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Somatic alterations in cellular DNA underlie almost all human cancers 1 . The prospect of targeted therapies 2 and the development of high-resolution, genome-wide approaches 3–8 are now spurring systematic efforts to characterize cancer genomes. Here we report a large-scale project to characterize copy-number alterations in primary lung adenocarcinomas. By analysis of a large collection oftumours(n 5371)usingdensesinglenucleotidepolymorphism arrays, we identify a total of 57 significantly recurrent events. We find that 26 of 39 autosomal chromosome arms show consistent large-scalecopy-numbergainorloss,ofwhichonlyahandfulhave been linked to a specific gene. We also identify 31 recurrent focal events, including 24 amplifications and 7 homozygous deletions. Only six of these focal events are currently associated with known mutations in lung carcinomas. The most common event, amplification of chromosome 14q13.3, is found in 12% of samples. On the basis of genomic and functional analyses, we identify NKX2-1 (NK2 homeobox 1, also called TITF1), which lies in the minimal 14q13.3 amplification interval and encodes a lineagespecific transcription factor, as a novel candidate proto-oncogene involved in a significant fraction of lung adenocarcinomas. More generally, our results indicate that many of the genes that are involved in lung adenocarcinoma remain to be discovered. A collection of 528 snap-frozen lung adenocarcinoma resection specimens, with at least 70% estimated tumour content, was selected by a panel of thoracic pathologists (Supplementary Table 1); samples were anonymized to protect patient privacy. Tumour and normal DNAs were hybridized to Affymetrix 250K Sty single nucleotide polymorphism (SNP)arrays. Genomic copy number foreach ofover 238,000 probe sets was determined by calculating the intensity ratio between the tumour DNA and the average of a set of normal DNAs 9,10 . Segmented copy numbers for each tumour were inferred with the GLAD (gain and loss analysis of DNA) algorithm 11 and normalized to a median of two copies. Each copy number profile was then subjected to quality control, resulting in 371 high-quality samples used for further analysis, of which 242 had matched normal

The aim of this study was to investigate adiponectin, leptin, and metabolic syndrome as predictors of the severity of obesity-related steatosis. By ultrasonography steatosis-positive (cases) subjects (n=141) were compared with controls (n=111). Demographic and anthropometric data and serum concentrations of adiponectin, leptin, and insulin were measured. The impact of several criteria of metabolic syndrome, serum adiponectin concentrations, and serum leptin concentrations were tested using a multivariate logistic regression analysis. The frequency of metabolic syndrome was higher in cases (44.0% versus 9.2%; P

To analyze the relationship between smoking and nonalcoholic fatty liver disease (NAFLD).This is a cross-sectional study of a healthy population, carried out in a check-up unit of a university hospital in Mexico City. We enrolled 933 subjects, 368 current smokers (cases) and 565 persons who had never smoked (controls). Demographic, metabolic and biochemical variables were measured in the two groups. NAFLD was determined by ultrasound and metabolic syndrome according to ATPIII.A total of 548 men (205 cases and 343 controls) and 337 women (114 cases and 223 controls) were included in the analysis. Statistical differences between cases and controls were observed only in high blood pressure prevalence (6.6% vs 11.3%, P0.05; cases and controls respectively), high-density lipoproteins (1.00 +/- 0.26 vs 1.06 +/- 0.28 mmol/L, P0.005), triglycerides (2.18 +/- 1.49 vs 1.84 +/- 1.1 mmol/L, P0.001), and erythrocyte sedimentation rate (11.3 +/- 9.3 vs 13.5 +/- 11.9 mm/h, P0.001). No differences were observed in the prevalence of NAFLD (22.27% vs 29.68%, P = NS) and metabolic syndrome (41.69% vs 36.74%, P = NS). Univariate analysis showed that smoking was not a risk factor for NAFLD (OR = 0.89, 95% CI 0.65-1.21).No differences in NAFLD prevalence were observed between current smokers and nonsmokers, and furthermore, no differences were observed in heavy smokers (more than 20 packs/year), indicating that there is no relationship between smoking and NAFLD.

Objective: Investigate the association between polymorphisms in the leptin receptor gene associated with obesity and gallstone disease. Design: We conducted a cross-sectional study, carried out at a tertiary setting. Subjects: We enrolled 97 subjects, comprising 54 subjects with gallstones (cases) and 43 controls (without gallstones). Measurements: Diet was assessed using a validated questionnaire for the Mexican population. Body mass index, waist circumference, serum glucose, insulin, leptin, lipids and lipoproteins levels were measured. Insulin resistance was calculated by HOMA-IR. Genomic DNA was isolated from lymphoblastoid cells, and Q223R and K656N polymorphisms in the leptin receptor gene were typed using polymerase chain reaction. Unconditional univariate logistic regression analysis was conducted to estimate the probability of gallstone disease associated with the polymorphisms as main effect. Results: Cases were different in gender (40.74% males in cases vs 74.41% in controls; p < 0.001), older (49.74 vs 44.83 years; p < 0.05), and had more body fat (32.34% vs 28.14%; p = 0.01). Individuals carrying the polymorphism Q223R exhibited a higher BMI (28.44 ± 6.6 kg/m2 vs 25.94 ± 3.67 kg/m2, p < 0.05) and waist circumference (96.7 ± 16.39 cm vs 89.2 ± 11.05 cm, p < 0.05). In univariate analysis, we did not observe a relation between the presence of a R223 or N656 genotype and gallstone disease in our population (OR = 0.78, 95% CI 0.35-1.73). Conclusion: Obesity-related leptin receptor polymorphisms are not associated with gallstones disease.

Summary The enzyme hypoxanthine-guanine phosphoribo-syltransferase (HPRT) catalyzes the reutilization ofhypoxanthine and guanine to the purine nucleotidesIMP and GMP, respectively. HPRT deficiency is an X-linked disorder characterized by uric acid over-production and variable neurologic impairment. Thecomplete deficiency of HPRT is diagnostic of Lesch-Nyhan syndrome manifested by choreoathetosis,spasticity, mental retardation, and self-injurious be-havior. In some HPRT-deficient patients the enzymedefect appeared to be “partial” and the neurologicsymptoms mild to severe (Kelley-Seegmiller syn-drome). This has prompted the classification ofHPRT deficiency in 2 distinct groups: Lesch-Nyhansyndrome and Kelley-Seegmiller syndrome, whichhas created much confusion. A spectrum of clinicalconsequences of HPRT deficiency has been recog-nized in small series of patients, but the completespectrum of the neurologic disorder has not been de-scribed in a single series of patients examined by thesame observers.We analyzed our experience with 22 patients be-longing to 18 different families with HPRT deficiencydiagnosed at “La Paz” University Hospital in Madridover the past 16 years. The clinical spectrum of theseHPRT-deficient Spanish patients was similar to thedifferent phenotypes occasionally reported in the lit-erature, in some cases diagnosed as Lesch-Nyhan“variants.” The clinical, biochemical, enzymatic, andmolecular genetic studies on these 22 patients al-lowed us to delineate a new classification of HPRTdeficiency. Based on the neurologic symptoms, de-pendency for personal care, HPRT activity in he-molysate and in intact erythrocytes, and predictedprotein size, patients were classified into 4 groups:Group 1 (2 patients), normal development with noneurologic symptoms, HPRT activity was detectablein hemolysates and in intact erythrocytes, and themutation did not affect the predicted protein size.Group 2 (3 patients) mild neurologic symptoms thatdid not prevent independent lives, HPRT activity wasdetectable in intact erythrocytes, and the protein sizewas normal. Group 3 (2 patients), severe neurologicimpairment that precluded an independent life, noresidual HPRT activity, and normal protein size.Group 4 (15 patients), clinical characteristics ofLesch-Nyhan syndrome (some may not show self-injurious behavior), no residual HPRT activity, and inmost (7 of 8 patients in whom the mutation could bedetected) the mutation affected the predicted pro-tein size.This classification of HPRT deficiency into 4groups may be more useful in terms of accuracy, re-producibility, assessment for treatment trials andprognosis. The study of this Spanish series allows usto conclude that HPRT deficiency may be manifestedby a wide spectrum of neurologic symptoms; theoverall severity of the disease is associated with mu-tations permitting some degree of residual enzymeactivity; and mutation analysis provides a valuable

AIM: To establish an association between the serum leptin levels and the development of gallstone disease (GD). METHODS: We carried out a non-matched case-controlled study in a university hospital in Mexico City. Two hundred and eighty-seven subjects were included: 97 cases with gallstones and 190 controls. Body mass index (BMI), fasting plasma leptin, insulin, serum lipid, and lipoprotein levels were measured. Insulin resistance was calculated by homeostasis model assessment (HOMA-IR). Unconditional logistic regression analysis (univariate and multivariate) stratified by BMI was used to calculate the risk of GD. RESULTS: The multivariate conditional regression analysis revealed a model for those patients with BMI

It has been suggested that genes related to Amerindian ancestry account for the high prevalence of gallstone disease (GD) observed in Mexican-Americans. The HLA-B39 is an allele found in higher frequency in Amerindians whereas HLA-B15 is rarely found. The aim of this study was to test the hypothesis that gallstone susceptibility genes are more prevalent in Mexicans with recent Amerindian ancestry.We carried out a prospective case-controlled study. Subjects were divided into those who had stones visible on gallbladder ultrasound (cases), and those whose ultrasounds were negative for gallstones (controls). Body mass index (BMI) was calculated, and serum lipids and lipoprotein, and glucose levels were measured. Class I HLA (HLA-B) typing was performed by PCR amplification of genomic DNA.Of the 1,101 subjects, 146 were classified as subjects with GD (cases) and 955 as subjects without GD (controls). Mean age of the cases was 53.5 +/- 12.5 yr versus 44.78 +/- 12.0 yr for the controls, p= 0.001. A family history of GD was observed in 48% of the cases versus 28.4% of the controls, p= 0.001. HLA-B39 was more frequently increased in GD subjects (0.162), compared with controls (0.063), p= 0.008. The odds ratio of having HLA-B39 was 2.8 and 95% (CI 95%= 1.3-6.3) for GD; HLA-B15 was more frequently increased in controls than in cases.The most prevalent HLA alleles detected in these GD cases are characteristic of Amerindian populations, supporting the role of genetics in the high prevalence of the development of GD in Mexican mestizos.

The present work aims to develop a growth medium to render a wild-type strain ofSaccharomyces cerevisiaepermeable to the antifungal drug Brefeldin A. In the current study, a synthetic medium containing 0.1% L-proline and supplemented with3.0×10−3% SDS is employed. When Brefeldin A is added to this medium, a wild-type strain shows increased growth sensitivity and a diminished transport of the amino acid L-leucine. Since Brefeldin A exerts its effect on the endoplasmic reticulum and the Golgi apparatus, the medium permits the study of the drug effect on the intracellular traffic of L-leucine permeases.

Stormer method is a multistep code with fixed step suitable for the numerical integration of second-order differential equations of the special form y''(x) = f(x,y(x)), y(x0) = y0, y'(x0) = y'0, (1) where the right hand side does not include the derivative of y [4, p.462].

Although it is possible to integrate a special second-order differential equation of the form y''(x) = f(x,y(x)), y(x0) = y0, y'(x0) = y'0 (1) by reducing it to a first order system and applying one of the methods available for those systems, it seems more natural to provide numerical methods to integrate (1) directly without using first derivatives.The advantage of these approaches has to be with the fact that they are able to exploit special information about ODES, and this results in an increase in efficiency (that is, high accuracy at low cost). For instance, it is well-known that Runge-Kutta-Nystrom methods for (1) involve a real improvement compared to standard Runge-Kutta methods, for a given number of stages [ 4, p. 285]. On the contrary, a linear k-step method for first-order ODEs becomes a 2k-step method for (1), [4, p. 461], increasing the computational work.

The goal of providing efficient general purpose numerical methods has been a central activity within the full scope of solving numerically differential equations. But lately there has been a certain change of trend, in the sense that some types of problems are so particular that it is better to develop their own special theory and techniques.

The purpose of this study was to evaluate the effects of the Scorpion Load Carriage System (SLCS) on biomechanics, oxygen consumption and performance on militarily relevant tasks, and to compare the SLCS to the MOLLE system. Eleven subjects completed testing.

O objectivo do presente estudo é analisar o impacto da presença de complicações crónicas específicas na qualidade de vida de indivíduos com diabetes. Foi avaliada uma amostra de conveniência de 316 sujeitos com diabetes, dos quais 44,6% do sexo masculino; com idades compreendidas entre os 16 e os 84 anos (M= 48,39; DP=16,90); 41,8% (n=132) apresenta diagnóstico de diabetes tipo 1 e a duração da doença varia entre 4 meses e 43 anos (M=13,66; DP=9,32). Destes doentes, 59,8% sofre de complicações crónicas da diabetes. Os resultados sugerem que, entre os indivíduos com diabetes, aqueles que sofrem de complicações crónicas demonstram ter uma qualidade de vida inferior à dos que não sofrem de sequelas. Esta diferença verifica-se quando são analisadas especificamente a microangiopatia, catarata, macroangiopatia, neuropatia autonómica (nomeadamente, disfunção sexual), doença cardíaca coronária, antecedentes de acidente vascular cerebral, hipertensão arterial e doença arterial periférica. Todavia, quando controlada a gravidade de complicações como a retinopatia e a nefropatia, constata-se que não existe uma relação linear entre a gravidade da complicação e o impacto na qualidade de vida, uma vez que, por vezes, doentes com níveis mais graves da complicação apresentam uma qualidade de vida superior à de indivíduos com níveis menos graves desta.

Four computer-typing accommodations were evaluated to determine if arm supports would enhance typing performance and/or comfort of adults with neurological disorders. Each of 12 adults (6 females and 6 males) participated in 7-min typing tasks under the following conditions: 1) without arm support, 2) Ergo Rest arm support, 3) custom arm support in fixed mode, and 4) custom arm support with movable mode. The objective dependent variables were speed, errors, and type of errors. The subjective dependent measures were reported body part discomfort, and arm support ratings. The data were found to be nonparametric in nature. Therefore, nonparametric techniques were employed to analyze the data. Only one of the objective dependent measures were statistically significant; Type of error (additional key). This result may be attributed to the wide range of disability manifestations among participants. Subjective dependent measures were found to be statistically significant, leading to the conclusion that typing with an arm support was more comfortable and easier than without an arm support.

Initial data mining of results from several on-going public cancer genomics initiatives has identified both known and novel somatic alterations across multiple cancer types. A challenge of validating putative cancer targets discovered by such studies is the identification of appropriate preclinical cell line models with relevant genetic alterations. Thus there is an urgent need for comprehensive mutation information across a large panel of cancer cell lines. Mutation discovery in cell lines brings its own unique challenges. Most cancer re-sequencing studies published to date typically use DNA derived from both primary tissue and matched DNA from a non-cancerous sample, often from peripheral blood or adjacent tissue. Cell lines present a challenge for discovering somatic mutations because a matched normal sample that can be used to filter for somatic mutations rarely exists. To this end we have implemented a filtering scheme that attempts to enrich for true-somatic mutations. To assess the effectiveness of the filtering pipeline, we compared filtered results with mutations discovered in matched normal cell lines derived from blood for 10 cancer cell lines. We estimate that filtering captures 68% of true somatic mutations. This rate is slightly improved or comparable over existing methods used in previous studies involving sequencing of cancer cell lines. Interestingly, we find that there is typically 10% of known true somatic mutations that are unable to be called by any of the mutation callers used in a “no-normal” context but are discoverable with a normal present. In such cases, the presence of the normal can add additional information that aids mutation calling. To genetically characterize a panel of cell lines for pharmacological studies, we performed exome sequencing of 223 cancer cell lines. Analysis of filtered mutation variants reveals that cell lines harbor an average of 270 nonsynonymous coding variants, an increase over typical amounts found in primary tumors. Furthermore we find that for several tumor lineages, the spectra of mutations observed reflect the mutation signatures identified by other large scale efforts in primary tumors. With regard to recurrently mutated oncogenes and tumor suppressors, we observe significant overlap with existing mutation data derived from primary tumor samples. Additionally, we identify relevant cell line models for several novel cancer driver genes reported in recent studies. As a resource for the scientific community, we have made both aligned read data as well as mutation calls in various formats freely available and easily accessible. Citation Format: Alex H. Ramos, Ruibang Luo, Jacob Feala, Binghang Liu, Lara Gong, Markus Warmuth, Ping Zhu, Peter Smith, Lihua Yu. Exome sequencing of tumor cell lines: Optimizing for cancer variants. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4269. doi:10.1158/1538-7445.AM2014-4269

Objective To evaluate the frequency of microalbuminuria and its relationship with several risk factors and left ventricular mass in a population of mildly hypertensive subjects attended in a primary-care setting. Design Cross-sectional study. Setting Eight primary-care centres. Patients Two hundred and twenty-three non-diabetic patients recently diagnosed with mild hypertension were included in the study. None of them had clinical evidence of target-organ damage or had received prior antihypertensive treatment. Interventions Subjects included in the study underwent clinical interview, measurement of blood pressure (BP) on three visits, blood analysis, measurement of albumin by immunonephelometry in three overnight urine collections, 24 h BP monitoring and M-mode and Doppler echocardiography. Main outcome measures Tobacco habit, clinic BP, body mass index, serum lipids and uric acid, glycaemia, urinary albumin excretion (UAE), ambulatory BP and left ventricular mass index. Results The frequency of microalbuminuria was 7.2%. Microalbuminuric patients were more likely to be men and to be characterized by higher ambulatory BP, body mass index and uric acid levels. Regression analysis demonstrated that male sex and 24 h systolic BP were determinants of UAE. Patients with white-coat hypertension showed lower UAE than did subjects with sustained hypertension. Although a certain relationship between UAE and left ventricular mass index was found, these variables were not significantly correlated. Conclusions A low proportion of mildly hypertensive patients attended in a primary care setting are microalbuminuric. In this population, UAE is an expression of BP values over 24 h and correlates with several risk factors.

This chapter will review the classification, dietary sources and metabolism, biological effects, and potential clinical effects of natural products, including phytoestrogen and herbs, and the medical alternative, raloxifene. Phytoestrogens, estrogen-like compounds found in plant products, have been shown to have both estrogenic and antiestrogenic properties. Following the publication of the Allen-Doisy bioassay for estrogens in 1923, plant extracts were first reported to exhibit estrogenic activity in 1926. By 1975, several hundred plants had been found to exhibit estrogenic activity on bioassay or to contain estrogenically active compounds. Phytoestrogens have been identified in bile, urine, semen, blood, and feces in humans and animals. Epidemiologic studies have suggested that consumption of a diet rich in phytoestrogen, commonly seen in Asian populations, especially Japan, has reduced the risk for the so-called Western diseases, i.e., cardiovascular disease and breast and endometrial cancers, as well as menopausal symptoms, especially hot flushes.

Because the exact etiology of functional, or idiopathic, hypothalamic amenorrhea (FHA) is still unknown, FHA remains a diagnosis of exclusion. The disorder may be stress induced. However, mounting evidence points to a metabolic/nutritional insult that may be the primary causal factor. We explored the thyroid, hormonal, dietary, behavior, and leptin changes that occur in FHA, as they provide a clue to the etiology of this disorder. Fourteen cycling control and amenorrheic nonathletic subjects were matched for age, weight, and height. The amenorrheic subjects denied eating disorders; only after further, detailed questioning did we uncover a higher incidence of anorexia and bulimia in this group. The amenorrheic subjects demonstrated scores of abnormal eating twice those found in normal subjects (P0.05), particularly bulimic type behavior (P0.01). They also expended more calories in aerobic activity per day and had higher fiber intakes (P0.05); lower body fat percentage (P0.05); and reduced levels of free T4 (P0.05), free T3 (P0.05), and total T4 (P0.05), without a significant change in rT3 or TSH. Cortisol averaged higher in the amenorrheics, but not significantly, whereas leptin values were significantly lower (P0.05). Bone mineral density was significantly lower in the wrist (P0.05), with a trend to lower BMD in the spine (P0.08). Scores of emotional distress and depression did not differ between groups. The alterations in eating patterns, leptin levels, and thyroid function present in subjects with FHA suggest altered nutritional status and the suppression of the hypothalamic-pituitary-thyroid axis or the alteration of feedback set-points in women with FHA. Both lower leptin and thyroid levels parallel changes seen with caloric restriction. Nutritional issues, particularly dysfunctional eating patterns and changes in thyroid metabolism, and/or leptin effects may also have a role in the metabolic signals suppressing GnRH secretion and the pathogenesis of osteopenia despite normal body weight. These findings suggest that the mechanism of amenorrhea and low leptin in these women results mainly from a metabolic/nutritional insult.

The effects of regulatory amounts of Ca2+ on the in situ structures of troponin C (TnC) and troponin I (TnI) in whole troponin have been investigated by neutron scattering. In separate difference experiments, 97% deuterated TnC and TnI within whole troponin were studied +/-Ca2+ in 41.6% 2H2O buffers in which protonated subunits were rendered "invisible". We found that the radius of gyration (Rg) of TnI decreased by approximately 10% upon addition of regulatory Ca2+ indicating that it was significantly more compact in the presence of Ca2+. The apparent cross-sectional radius of gyration (Rc) of TnI increased by about 9% when regulatory Ca2+ was bound to TnC. Modeling studies showed that the high-Q scattering patterns of TnI could be fit by a TnI which consisted of two subdomains: one, a highly oblate ellipsoid of revolution containing about 65% of the mass and the other, a highly prolate ellipsoid of revolution consisting of about 35% of the mass. No other fits could be found with this class of models. Best fits were achieved when the axes of revolution of these ellipsoids were steeply inclined with respect to each other. Ca2+ addition decreased the center of mass separation by about 1.5 nm. The Rg of TnI, its high-Q scattering pattern, and the resultant structure were different from previous results on neutron scattering by TnI in the (+Ca2+) TnC.TnI complex. The Rg of TnC indicated that it was elongate in situ. The Rg of TnC was not sensitive to the Ca2+ occupancy of its regulatory sites. However, Rc increased upon Ca2+ addition in concert with expectations from NMR and crystallography of isolated TnC. The present observations indicate that TnI acts like a molecular switch which is controlled by smaller Ca2+-induced changes in TnC.

Primary gout is characterized by increased plasma and decreased urinary concentrations of hypoxanthine, xanthine and uric acid. To examine whether lead could explain the disturbance of purine metabolism in gout, we determined hypoxanthine, xanthine and uric acid metabolism and 5-day cumulative urinary lead excretion rates after an EDTA (calcium disodium edetate) test in 27 patients with primary gout and reduced creatinine clearance (C(cr)) and in 50 patients with gout and normal C(cr). The results were compared to those obtained in 26 normal subjects matched for age. All gout patients evidenced a marked renal underexcretion of hypoxanthine, xanthine and uric acid relative to their increased plasma levels. Purine metabolism was remarkably similar in both gout groups except for a significantly lower uric acid excretion in patients with reduced C(cr). Blood lead levels and cumulative lead excretion rates were significantly higher in gout patients with renal failure as compared to patients with normal C(cr). Fourteen patients (52%) with renal insufficiency and 6 (12%) with normal C(cr) showed increased lead excretion rates (95% Cl for the difference, 29-51%, p < 0.001). Mobilizable lead was not significantly correlated with serum or urinary purine concentrations. Hypoxanthine, xanthine and uric acid underexcretion was similar in gout patients with increased or normal cumulative lead excretion rates. The prevalence of atheromatosis and arterial hypertension together was significantly higher in gout patients with renal failure than in patients with normal C(cr) (81 vs. 60%, 95% Cl for the difference, 11-31%, p < 0.005). These results indicate that lead is not a significant contributor to the renal underexcretion of purines in gout. An increased mobilizable lead is not by itself evidence that lead is the cause of the renal insufficiency in patients with primary gout. Atheromatous nephropathy and/or nephroangiosclerosis may explain impaired renal function in patients with primary gout.

Characterizing the genomic evolution of cancer is critical to understanding disease progression and identifying potential therapeutic targets. By examining the clonal hierarchy of genomic lesions in common tumors, it would be possible to reconstruct the path of oncogenic events that drive carcinogenesis. Reliable assessment of such paths from high-throughput genome sequencing data is complicated by the admixture of normal DNA in tumor samples and by reduced data signal for highly subclonal events. We introduce an approach that exploits individuals’ genetic background by using the abundant germline SNP genotype data provided by whole genome sequence coverage to assess the clonality of genomic alterations, including copy number changes, rearrangements, and point mutations. We developed a novel algorithm, CLONET (CLONality Estimate in Tumors), which analyzes patient-specific heterozygous SNP loci (informative SNPs) and mono-allelic somatic deletions to assess levels of stromal DNA admixture and infer the clonal status of each aberration. For every mono allelic deletion, CLONET assesses the allelic fractions of informative SNPs to determine the apparent proportion of normal cells DNA. Next, through a conservative use of simulation-based error estimates, deletions with the lowest proportions of normal DNA reads are considered clonal. For point mutations, the tumor allelic fraction is corrected for stromal DNA admixture level and subclonality is inferred when it differs significantly from the expected value for clonal lesions. Similarly, the proportions of reads that span each side of a putative breakpoint involved in a rearrangement are matched against the expected values. CLONET also addresses tumor aneuploidy by searching for chromosomes with coverage and allelic fractions of informative SNPs not consistent with a diploid genome. CLONET was tested on 55 whole genome sequences from prostate cancers, a highly heterogeneous tumor type, to catalogue the accumulation of somatic alterations during oncogenesis and progression. In 98% of the cases CLONET made confident assessment of admixture and clonality. We observed consistent clonal lesions involving NKX3-1, the 3Mb region between TMPRSS2 and ERG and FOXP1, as well as early point mutations in SPOP and FOXA1. Overall, we observed a higher rate of subclonal protein-coding point mutation versus deletions (p-value < 10−7). We validated this approach by IHC and FISH for predicted clonal and sub-clonal events. A predicted subclonal homozygous deletion of CHD1 was confirmed by FISH that demonstrated the presence of both nuclei with homozygous and with hemizygous deletion of CHD1. Finally, to assess the general validity of CLONET, we analyzed data from 53 additional tumor genomes, including 25 melanomas and 28 lung adenocarcinomas. In summary, our results imply the existence of consensus paths of tumor carcinogenesis that favor dysregulation of cancer genes in a defined sequence. Citation Format: Davide Prandi, Sylvan C. Baca, Michael S. Lawrence, Juan Miguel Mosquera, Alessandro Romanel, Yotam Drier, Kyung Park, Naoki Kitabayashi, Theresa Y. MacDonald, Eliezer Van Allen, Gregory V. Kryukov, Jean-Philippe Theurillat, T. David Soong, Elizabeth Nickerson, Daniel Auclair, Ashutosh Tewari, Himisha Beltran, Robert C. Onofrio, Gunther Boysen, Candace Guiducci, Christopher E. Barbieri, Kristian Cibulskis, Andrey Sivachenko, Scott L. Carter, Gordon Saksena, Douglas Voet, Alex H. Ramos, Wendy Winckler, Michelle Cipicchio, Kristin Ardlie, Philip W. Kantoff, Michael F. Berger, Stacey B. Gabriel, Todd R. Golub, Matthew Meyerson, Eric S. Lander, Olivier Elemento, Gad Getz, Francesca Demichelis, Mark A. Rubin, Levi A. Garraway. Dissecting the clonal hierarchy of cancer-driving genomic lesions. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4017. doi:10.1158/1538-7445.AM2013-4017

A yeast mutant defective in permeases S1 and S2 which transport L-leucine was isolated from a parental strain already deficient in the general amino acid permease, GAP1. The mutant was selected as a spontaneous, trifluoroleucine-resistant (TFLR) strain. Full resistance depended upon the presence of two unlinked mutant genes designated let1 and let2. The let1 mutation completely inactivates the high-affinity leucine transport system defined kinetically as S1. Although the let2 mutation caused a marked decrease in the Jmax of the low-affinity transport system, S2, residual leucine transport in the let1 let2 gap1 mutant had the same KT as in the LET1 LET2 gap1 parent. The mutant exhibited a marked decrease in growth on minimal medium containing leucine, isoleucine or valine as a sole nitrogen source. Moreover, assimilation of methionine, phenylalanine, serine and threonine was decreased, whereas basic and acidic amino acids supported normal growth. This indicates that at least one of the leucine permeases has a fairly broad, but still limited, specificity. Reversion of the gap1 gene restored leucine transport. The revertant was sensitive to TFL when grown on proline but resistant when NH4+ was the nitrogen source. The previously published mutations (shr3, aat1, lup1 or raa) would not be related to either LET1 or LET2.

The polyene antibiotic amphotericin B (AmB) is known to form two types of ionic channels across sterol-containing liposomes, depending on its concentration and time after mixing (Cohen, 1992). In the present study, it is shown that AmB only kills unicellular Leishmania promastigotes (LPs) when aqueous pores permeable to small cations and anions are formed. Changes of membrane potential across ergosterol-containing liposomes and LPs were followed by fluorescence changes of 3,3′ dipropylthiadicarbocyanine (DiSC3(5)). In KCl-loaded liposomes suspended in an iso-osmotic sucrose solution, low AmB concentrations (≤0.1 μm) induced a polarization potential, indicating K+ leakage, but no movement of cations and anions was allowed until AmB concentrations greater than 0.1 μm were added. In agreement with these data, it was found that AmB altered the negative membrane potential held across LPs in a manner consistent with the differential cation/anion selectivity exhibited by the channels formed in liposomes. Thus, LPs suspended in an iso-osmotic sucrose solution did not exhibit any AmB-induced membrane depolarization effect brought about by efflux of anions until 0.1 μm or higher AmB concentrations were added. By contrast, LPs suspended in an iso-osmotic NaCl solution and exposed to 0.05 μm AmB exhibited a nearly total collapse of the negative membrane potential, indicating Na+ entry into the cells. The concentration dependence of the AmB-induced permeability to different salts was also measured across vesicles derived from the plasma membrane of leishmanias (LMVs), by using a rapid mixing technique. At concentrations above 0.1 μm, AmB induced the formation of aqueous pores across LMVs with a positive cooperativity, yielding Hill coefficients between 2 to 3. Measured anion selectivity across such aqueous pores followed the sequence: SCN > NO3 > Cl > I > Br > acetate (SO2− 4 being impermeable). Cell killing by AmB was followed by fluorescence changes of the DNA-binding compound ethidium bromide (EB). At low concentrations (≤0.1 μm), AmB was found to be nonlethal against LPs but, above this concentration, leishmanias were rapidly killed. The rate and extent of such an effect were found to be dependent on the type of cation and anion present in the external aqueous solution. For both NH+ 4 and Na+ salts, the measured rank order of AmB cell killing followed the same sequence that was determined for AmB-induced salt permeation across LMVs. Further, replacement of either extracellular Na+ by choline or Cl− by SO2− 4, or its partial substitution by sucrose, in iso-osmotic conditions, led to a complete inhibition of the killing effect exerted by otherwise lethal AmB concentrations. Finally, it was shown that tetraethylammonium (TEA+), an organic cation that is known to block AmB-induced salt permeation across LMVs was able to retard the time lag observed for EB incorporation across LPs, indicating that this parameter can be taken to represent the time taken for salt accumulation inside the parasites. The present results thus indicate clearly that low AmB concentrations (≤0.1 μm) were able to form across LPs, cation channels that collapsed the parasite membrane potential but are not lytic. At high concentrations (

In 1994, Microbiology Laboratories of ten Portuguese hospitals analysed isolated microorganisms found in blood and urine samples and studied antimicrobial susceptibilities of the most frequent bacterial pathogens. From 63780 blood samples, the most frequent were Staphylococcus spp. and from 69189 urine samples significant numbers of Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa and Candida spp. were isolated. Escherichia coli strains (c.7000) revealed a low percentage of resistance to antibiotics with the exceptions of ampicillin (48%) and co-trimoxazol (25%). Klebsiella pneumoniae isolates (c.2000) revealed important resistance to ampicillin (98%), cephalotin (31%), co-trimoxazol (38%) and gentamicin (28%), while values for 3rd generation cephalosporins varied among hospitals, with several strains showing phenotype of extended-spectrum beta-lactamase. A great variation in resistance values of P. aeruginosa (c.4000) was found in relation to the antibiotics as well as to the hospitals. Resistance to methicillin in S. aureus (c.6000) was high, reaching an average of 47%, and it was even higher with S. epidermidis (c.3000) and S. haemolyticus (c.650). Only vancomycin was always active against these strains. In E. faecalis (c.2500) resistance was of 2% to ampicillin, 35% to gentamicin, 45% to streptomycin and 1% to vancomycin. E. faecium isolates (c.300) showed the most worrying results with 70% resistance to ampicillin, 42% to gentamicin, 59% to streptomycin and 9% (30 strains isolated in 5 hospitals) to vancomycin. Vancomycin resistant strains were also resistant to all other antibiotics.

The aim of this work was to characterize the 4-aminobutyric acid (GABA) transport in the Saccharomyces cerevisiae D27 strain, followed by the study of the relationship between 5-aminolevulinic acid (ALA) and GABA transport systems. It was found that the general amino acid permease (GAP) is not active in D27 strain, suggesting that GABA incorporation should be mediated by PUT4 and UGA4 permeases. However, after kinetic studies only one system was detected. It was also shown that GABA uptake is competitively inhibited by ALA. GABA incorporation is regulated by the carbon source but not by the nitrogen source. When cells were grown in the presence of GABA, its entrance was very low.

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (D-Val-Leu-Arg-Nan) at five different concentrations (10-20 microM) by human urinary kallikrein was studied in the absence and in the presence of increasing concentrations of basic pancreatic trypsin inhibitor (BPTI) (1.35-9.15 nM). The data indicate that the inhibition of human urinary kallikrein by BPTI is not a simple competitive inhibition as reported by others, but that it is a competitive inhibition of the parabolic type, with two inhibitor molecules binding to one enzyme molecule, with the formation of a ternary enzymatic complex. Statistical analysis of the experimental data supports the kinetic model proposed. The calculated values of the constants Ki and Kii were 16.20 nM and 1.10 nM, respectively. It is noteworthy that the KiiKi, i.e., the second BPTI molecule binds to the enzyme with a larger affinity suggesting that this second binding site was probably created or positively modulated as a consequence of the binding of the first BPTI molecule.

We report a 59-year-old female with a dissecting pseudoaneurysm of the allograft hepatic artery, as a delayed complication of percutaneous transluminal angioplasty (PTA). PTA of a severe anastomotic stenosis was successful, but complicated by a dissection involving the allograft hepatic artery. A large dissecting pseiidoaneurysm developed and was incidentally detected during routine sonographic evaluation 14 months after PTA. Because of the extent of the pseudoaneurysm, percutaneous repair or surgical reconstruction was considered impossible. The patient underwent successful retransplantation 1 week after diagnosis. Key words: Liver, transplantation-Hepatic arteries, stenosis or obstruction-Transluminal angioplasty-Aneurysm, hepatic

We compared leishmanicidal activity of five formulations of amphotericin B (AmB). AmB used as Fungizone immediately caused cell lysis, at lower concentrations partial, and at higher concentrations total; the non-lysed cells reassuming growth. AmB formulated with egg-lecithin and bile salt at doses equivalent to those of Fungizone was not active, whereas at higher doses it resulted in a delayed but complete inhibition of cell growth. The activity of liposomal AmB was comparable to that of Fungizone. The time of initial leishmanicidal activity of the five formulations tested can be ascribed to the rate of free, monomeric AmB delivered. Features that are characteristic of the anticellular effect of AmB formulated with egg lecithin and bile salts, a delay in activity, a requirement for higher concentration and total inhibition of cell growth, were also noted.

Today, more than 55% of the world's breast cancer cases are diagnosed in low and middle-income countries and in 2020, more that 70% of the cases will come from the developing nations. In Mexico, breast cancer-specific mortality doubled during the past 20 years, representing the second-leading cause of death in women between 30 and 59 years and the leading cause of cancer related death in the female population. According to statistics, in Mexico a woman dies due to breast cancer every two hours. Even though breast cancer represents a major public health problem in the developing world, knowledge about the genetic and genomic structure of breast tumors in Mexican or Latin American populations is very limited. In the past four years, we have participated in the Slim Initiative of Genomic Medicine (SIGMA) Project, a collaboration between the Carlos Slim Institute of Health, the Broad Institute, and the National Institute of Genomic Medicine in Mexico city. The goal of the SIGMA project is to characterize the genomic basis of common diseases, including several types of cancer. This effort has focused on the application of whole genome and whole exome sequencing of human tumors. In the case of breast cancer, we have analyzed the whole genomes of 22 tumor/normal tissue pairs and the whole exomes of 103 tumor/normal tissues from Mexican and Vietnamese patients. Sequence analysis led to the novel identification of potential loss of function mutations of the CBFB transcription factor, and deletions of its partner RUNX1, an event which has never been previously reported in breast tumors or in any other epithelial tumor. Of clinical relevance, we also identified a somatic translocation involving MAGI3 and AKT3 in a triple negative breast tumor. Ectopic expression of the fusion transcrip leads to constitutive phosphorylation of downstream GSK and loss of contact inhibition. Most importantly, the activity of the fusion protein can be abrogated by an ATP-competitive small molecule inhibitor of AKT, potentially representing a new therapeutic avenue for these patients. In parallel with sequencing, we have also been working on the analysis of somatic DNA copy number aberrations, messenger RNA expression, and microRNA expression patterns in tumors from Mexican patients. Intrinsic breast cancer sub-typing in 125 tumors from Mexican patients showed that 13.6% of the tumors were basal-like, 16.8% were Her2-enriched, 24.8% Luminal A, 34.4% Luminal B and 10.4 normal-like. With microRNA expression, we have identified a group of microRNAs whose role in breast cancer has not been previously described and are currently analyzing differential microRNA expression across tumor sub-types, in particular triple negative tumors, where we have been able to identify at least three different tumor sub-groups based on microRNA expression patterns. Citation Format: Shantanu Banerji, Kristian Cibulskis, Claudia Rangel-Escareño, Kristin K. Brown, Scott L. Carter, Abbie M. Frederick, Michael S. Lawrence, Andrey Y. Sivachenko, Carrie Sougnez, Lihua Zou, Maria L. Cortes, Juan C. Fernandez-Lopez, Shouyong Peng, Kristin G. Ardlie, Daniel Auclair, Veronica Bautista-Piña, Fujiko Duke, Joshua Francis, Joonil Jung, Antonio Maffuz-Aziz, Robert C. Onofrio, Melissa Parkin, Nam H. Pho, Valeria Quintanar-Jurado, Alex H. Ramos, Rosa Rebollar-Vega, Sergio A. Rodríguez-Cuevas, Sandra L. Romero-Cordoba, Steven E. Schumacher, Nicolas Stransky, Kristin M. Thompson, Laura Uribe-Figueroa, Jose Baselga, Rameen Beroukhim, Kornelia Polyak, Dennis C. Sgroi, Andrea L. Richardson, Gerardo Jimenez-Sánchez, Eric S. Lander, Stacey B. Gabriel, Levi A. Garraway, Todd R. Golub, Jorge Meléndez-Zajgla, Alex Toker, Gad Getz, Matthew Meyerson, Alfredo Hidalgo-Miranda. Molecular profiling of breast cancer in Mexico: Identification of novel therapeutic targets through whole genome sequencing analysis. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr PL07-01.

Medulloblastoma is a pediatric brain tumor with poor overall survival and adverse long-term effects from current surgical and radiation treatments. Our group has recently used expression and copy number analysis to define six medulloblastoma subtypes, c1-c6. To assess whether these subgroups contain somatic mutations that encode potential therapeutic or diagnostic targets, we performed whole exome hybrid capture and Illumina sequencing of 94 tumor/normal pairs. For each sample, we sequenced 193,094 exons from 18,863 genes to 126X average coverage. Tumors contained a median 5 silent and 11 non-silent candidate mutations, corresponding to 0.34 non-silent mutations per megabase, a low mutation rate consistent with other pediatric tumors. Overall, 16 genes were mutated at statistically significant frequency and several clustered within subtypes. Eleven tumors harbored mutually-exclusive, likely loss-of-function missense mutations within the helicase domains of candidate oncogene DDX3X (n=7) or SMARCA4 (n=4). These mutations were found in five of seven c6 tumors (3 DDX3X, 2 SMARCA4) of which four had known beta-catenin mutations. Two SMARCA4 mutations were identical and two affected adjacent residues. None of the DDX3X mutations were recurrent, however several are proximal when mapped to a tertiary protein model. To confirm these variants, we are performing deep sequencing of these genes using multiplex PCR (Fluidigm) followed by single-molecule real-time sequencing (PacBio). We are also characterizing the functional effect of DDX3X and SMARCA4 mutations alone and in combination with beta-catenin mutations in medulloblastoma cell lines. Other genes with subtype-associated, loss-of-function mutations include DULLARD in 2 of 9 c1 tumors, PTCH1 in 5 of 16 c3 tumors, and MLL2 in 2 of 3 tumors without any copy number alterations as well as a c1 tumor and two c3 tumors. Several genes are mutated across subtypes, notably tumor suppressors TP53 (n=3), GPS2 (n=3) and SOCS4 (n=2). 19 chromatin remodeling genes including cancer genes BRCA2, KDM5/6A, CREBBP, EP300, BRD4, and MLL3/4 are mutated across 17 tumors from all subtypes. While we have uncovered several subtype-associated mutations, 88% of mutated genes are only altered in a single tumor. This analysis demonstrates the diversity of somatic mutation in medulloblastoma, even within copy number subtypes. To better understand infrequently mutated genes, we will attempt to assemble them into commonly altered gene sets and pathways. We also intend to investigate potentially pathogenic germline variations in each case. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4868. doi:1538-7445.AM2012-4868

The authors report on six pediatric liver transplant recipients for whom allograft tolerance occurred shortly after transplantation (ie, less than 1.5 years). All the patients had associated life-threatening viral complications. They are currently immunocompetent. The tolerant state may be related to the development of a TH2 cytokine pattern.