belongs to the family equenced  at 15,38equenced  includinequenced  and releHumans, so far as is known, are the only natural host of the mumps virus although animal species including hamster, mouse, developing chick embryo and non-human primates can be infected  under laet al. [Mumps is highly infectious and Philip et al.  in a uniet al.  in epideet al. . Typicalversus control groups could be achieved and that mumps occurring in previously vaccinated individuals was of reduced severity [Mumps vaccination was first practiced during the 1940s using both live attenuated and inactivated vaccine preparations and it was shown that a significant reduction in the incidence of mumps in vaccinated severity . The firseverity . A numbeseverity ,19.In England and Wales, a national immunization program using one dose of a combined measles, mumps and rubella (MMR) vaccine given to children 12–15 months of age was commenced in 1988 and in 1996 a second dose was added, given at school entry . Many coThe detection of mumps specific IgM in oral fluids using capture radioimmunoassay has been available since the early 1990s  and in EIn England and Wales the Oracol oral fluid collection device is frequently used for MMR surveillance ,34. The Testing of oral fluid for the presence of specific antibody IgG or IgM requires the use of highly sophisticated assay methodologies because the amount of specific antibody in the oral fluid sample is much less that that found within a comparable blood sample . InitialMumps oral fluid testing offers an effective means for screening the incidence of mumps and estimating levels of vaccine coverage ,45 essenvice versa. Rubella frequently occurred in epidemics, had an incubation period of 14–21 days and produced confluent indistinct papules of a rose-red colour following no or an indistinct prodrome lasting one day. The potential linkage of rubella infection during pregnancy with congenital cataract and failure to thrive of the newborn combined with a high risk of congenital heart defects was first proposed by a Sydney based ophthalmologist, Sir Norman McAlister Gregg in 1941 [First described in 1814 and initially known as German Measles (Rötheln) until 1866 when the term “rubella” was proposed, this infection was viewed as an intermediate between measles and scarlatina . By the  in 1941 . In the  in 1941 ,55. A li in 1941  confirme in 1941  reportedNowadays, with the availability of enhanced techniques for rubella virus cultivation , plus thThe clinical and laboratory diagnosis of rubella and congenital rubella syndrome together with the development of rubella vaccines ,67 has bLaboratory methodologies for diagnosing rubella and measuring immunity have been reviewed elsewhere ,73 EvideThe role of oral fluid assays for rubella IgM detection is more established ,83 and tOral fluids, if collected soon enough following onset of illness and appropriately stored prior to testing are a valuable source of rubella virus genome which can be amplified by reverse transcription polymerase chain reaction (RT-PCR) and used to complement the results of antibody testing or sequenced so that the spread of virus can be tracked ,90,91. IPrimary infection with varicella zoster virus (VZV) manifests as varicella (chickenpox) and reactivation later in life produces herpes zoster (shingles). In the UK, traditionally, most cases of varicella occurred in 5–14 year olds; however, in recent years there has been a sharp increase in the prevalence of varicella in 1–4 year olds so that most cases, now, are reported in children aged 0–5 years ,95. PrimAn effective vaccine against varicella has been available for a number of years and universal childhood vaccination is undertaken in a number of countries  although not the UK . The UK Detection of VZV IgG in serum, using appropriately validated methods , has been shown ,105 to cDetection of VZV IgG using oral fluid samples removes the biases and limitations associated with blood sampling or the need to rely upon uncertain recall of no history of chickenpox. Such an approach can be used to identify susceptible individuals as part of targeted vaccination initiatives. In a study of adolescents  use of aThis review shows that oral fluid sampling and analysis can be of significant value in supporting disease control programmes and monitoring the impact of interventions such as vaccination. The technology for undertaking oral fluid analysis is currently highly specialised and only available at a limited number of laboratories and although such testing is of great value the lack of availability of more readily accessible assays can be seen as a drawback to deriving the full benefits of collecting these samples . There i


Page 2: The final sentence in the Funding Information should read as follows. “This work was funded by U.S. Department of Energy (DOE) under grant DE-AC02-05CH11231.”Volume 4, no. 1,


Scott (Herbert) Federhen passed away on May 5, 2016, after a short illness. Scott was well known for heading the National Center for Biotechnology Information’s (NCBI’s) Taxonomy project, which he started in 1991. Under his leadership it became an international standard for nomenclature and classification.Born in 1952, Scott obtained a MS degree in Computer Science, focused on denotational semantics of AI languages, from University of Maryland in 1980. An MS in Biology from MIT followed, with a focus on control structures in biological systems. He joined NCBI in 1990, shortly after its establishment. His dual training in both Computer Sciences and Biology served him well as he oversaw tremendous growth in the NCBI Taxonomy Database, developed numerous submission and database enhancements, and collaborated regularly with many other NCBI groups and members of the International Nucleotide Sequence Database Collaboration (INSDC). The INSDC continues to follow the NCBI Taxonomy Database classification.Under Scott’s guidance, the NCBI Taxonomy group was occasionally involved in scientific controversy, when underlying molecular data supported taxonomic changes. One example is the early inclusion of birds (Aves) under the Dinosauria in the NCBI classification. Scott also spearheaded NCBI’s efforts with the Barcode of Life project, an international initiative to identify and archive the barcode (a short genetic sequence from a standard part of the genome) for every species of life.Recently, Scott was instrumental in greatly expanding the annotation of type material in the NCBI taxonomic database. This allowed for improved taxonomic accuracy. Almost a year to the day before his death, he presented a proposal he developed for a groundbreaking change in how taxonomic names would be treated at NCBI. His sly sense of humor is evidenced in the title: “A Modest Proposal for making the Genomes of GenBank beneficial to the Publick, and preventing them from being a Burthen to the Curators and Taxonomists” (a nod to Johnathan Swift’s famous satire). The group he invited to the workshop included members of the International Committee on Systematics of Prokaryotes (ICSP) and the Judicial Commission on Prokaryotic Nomenclature, as well as bioinformaticians and taxonomists working with genomic similarity measures. Scott proposed a set of measures to represent a scaffold of reliably identified sequences that could be used to find and correct misidentified genomes. The invitees also agreed on a set of principles that will guide future efforts. It is anticipated that this project will continue and expand, but its inception was a testament to Scott’s drive and vision.Scott Federhen will be remembered as an adventurous eater, kind colleague and devoted Red Sox fan with an exceptionally broad intellectual range. He leaves behind his wife, Sheree, a daughter, Erica, a son, Luke, and many friends.


Wilms’ tumour (WT) is seldom seen in a neonate and prenatal diagnosis is rare. We present a case of antenatally diagnosed left sided WT with features of hydrops foetalis in a girl baby. Emergency LSCS was done at 34 weeks of gestation for foetal distress. Patient required mechanical ventilation for birth asphyxia and congestive cardiac failure. After stabilization, gross total resection of the tumour was done on day 4 of life. Histopathology HPE confirmed classical WT (stage I). Unfortunately, the patient died on the second postoperative day despite all supportive measures. Neonatal Wilms’ tumour (WT) is rare; the most common neonatal renal tumour being Congenital Mesoblastic Nephroma  comprising 66.7% of all neonatal renal masses . Only 0.12% of all Wilms’ tumours in NWTS 1-3 study were less than 30 days old vis-a-vis 0.54% CMN [2]. We came across an antenatally diagnosed WT, the details of whom are described here. A 20-year-old second gravida mother was referred to us at 29+5 weeks of gestation for prenatal ultrasonography (USG) findings of polyhydramnios associated with a heterogeneous left renal mass measuring 3.5 x 5.5 x 4.7 cms with solid and cystic components. She had had a previous LSCS at 28 weeks for pre-eclampsia with placenta abruption. During the current pregnancy, she had malaria in the first trimester and varicella in the second trimester. Scans during early pregnancy did not reveal any anomaly. Serial USG done 4 weeks later showed increase in size of the tumour to 9.8 x 8.7 cms along with features of hydrops foetalis , polyhydramnios and foetal distress. Hence, emergency LSCS was done at 34 weeks and a 3420 G girl baby was born.The newborn required resuscitation for poor APGAR scores which was followed by mechanical ventilation. On examination, there was generalised anasarca, anaemia and a large retroperitoneal tumour on the left side. There was no haematuria or hypertension. Postnatal USG confirmed the large left renal mass crossing the midline associated with ascites. Ascitic tap revealed transudative fluid. Laboratory investigations showed anaemia, leucocytosis and thromcytopenia. Peripheral smear showed the presence of some nucleated red blood cells. She required platelet transfusion for progressive decrease in platelet counts. After optimising the child, guided fine needle aspiration cytology (FNAC) was done which showed evidence of small round blue cell tumour. Following this, left nephroureterectomy was done on day 4 of life . Gross total resection of a 12x10 cm left renal tumour was done without any tumour rupture/spillage. The opposite kidney was normal. The child was kept on ventilator in the neonatal intensive care unit. Postoperatively, the condition of the baby deteriorated despite all possible measures and she died on the second postoperative day. Histopathology (HPE)  report showed evidence of Wilms’ tumour with areas of focal necrosis, haemorrhage, cystic changes and capsular infiltration without any breach; perinephric fat was free of tumour. Renal pelvis, hilum and ureter were free of the tumour. Antenatally diagnosed solid renal masses could be CMN, suprarenal neuroblastoma, adrenal haemorrhage, Wilms’ tumour or retroperitoneal teratoma; of which CMN is the commonest . Prenatal detection of Wilms’ tumour is extremely rare . Besides ultrasonography, foetal magnetic resonance imaging (MRI) has been found to be helpful in resolving the diagnostic dilemma of foetal renal masses [7-10]. Imaging guided FNAC, when feasible can confirm the diagnosis [4]. Unfortunately, there isn’t much scope of prenatal intervention for foetal renal tumours and definitive intervention is usually deferred until birth.An antenatally detected renal tumour may be associated with polyhydramnios, hydrops foetalis and acute foetal distress [4]; of which polyhydramnios is the commonest while hydrops is rare. Our patient had evidence of foetal hydrops and deranged coagulation parameters. The rapidly growing tumour  could be held responsible for the hydrops; due to compression of the infradiaphragmatic great vessels. In addition, vascular shunting into the tumour could also have led to circulatory failure and hydrops. Worsening of hydrops and rapid increase in the tumour size on serial USG made us suspicious about the malignant nature of the mass. As was seen in our case, foetal hydrops, when present, is a poor prognostic factor and often leads to demise of the newborn.The management of this patient posed a major challenge. Prematurity, worsening hydrops, haemodynamic instability, cardiomegaly and the malignancy per se made the survival chances dismal. Early surgery after optimum stabilization was undertaken in an attempt to improve the circulatory physiology by reducing the tumour burden, though in vain. As per the HPE and staging, it was a localized WT with good prognosis. Associated hydrops and polyhydramnios worsen the prognosis.Had she survived, her further management would again have been a matter of concern. There are no clear guidelines on the use of adjunctive therapy in neonates and infants. Radiotherapy does not have documented safety in infants and chemotherapy in regular doses is toxic and poorly tolerated. In our patient, since the tumour weight was less than 550 gram and HPE was favourable, we would not have given any further chemotherapy and would have kept her on close follow-up instead.Although neonatal WT usually has a good prognosis, close follow-up is essential as adjuvant therapy is often impractical and recurrence is likely to cause further morbidity and mortality. In a resource challenged nation like ours, foetal diagnostic tests are far from accurate, there is a lack of definite algorithm for antenatal management and foetal surgery is still in its early days.Source of Support: NilConflict of Interest: None


The images for figures Figs The affiliation for the fifth author is incorrect. Ping-Chin Lee is not affiliated with #2 but with #9 Faculty of Science and Natural Resources, University Malaysia Sabah, Jalan UMS, Kota Kinabalu, 88400, Sabah, Malaysia.In the Funding section, the incorrect author is attributed to the grant. The following grant should be attributed to C-C Ng.: High Impact Research Grant (UMMOHE UM.C/625/1/HIR/MOHE/ CHAN-02 H-50001-A000023), University of Malaya, to C-C Ng.


The ability to measure cellular proliferation non-invasively in renal cell carcinoma may allow prediction of tumour aggressiveness and response to therapy. The aim of this study was to evaluate the uptake of 18F-fluorothymidine (FLT) PET in renal cell carcinoma (RCC), and to compare this to 18F-fluorodeoxyglucose (FDG), and to an immunohistochemical measure of cellular proliferation (Ki-67).Twenty seven patients  with newly diagnosed renal cell carcinoma suitable for resection were prospectively enrolled. All patients had preoperative FLT and FDG PET scans. Visual identification of tumour using FLT PET compared to normal kidney was facilitated by the use of a pre-operative contrast enhanced CT scan. After surgery tumour was taken for histologic analysis and immunohistochemical staining by Ki-67.P<0.0001) in RCC. Ki-67 proliferative index was mean ± SD of 13.3%±9.2 (range 2.2% - 36.3%).The SUVmax  mean±SD for FLT in tumour was 2.59±1.27, compared to normal kidney (2.47±0.34). The mean SUVmax for FDG in tumour was similar to FLT (2.60±1.08). There was a significant correlation between FLT uptake and the immunohistochemical marker Ki-67 (r=0.72, There is detectable uptake of FLT in primary renal cell carcinoma, which correlates with cellular proliferation as assessed by Ki-67 labelling index. This finding has relevance to the use of FLT PET in molecular imaging studies of renal cell carcinoma biology. The ability to measure proliferation in vivo has several benefits, including the detection of tumour, the non-invasive assessment of tumour grade  with ur grade , and evaur grade -7. A numur grade , 8-17. Hur grade .1). This leads to intracellular trapping of FLT. The activity of TK1 varies during the cell cycle, being highest during the late G1 and S phases of the cell cycle, when preparation for DNA synthesis is occurring. During G0 (quiescent) phase there is very low or no activity of TK1. Therefore proliferating tissue, such as tumour, is expected to have uptake of FLT. In addition, malignant cells have been shown to have deregulated TK1 activity, leading to increased phosphorylation and accumulation of FLT  is the improved ability to distinguish inflammation from tumour. FDG PET reflects glucose metabolism, which may be increased in both benign inflammatory and malignant tissue. Animal studies comparing FLT with FDG in acutely inflamed or infected tissue have shown that FLT PET is less likely to be falsely positive in these situations , and establish whether a correlation exists between FLT uptake, morphologic changes in tumour and cellular proliferation by Ki-67 immunohistochemistry staining in RCC.The purpose of this study was to determine whether cellular proliferation can be quantitatively imaged in primary RCC by FLT PET. A secondary aim was to compare the uptake of FLT to FDG as measured by maximum standardized uptake values  for both PET scans were acquired on the Gemini PET/CT  or Allegro PET  scanner. Attenuation correction was performed from low dose CT scan (Gemini PET scanner), or 137Cs point source (Allegro PET scanner).Synthesis of FDG and FLT occurred on site in our radiochemistry facilities, using an in-house cyclotron  and radiochemistry synthesis techniques as previously described , 25. FDGmax of normal kidney. A visual grading system was also used to assess tumours. A score of 0 represented tumour uptake less than normal kidney, 1 representing an isointense tumour, and 2-4 uptake being mild, moderate or markedly greater than normal tissue.CT scans and patient history were available to two nuclear medicine specialists. The CT scans were used to identify the region of interest (ROI) for analysis and calculation of standardized uptake values. To improve the accuracy in identifying tumour from excreted radiotracer delayed phase, CT scans were used to exclude the collecting system from the region of interest. The contralateral kidney was used to represent the SUVThe diagnosis of disease was based on histopathology obtained via surgical resection. To determine Ki-67 staining, paraffin blocks tumour were cut into 4 μm sections, dewaxed, and antigen was retrieved in citrate buffer at 100 ºC water bath for 20 minutes. They were then stained with a monoclonal antibody to Ki-67  in a 1:50 dilution. Positive and negative controls were obtained, either through the use of highly proliferating reference tissue, or by using an IgG-matched control antibody, or omission of the primary antibody. Areas with the greatest density of staining were chosen for analysis. At 400x magnification, image acquisition software  was used to assist in manually counting at least 500 cells, in three fields within the region of interest. Nuclei with any staining for Ki-67 were considered positive. The Ki-67 proliferative index was defined as the percentage of Ki-67 stained nuclei per total nuclei.P value of <0.05 was considered to be statistically significant.Paired t-tests were used to compare FLT and FDG standardized uptake values. Linear regression analysis was used to compare Ki-67 uptake and SUV. A Twenty seven patients  with suspected primary renal cell carcinoma were recruited into the study. Post operative histology revealed sixteen clear cell RCCs, five papillary RCCs, and one each of sarcomatoid, chromophobe, transitional cell carcinoma (TCC) and oncocytoma. Two patients were found to have benign pathology. Patient characteristics are summarized in max of 2.59±1.27 (range 0.9 – 7.6) , but this did not reach statistical significance. Five tumours had visually discernable uptake greater than the contralateral kidney  , which had a mean SUVmax of 2.94±1.59). However, this did not reach statistical significance (P=0.09). Size and histological RCC subtype did not correlate with FLT uptake.FLT showed uptake in the majority of RCC tumours with a mean tumour SUV9 – 7.6) . The meae 2 – 4) . Nine tumax was 2.60±1.08 (range 1.0 - 6.0), and was again found to be slightly greater than that of normal kidney, which had a mean SUVmax of 2.20±0.54 (range 0.8 - 2.9). There was a greater uptake ratio for tumour to normal tissue in FDG compared to FLT, being 1.21 for FDG, and 1.04 for FLT, although this was not statistically significant (P=0.25). A significant difference between the mean SUVmax for low grade tumours at 2.25±0.99, compared to high grade tumours at 3.03±1.14 (P=0.03) was observed. There was significant correlation between the FLT SUVmax and FDG SUVmax .FDG PET showed low grade uptake in RCC tumours . The meamax and Ki-67 . Tumour stage, histological subtype and size failed to show a correlation with Ki-67 labelling index.Ki-67 proliferative index correlated with tumour grade. Low to moderate grade tumours had 8.9 % of cells positive for Ki-67, and high grade tumours 17.8% , which was a similar finding for FDG SUVmax as well. The relationship between FDG uptake and proliferation in RCC as measured by FLT PET and Ki-67 proliferative index in renal cell carcinoma has not been previously reported, and our results indicate that the low glucose metabolic rate of primary RCC is matched by a low level of proliferation. Interestingly, one patient in our study had metastatic lesions identified, which all showed prominent uptake of FDG and FLT, highlighting the underlying biologic differences which may exist between primary and metastatic RCC. In many other tumour types, there has also been correlation between FLT uptake, tumour grade and even prognosis, as described in a recent review (FDG has been reported to have a sensitivity between 31 and 94% in the assessment of primary RCC -36. We ft review . Howevert review .FLT uptake in primary RCC is detectable and correlates with cellular proliferation on Ki-67 biopsy analysis. FLT uptake also correlates with FDG uptake in primary RCC. Further studies are required to define the role of FLT PET in assessing RCC response to treatment and in prognostic assessment, particularly in metastatic disease.


The Funding Statement in the original article is missing the grant number for the KINDRED project. The correct funding statement is as follows:This work was supported by an ANR  grant (LeishApo). The research leading to these results has also received funding from the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED). JE thanks the Canada Research Chair program for financial assistance. VR is supported by a doctoral fellowship from FCT (Fundação para a Ciência e Tecnologia); code SFRH/BD/64064/2009. RS is supported by Programa Ciência – financed by Programa Operacional Potencial Humano POPH – QREN– Tipologia 4.2 – Promocão do Emprego Científico, co-funded by Fundo Social Europeu and National funding from Ministry of Science, Technology and Higher Education (MCTES). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.