Clonality and evolutionary history of rhabdomyosarcoma.

To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS

Mouse models for preeclampsia: disruption of redox-regulated signaling

The concept that oxidative stress contributes to the development of human preeclampsia has never been tested in genetically-defined animal models. Homozygous deletion of catechol-Omethyl transferase (Comt-/-) in pregnant mice leads to human preeclampsia-like symptoms (high blood pressure, albuminurea and preterm birth) resulting from extensive vasculo-endothelial pathology, primarily at the utero-fetal interface where maternal cardiac output is dramatically increased during pregnancy. Comt converts estradiol to 2-methoxyestradiol 2 (2ME2) which counters angiogenesis by depleting hypoxia inducible factor-1 alpha (HIF-1 alpha) at late pregnancy. We propose that in wild type (Comt++) pregnant mice, 2ME2 destabilizes HIF-1 alpha by inhibiting mitochondrial superoxide dismutase (MnSOD). Thus, 2ME2 acts as a pro-oxidant, disrupting redox-regulated signaling which blocks angiogenesis in wild type (WT) animals in physiological pregnancy. Further, we suggest that a lack of this inhibition under normoxic conditions in mutant animals (Comt-/-) stabilises HIF-1 alpha by inactivating prolyl hydroxlases (PHD). We predict that a lack of inhibition of MnSOD, leading to persistent accumulation of HIF-1 alpha, would trigger inflammatory infiltration and endothelial damage in mutant animals. Critical tests of this hypothesis would be to recreate preeclampsia symptoms by inducing oxidative stress in WT animals or to ameliorate by treating mutant mice with Mn-SOD-catalase mimetics or activators of PHD

Prospective comparison of novel dark blood late gadolinium enhancement with conventional bright blood imaging for the detection of scar

BACKGROUND: Conventional bright blood late gadolinium enhancement (bright blood LGE) imaging is a routine cardiovascular magnetic resonance (CMR) technique offering excellent contrast between areas of LGE and normal myocardium. However, contrast between LGE and blood is frequently poor. Dark blood LGE (DB LGE) employs an inversion recovery T2 preparation to suppress the blood pool, thereby increasing the contrast between the endocardium and blood. The objective of this study is to compare the diagnostic utility of a novel DB phase sensitive inversion recovery (PSIR) LGE CMR sequence to standard bright blood PSIR LGE. METHODS: One hundred seventy-two patients referred for clinical CMR were scanned. A full left ventricle short axis stack was performed using both techniques, varying which was performed first in a 1:1 ratio. Two experienced observers analyzed all bright blood LGE and DB LGE stacks, which were randomized and anonymized. A scoring system was devised to quantify the presence and extent of gadolinium enhancement and the confidence with which the diagnosis could be made. RESULTS: A total of 2752 LV segments were analyzed. There was very good inter-observer correlation for quantifying LGE. DB LGE analysis found 41.5% more segments that exhibited hyperenhancement in comparison to bright blood LGE (248/2752 segments (9.0%) positive for LGE with bright blood; 351/2752 segments (12.8%) positive for LGE with DB; p < 0.05). DB LGE also allowed observers to be more confident when diagnosing LGE (bright blood LGE high confidence in 154/248 regions (62.1%); DB LGE in 275/324 (84.9%) regions (p < 0.05)). Eighteen patients with no bright blood LGE were found to have had DB LGE, 15 of whom had no known history of myocardial infarction. CONCLUSIONS: DB LGE significantly increases LGE detection compared to standard bright blood LGE. It also increases observer confidence, particularly for subendocardial LGE, which may have important clinical implications

Identifying uncertainties in Arctic climate change projections

Wide ranging climate changes are expected in the Arctic by the end of the 21st century, but projections of the size of these changes vary widely across current global climate models. This variation represents a large source of uncertainty in our understanding of the evolution of Arctic climate. Here we systematically quantify and assess the model uncertainty in Arctic climate changes in two CO2 doubling experiments: a multimodel ensemble (CMIP3) and an ensemble constructed using a single model (HadCM3) with multiple parameter perturbations (THC-QUMP). These two ensembles allow us to assess the contribution that both structural and parameter variations across models make to the total uncertainty and to begin to attribute sources of uncertainty in projected changes. We find that parameter uncertainty is an major source of uncertainty in certain aspects of Arctic climate. But also that uncertainties in the mean climate state in the 20th century, most notably in the northward Atlantic ocean heat transport and Arctic sea ice volume, are a significant source of uncertainty for projections of future Arctic change. We suggest that better observational constraints on these quantities will lead to significant improvements in the precision of projections of future Arctic climate change

Incorporating prior knowledge improves detection of differences in bacterial growth rate

BACKGROUND: Robust statistical detection of differences in the bacterial growth rate can be challenging, particularly when dealing with small differences or noisy data. The Bayesian approach provides a consistent framework for inferring model parameters and comparing hypotheses. The method captures the full uncertainty of parameter values, whilst making effective use of prior knowledge about a given system to improve estimation. RESULTS: We demonstrated the application of Bayesian analysis to bacterial growth curve comparison. Following extensive testing of the method, the analysis was applied to the large dataset of bacterial responses which are freely available at the web-resource, ComBase. Detection was found to be improved by using prior knowledge from clusters of previously analysed experimental results at similar environmental conditions. A comparison was also made to a more traditional statistical testing method, the F-test, and Bayesian analysis was found to perform more conclusively and to be capable of attributing significance to more subtle differences in growth rate. CONCLUSIONS: We have demonstrated that by making use of existing experimental knowledge, it is possible to significantly improve detection of differences in bacterial growth rate

RNAi-mediated COPS3 gene silencing inhibits metastasis of osteogenic sarcoma cells

Metastatic disease is the primary cause of mortality among patients with osteogenic sarcoma (OGS). In this study, we aimed to identify the relationship of COPS3 gene expression to metastasis. Immunohistochemical staining for COPS3 was performed on 65 OGS samples (37 without and 28 with metastatic disease); 18.9% (7/37) of specimens from patients with no metastasis and 57.1% (16/28) of specimens from patients with metastasis showed intense staining of COPS3. Comparison of COPS3 expression between a poorly metastatic osteosarcoma cell line (SAOS-2) and highly metastatic osteosarcoma cell line (HOS) showed stronger expression of COPS3 in HOS cells. Inhibiting COPS3 function by siRNA resulted in reduced proliferation and migration of HOS cells. Inhibition of COPS3 gene downregulated expression of the MAPK signaling pathway, which has an important role in metastasis of OGS. Our results suggested that overexpression of the COPS3 gene might have important roles in metastasis of osteosarcoma cells

Applying refinement to the use of mice and rats in rheumatoid arthritis research

Rheumatoid arthritis (RA) is a painful, chronic disorder and there is currently an unmet need for effective therapies that will benefit a wide range of patients. The research and development process for therapies and treatments currently involves in vivo studies, which have the potential to cause discomfort, pain or distress. This Working Group report focuses on identifying causes of suffering within commonly used mouse and rat ‘models’ of RA, describing practical refinements to help reduce suffering and improve welfare without compromising the scientific objectives. The report also discusses other, relevant topics including identifying and minimising sources of variation within in vivo RA studies, the potential to provide pain relief including analgesia, welfare assessment, humane endpoints, reporting standards and the potential to replace animals in RA research

Cloning, expression and characterization of l-asparaginase from Withania somnifera L. for large scale production

l-Asparaginase (E.C. 3.5.1.1) is used as a therapeutic agent in the treatment of acute childhood lymphoblastic leukemia. It is found in a variety of organisms such as microbes, plants and mammals. In plants, l-asparaginase enzymes are required to catalyze the release of ammonia from asparagine, which is the main nitrogen-relocation molecule in these organisms. An Indian medicinal plant, Withania somnifera was reported as a novel source of l-asparaginase. l-Asparaginase from W. somnifera was cloned and overexpressed in E. coli. The enzymatic properties of the recombinant enzyme were investigated and the kinetic parameters (Km, kcat) for a number of substrates were determined. The kinetic parameters of selected substrates were determined at various pH and the pH- and temperature-dependence profiles were analyzed. WA gene successfully cloned into E. coli BL21 (DE3) showed high asparaginase activity with a specific activity of 17.3 IU/mg protein

Cardiac Structural and Functional Consequences of Amyloid Deposition by Cardiac Magnetic Resonance and Echocardiography and Their Prognostic Roles

OBJECTIVES: This cross-sectional study aimed to describe the functional and structural cardiac abnormalities that occur across a spectrum of cardiac amyloidosis burden and to identify the strongest cardiac functional and structural prognostic predictors in amyloidosis using cardiac magnetic resonance (CMR) and echocardiography. BACKGROUND: Cardiac involvement in light chain and transthyretin amyloidosis is the main driver of prognosis and influences treatment strategies. Numerous measures of cardiac structure and function are assessed by multiple imaging modalities in amyloidosis. METHODS: Three hundred twenty-two subjects (311 systemic amyloidosis and 11 transthyretin gene mutation carriers) underwent comprehensive CMR and transthoracic echocardiography. The probabilities of 11 commonly measured structural and functional cardiac parameters being abnormal with increasing cardiac amyloidosis burden were evaluated. Cardiac amyloidosis burden was quantified using CMR-derived extracellular volume. The prognostic capacities of these parameters to predict death in amyloidosis were assessed using Cox proportional hazards models. RESULTS: Left ventricular mass and mitral annular plane systolic excursion by CMR along with strain and E/e' by echocardiography have high probabilities of being abnormal at low cardiac amyloid burden. Reductions in biventricular ejection fractions and elevations in biatrial areas occur at high burdens of infiltration. The probabilities of indexed stroke volume, myocardial contraction fraction, and tricuspid annular plane systolic excursion (TAPSE) being abnormal occur more gradually with increasing extracellular volume. Ninety patients (28%) died during a median follow-up of 22 months (interquartile range: 10 to 38 months). Univariable analysis showed that all imaging markers studied significantly predicted outcome. Multivariable analysis showed that TAPSE (hazard ratio: 1.46; 95% confidence interval: 1.16 to 1.85; p < 0.01) and indexed stroke volume (hazard ratio: 1.24; 95% confidence interval: 1.04 to 1.48; p < 0.05) by CMR were the only independent predictors of mortality. CONCLUSIONS: Specific functional and structural abnormalities characterize different burdens of cardiac amyloid deposition. In a multimodality imaging assessment of a large cohort of amyloidosis patients, CMR-derived TAPSE and indexed stroke volume are the strongest prognostic cardiac functional markers