Hypoxia-mediated repression of pyruvate carboxylase drives immunosuppression.

BACKGROUND: Metabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression. METHODS: We used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity. RESULTS: PC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors. CONCLUSIONS: We present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity.

Picky eaters: selective autophagy in plant cells.

Autophagy, a fundamental cellular process, plays a vital role in maintaining cellular homeostasis by degrading damaged or unnecessary components. While selective autophagy has been extensively studied in animal cells, its significance in plant cells has only recently gained attention. In this review, we delve into the intriguing realm selective autophagy in plants, with specific focus on its involvement in nutrient recycling, organelle turnover, and stress response. Moreover, recent studies have unveiled the interesting interplay between selective autophagy and epigenetic mechanisms in plants, elucidating the significance of epigenetic regulation in modulating autophagy-related gene expression and finely tuning the selective autophagy process in plants. By synthesizing existing knowledge, this review highlights the emerging field of selective autophagy in plant cells, emphasizing its pivotal role in maintaining nutrient homeostasis, facilitating cellular adaptation, and shedding light on the epigenetic regulation that governs these processes. Our comprehensive study provides the way for a deeper understanding of the dynamic control of cellular responses to nutrient availability and stress conditions, opening new avenues for future research in this field of autophagy in plant physiology.

Low-Cost, High-Pressure-Synthesized Oxygen-Entrapping Materials to Improve Treatment of Solid Tumors.

Tumor hypoxia drives resistance to many cancer therapies, including radiotherapy and chemotherapy. Methods that increase tumor oxygen pressures, such as hyperbaric oxygen therapy and microbubble infusion, are utilized to improve the responses to current standard-of-care therapies. However, key obstacles remain, in particular delivery of oxygen at the appropriate dose and with optimal pharmacokinetics. Toward overcoming these hurdles, gas-entrapping materials (GeMs) that are capable of tunable oxygen release are formulated. It is shown that injection or implantation of these materials into tumors can mitigate tumor hypoxia by delivering oxygen locally and that these GeMs enhance responsiveness to radiation and chemotherapy in multiple tumor types. This paper also demonstrates, by comparing an oxygen (O2 )-GeM to a sham GeM, that the former generates an antitumorigenic and immunogenic tumor microenvironment in malignant peripheral nerve sheath tumors. Collectively the results indicate that the use of O2 -GeMs is promising as an adjunctive strategy for the treatment of solid tumors.

Unraveling the genetics of tomato fruit weight during crop domestication and diversification.

KEY MESSAGE: Six novel fruit weight QTLs were identified in tomato using multiple bi-parental populations developed from ancestral accessions. Beneficial alleles at these loci arose in semi-domesticated subpopulations and were likely left behind. This study paves the way to introgress these alleles into breeding programs. The size and weight of edible organs have been strongly selected during crop domestication. Concurrently, human have also focused on nutritional and cultural characteristics of fruits and vegetables, at times countering selective pressures on beneficial size and weight alleles. Therefore, it is likely that novel improvement alleles for organ weight still segregate in ancestral germplasm. To date, five domestication and diversification genes affecting tomato fruit weight have been identified, yet the genetic basis for increases in weight has not been fully accounted for. We found that fruit weight increased gradually during domestication and diversification, and semi-domesticated subpopulations featured high phenotypic and nucleotide diversity. Columella and septum fruit tissues were proportionally increased, suggesting targeted selection. We developed twenty-one F2 populations with parents fixed for the known fruit weight genes, corresponding to putative key transitions from wild to fully domesticated tomatoes. These parents also showed differences in fruit weight attributes as well as the developmental timing of size increase. A subset of populations was targeted for QTL-seq, leading to the identification of six uncloned fruit weight QTLs. Three QTLs, located on chromosomes 1, 2 and 3, were subsequently validated by progeny testing. By exploring the segregation of the known fruit weight genes and the identified QTLs, we estimated that most beneficial alleles in the newly identified loci arose in semi-domesticated subpopulations from South America and were not likely transmitted to fully domesticated landraces. Therefore, these alleles could be incorporated into breeding programs using the germplasm and genetic resources identified in this study.

QTL-seq for identification of loci associated with resistance to Phytophthora crown rot in squash.

Phytophthora capsici Leonian, the causal agent of foliar blight, root rot, fruit rot and crown rot syndromes in squash (Cucurbita moschata), is a devastating pathogen worldwide. Resistance to Phytophthora crown rot in University of Florida breeding line #394-1-27-12 (C. moschata) is conferred by three independent dominant genes (R1R2R3). Availability of DNA markers linked to R1R2R3 genes would allow efficient breeding for Phytophthora crown rot resistance through marker-assisted selection (MAS). The goal of the current study was to identify quantitative trait loci (QTLs) associated with resistance to Phytophthora crown rot in an F2 population (n = 168) derived from a cross between #394-1-27-12 (R) and Butter Bush (S) using QTL-seq bulk segregant analysis. Whole-genome resequencing of the resistant (n = 20) and susceptible (n = 20) bulk segregants revealed ~900,000 single nucleotide polymorphisms distributed across C. moschata genome. Three QTLs significantly (P < 0.05) associated with resistance to Phytophthora crown rot were detected on chromosome 4 (QtlPC-C04), 11 (QtlPC-C11) and 14 (QtlPC-C14). Several markers linked to these QTLs are potential targets for MAS against Phytophthora crown rot in C. moschata. The present study reports the first QTLs associated with Phytophthora crown rot resistance in C. moschata.

Genomic Evidence for Complex Domestication History of the Cultivated Tomato in Latin America.

The process of plant domestication is often protracted, involving underexplored intermediate stages with important implications for the evolutionary trajectories of domestication traits. Previously, tomato domestication history has been thought to involve two major transitions: one from wild Solanum pimpinellifolium L. to a semidomesticated intermediate, S. lycopersicum L. var. cerasiforme (SLC) in South America, and a second transition from SLC to fully domesticated S. lycopersicum L. var. lycopersicum in Mesoamerica. In this study, we employ population genomic methods to reconstruct tomato domestication history, focusing on the evolutionary changes occurring in the intermediate stages. Our results suggest that the origin of SLC may predate domestication, and that many traits considered typical of cultivated tomatoes arose in South American SLC, but were lost or diminished once these partially domesticated forms spread northward. These traits were then likely reselected in a convergent fashion in the common cultivated tomato, prior to its expansion around the world. Based on these findings, we reveal complexities in the intermediate stage of tomato domestication and provide insight on trajectories of genes and phenotypes involved in tomato domestication syndrome. Our results also allow us to identify underexplored germplasm that harbors useful alleles for crop improvement.

Euglycemic diabetic ketoacidosis associated with sodium-glucose cotransporter-2 inhibitor use: a case report and review of the literature.

BACKGROUND: The sodium-glucose cotransporter-2 (SGLT2) inhibitors are the newest class of anti-hyperglycemic medications used in the treatment of diabetes mellitus. Their increasing use has been driven by their apparent cardiovascular and renal benefits. They have been associated with a small but significantly increased risk of diabetic ketoacidosis (DKA). Many of the cases of DKA associated with SGLT2 inhibitor use present with normal or minimally elevated serum glucose levels, often delaying the diagnosis. CASE PRESENTATION: A 44-year-old woman with diabetes mellitus presented to our emergency department complaining of 3 days of generalized weakness. The SGLT2 inhibitor canagliflozin had been added to her medication regimen 4 weeks earlier, and she had stopped using insulin 2 weeks prior to presentation. Laboratory evaluation revealed a metabolic acidosis with an elevated anion gap and the presence of serum acetone, despite a minimally elevated serum glucose of 163 mg/dL. The patient was treated for euglycemic DKA with intravenous infusions of insulin and dextrose, with resolution of her symptoms in 3 days. CONCLUSIONS: The SGLT2 inhibitors are a novel class of anti-hyperglycemic medications that are being used with increasing frequency in the treatment of diabetes mellitus. They are associated with a small but significantly increased risk of DKA. Many of the patients presenting with DKA associated with SGLT2 inhibitor use will have normal or minimally elevated serum glucose levels. This unusual presentation of DKA can be diagnostically challenging.

Levels of empathy, empathy decline and differences between genders in medical students of Cartagena (Colombia) / Niveles de empatía, declinación empática y diferencias entre géneros en estudiantes de medicina en Cartagena, Colombia

INTRODUCTION: Empathy helps to develop an appropriate doctor-patient relationship, which, according to the literature is susceptible to decline. Therefore, the purpose of the present study was to estimate the levels of empathy in general, and the potential for the growth of empathy in medical students. MATERIALS AND METHODS: This exploratory and cross-sectional study included students from the first to fifth academic year of the University Corporation Rafael Nunez (Cartagena, Colombia) (N = 971, n = 756, 77.86% of the population studied). Participants were given the Jefferson Scale of Empathy, Spanish version for medical students, validated and adapted in Colombia. A bifactorial variance analysis (model III) was used to find the mean differences between courses and genders, and the interaction between these two factors. Data was described using simple arithmetic graphs and processed with SPSS 20.0 to estimate the total potential for growth. RESULTS: Differences were found between academic years and gender in empathy in general and in the component of "compassionate care". CONCLUSION: The levels of empathy are relatively low, and the behavior of empathy levels does not match the concept of empathy decline. There are no gender differences and there is a considerable potential for growth of empathy, as well as its components

INTRODUCCIÓN: La empatía permite la adecuada relación médico-paciente y se plantea que es susceptible de disminuir según lo informado en la literatura. El propósito del presente estudio fue estimar los niveles de empatía en general y el potencial para el crecimiento empático en estudiantes de medicina. MATERIALES Y MÉTODOS: Este estudio exploratorio y transversal, incluyó estudiantes desde el primero al quinto año académico de la Corporación Universitaria Rafael Núñez, Cartagena, Colombia (n = 971, n = 756, 77,86% de la población estudiada). Los participantes recibieron la escala de empatía de Jefferson, versión en español para estudiantes de medicina, validada y adaptada en Chile. Se aplicó un análisis de varianza bifactorial (modelo III) para encontrar diferencias de medias entre cursos, géneros y la interacción entre estos 2 factores. Los datos se describieron usando gráficos aritméticos simples y se procesaron con SPSS® Statistics V.20.0 para estimar el potencial total de crecimiento. RESULTADOS: Se encontraron diferencias entre los años académicos y el género en la empatía en general, y en el componente de «atención compasiva». CONCLUSIÓN: Los niveles de empatía son relativamente bajos, y el comportamiento de los niveles de empatía no coincide con el concepto de disminución empática. No hay diferencias de género, y la estimación del potencial de crecimiento de la empatía y la de sus componentes plantea la necesidad de un urgente cambio curricular

Video games as a method of training basic laparoscopic skills. / Videojuegos como método de entrenamiento para las habilidades quirúrgicas laparoscópicas básicas.

INTRODUCTION: The use of video games has been proposed as an alternative to shorten the learning curve of basic laparoscopic skills. However, it is not yet clar how useful this practice is. METHODS: We searched in Epistemonikos, the largest database of systematic reviews in health, which is maintained by screening multiple information sources, including MEDLINE, EMBASE, Cochrane, among others. We extracted data from the systematic reviews, reanalyzed data of primary studies, conducted a meta-analysis and generated a summary of findings table using the GRADE approach. RESULTS AND CONCLUSIONS: We identified three systematic reviews including eight primary studies, of which four were randomized trials. We concluded video games training could help shorten the learning curve of basic laparoscopic visuospatial skills measured in a virtual platform, but the certainty of the available evidence is low.

INTRODUCCIÓN: El uso de videojuegos ha sido propuesto como alternativa para acortar la curva de aprendizaje de las habilidades laparoscópicas básicas. Sin embargo, aún no está clara su real utilidad. MÉTODOS: Realizamos una búsqueda en Epistemonikos, la mayor base de datos de revisiones sistemáticas en salud, la cual es mantenida mediante el cribado de múltiples fuentes de información, incluyendo MEDLINE, EMBASE, Cochrane, entre otras. Extrajimos los datos desde las revisiones identificadas, analizamos los datos de los estudios primarios, realizamos un metanálisis y preparamos una tabla de resumen de los resultados utilizando el método GRADE. RESULTADOS Y CONCLUSIONES: Se identificaron tres revisiones sistemáticas que en conjunto incluyeron ocho estudios primarios, de los cuales cuatro son ensayos aleatorizados. Se concluyó que el entrenamiento mediante el uso de videojuegos podría ayudar a acortar la curva de aprendizaje de habilidades visuoespaciales laparoscópicas básicas medido en una plataforma virtual, sin embargo la certeza de la evidencia disponible es baja.

Acupuncture for rheumatoid arthritis. / Acupuntura para el tratamiento de la artritis reumatoide.

INTRODUCTION: Rheumatoid arthritis is the most common inflammatory arthritis worldwide. Chronic pain leads patients to use complementary therapies, including acupuncture. METHODS: To answer this question we used Epistemonikos, the largest database of systematic reviews in health, which is maintained by screening multiple information sources, including MEDLINE, EMBASE, Cochrane, among others. We extracted data from the systematic reviews, reanalyzed data of primary studies, conducted a meta-analysis and generated a summary of findings table using the GRADE approach. RESULTS AND CONCLUSIONS: We identified 7 systematic reviews including 20 studies overall, all of them randomized trials. We concluded the use of acupuncture probably has little or no impact in rheumatoid arthritis.

INTRODUCCIÓN: La artritis reumatoide es la artritis inflamatoria más común a nivel mundial. El dolor crónico lleva a pacientes a usar terapias complementarias, entre las cuales se ha planteado la acupuntura. MÉTODOS: Para responder esta pregunta utilizamos Epistemonikos, la mayor base de datos de revisiones sistemáticas en salud, la cual es mantenida mediante búsquedas en múltiples fuentes de información, incluyendo MEDLINE, EMBASE, Cochrane, entre otras. Extrajimos los datos desde las revisiones identificadas, reanalizamos los datos de los estudios primarios, realizamos un metanálisis y preparamos una tabla de resúmen de los resultados utilizando el método GRADE. RESULTADOS Y CONCLUSIONES: Identificamos siete revisiones sistemáticas que en conjunto incluyeron 20 estudios primarios, todos correspondientes a ensayos aleatorizados. Concluimos que el uso de acupuntura probablemente tiene un impacto mínimo o nulo en la artritis reumatoide.

Validation of a self-reported work disability questionnaire for ulcerative colitis.

Ulcerative colitis (UC) may severely limit patients' capacity to work. Recently, we validated a work disability questionnaire (WDQ) for Crohn disease. As UC shares clinical characteristics with Crohn disease, we hypothesized that the questionnaire might also be useful for UC. The study was aimed to validate the WDQ for use in UC.Consecutive patients with UC (n = 142, 67 women; age 48 ±â€Š1) completed the UC-WDQ and the inflammatory bowel disease questionnaire-9 (IBDQ-9), and EuroQoL-5D quality-of-life questionnaires. Validation of the UC-WDQ included an assessment of its construct validity, including: discriminant validity, convergent validity, and reproducibility (test-retest). We also calculated the intraclass correlation and the Cronbach alpha.The UC-WDQ is a valid and reliable tool for measuring work disability in patients with UC.

Fruit weight is controlled by Cell Size Regulator encoding a novel protein that is expressed in maturing tomato fruits.

Increases in fruit weight of cultivated vegetables and fruits accompanied the domestication of these crops. Here we report on the positional cloning of a quantitative trait locus (QTL) controlling fruit weight in tomato. The derived allele of Cell Size Regulator (CSR-D) increases fruit weight predominantly through enlargement of the pericarp areas. The expanded pericarp tissues result from increased mesocarp cell size and not from increased number of cell layers. The effect of CSR on fruit weight and cell size is found across different genetic backgrounds implying a consistent impact of the locus on the trait. In fruits, CSR expression is undetectable early in development from floral meristems to the rapid cell proliferation stage after anthesis. Expression is low but detectable in growing fruit tissues and in or around vascular bundles coinciding with the cell enlargement stage of the fruit maturation process. CSR encodes an uncharacterized protein whose clade has expanded in the Solanaceae family. The mutant allele is predicted to encode a shorter protein due to a 1.4 kb deletion resulting in a 194 amino-acid truncation. Co-expression analyses and GO term enrichment analyses suggest association of CSR with cell differentiation in fruit tissues and vascular bundles. The derived allele arose in Solanum lycopersicum var cerasiforme and appears completely fixed in many cultivated tomato's market classes. This finding suggests that the selection of this allele was critical to the full domestication of tomato from its intermediate ancestors.

Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene.

Selection for inflorescence architecture with improved flower production and yield is common to many domesticated crops. However, tomato inflorescences resemble wild ancestors, and breeders avoided excessive branching because of low fertility. We found branched variants carry mutations in two related transcription factors that were selected independently. One founder mutation enlarged the leaf-like organs on fruits and was selected as fruit size increased during domestication. The other mutation eliminated the flower abscission zone, providing "jointless" fruit stems that reduced fruit dropping and facilitated mechanical harvesting. Stacking both beneficial traits caused undesirable branching and sterility due to epistasis, which breeders overcame with suppressors. However, this suppression restricted the opportunity for productivity gains from weak branching. Exploiting natural and engineered alleles for multiple family members, we achieved a continuum of inflorescence complexity that allowed breeding of higher-yielding hybrids. Characterizing and neutralizing similar cases of negative epistasis could improve productivity in many agricultural organisms. VIDEO ABSTRACT.

Development and Validation of the Short Crohn's Disease Work Disability Questionnaire.

BACKGROUND: The aim was to develop and validate a self-reported short Crohn's disease work disability questionnaire (sCDWDQ). METHODS: (1) Development of a shortened questionnaire-Patients' responses to the validation process (n = 108) of a previously developed, 16-item Spanish Crohn's disease work disability questionnaire (CDWDQ) were analyzed using the Rasch model for multiple response items. After this process, a 9-item sCDWDQ was obtained. (2) Validation phase-The validation assessed the questionnaire's convergent validity, discriminant validity, test-retest reproducibility, and internal consistency. Spearman rank correlation, t test, intra-class correlation and Cronbach's alpha were used for the analysis. RESULTS: One hundred fifty-one patients were included in the validation phase. (1) Convergent validity was confirmed by correlations between the sCDWDQ and clinical activity (r = 0.66, P < 0.01), the short inflammatory bowel disease questionnaire IBDQ-9 (r = 0.74, P < 0.001), Euroqol-5D (r = 0.63, P < 0.01), the EuroQol-5D visual analog scale (r = 0.54, P < 0.01), and overall work impairment (r = 0.66, P < 0.01); (2) Discriminant validity-sCDWDQ scores were higher in patients with active disease (20.1 ± 6.3 versus 13.0 ± 3.8 inactive, P < 0.001), in those requiring previous sick leave (19.6 ± 6.9 versus no sick leave 14.2 ± 4.8, P < 0.01) and in those requiring hospitalization (20.0 ± 7.3 [n = 29] versus no hospitalization 14.1 ± 7.3 [n = 90], P < 0.01); (3) Internal consistency was also good (Cronbach's alpha = 0.92); and (4) Reproducibility-sCDWDQ measures obtained 2 weeks apart showed an excellent intraclass correlation coefficient of 0.92 (95% confidence interval, 0.90-0.94). CONCLUSIONS: The self-reported sCDWDQ appears to be a simple, valid, and reliable tool for measuring work disability in Crohn's disease.

IBD-related work disability in the community: Prevalence, severity and predictive factors. A cross-sectional study.

BACKGROUND AND AIMS: Data on the prevalence of work disability in patients with inflammatory bowel disease (IBD) are heterogeneous. As most studies have been performed in selected, often severe, IBD patients, the true prevalence of disability in the community remains controversial. The aim of this cross-sectional study was to evaluate the prevalence and severity of disability and its predictive factors in a community-based IBD population. PATIENTS AND METHODS: Patients recorded in the community-based IBD register at the Hospital Universitario de Burgos were contacted. After informed consent they completed a set of questionnaires including demographic, clinical, disability and quality of life data. The statistical study was performed using SPSS 21. RESULTS: A total of 293 patients were included - 151 Crohn's disease (CD), 142 ulcerative colitis (UC), 137 female, mean age: 45 ± 11 years, mean time since diagnosis: 10.6 ± 11 years. Twelve patients (4.1%) had a work-disability pension. In addition, 93 (32%) of all patients had an officially recognized disability degree, which was generally moderate (n = 73, 25%) or severe (N = 16, 5%). Age, time since IBD diagnosis, CD, perianal disease, incontinence, active disease, the need for anti-TNF or psychological treatment, previous surgeries and the number of diagnostic tests and medical visits in the previous year were predictors of disability. Major predictors of qualifying for a disability pension were age, IBD activity, incontinence, need for biological drugs and ostomy. CONCLUSION: Mild to moderate work disability is frequent in IBD. However, only a minority of patients develop severe disability qualifying them for a pension.

Algunos factores de riesgo de la Hipertensión Arterial en 3 consultorios. Reparto Antonio Guiteras, 2007 / Some risk factors of Hypertension in three medical consults. Antonio Guiteras Repart. 2007

Con el objetivo de determinar algunos factores de riesgo de la Hipertensión Arterial, se realizó un estudio descriptivo de los pacientes hipertensos de 3 consultorios médicos de la familia del Reparto Antonio Guiteras del municipio Bayamo, en el período comprendido entre el 1ro de Enero y 31 de Diciembre del 2007. El universo de trabajo estuvo conformado por los 305 pacientes hipertensos pertenecientes al área estudiada. La información se obtuvo mediante la aplicación de encuestas y entrevistas personales y revisión de historias clínicas personales y familiares donde se recogieron todas las variables utilizadas en el estudio. Los datos se procesaron por el método de conteo simple. El mayor número de hipertensos se encontró en el intervalo de 40-49 años (34,75 por ciento), seguido del intervalo entre 50-59 años (22,29 por ciento), representando estos dos intervalos de edades el 57,04% de los casos estudiados. El sexo más afectado fue el femenino con 163 pacientes (53,5 por ciento). El mayor número de pacientes se encontraron en el estadio I con 195 casos (63,9 por ciento) y el menos afectado el estadio IV con 7 pacientes el (2,2 por ciento).Otros factores de riesgo determinados fueron: el sedentarismo con 70 pacientes el (22,95 por ciento), seguido de la obesidad con 30 pacientes el (9,83 por ciento), el estrés con 27 casos el (8,85 por ciento) y el tabaquismo con 24 pacientes el (7,87 por ciento)(AU)

With the objective to determine some risk factors of hypertension it was performed a descriptive research of hypertensive patients from three medical consults of Antonio Guiteras Repart in Bayamo, during the period between January 1rst and December 31, 2007. The universe was made by the 305 hypertensive patients belonging to the studied area. The information was obtained through the application of personal surveys and interviews and revision of personal and family clinical history including the variables applied in the research. The data was processed through the simple count. The higher number of hypertensive patients appeared between 40 to 49 years (34.75 percent), followed by the ages 50-59 years (22.29 percent). Representing 57.04 percent of the studied cases. The most affected sex was the female, with 163 patients (53.5 percent). The greatest number of patients were in the stage I with 195 cases (63.9 percent) and the less affected was the stage IV with 7 patients (2.2 percent). Other risk factors were: sedentarism with 70 patients (22.95 percent), obesity with 30 patients (9.83 percent), stress with 27 cases (8.85 percent) and tobacco addiction with 24 patients (7.87 percent)(EU)

Subtype-specific genomic alterations define new targets for soft-tissue sarcoma therapy.

Soft-tissue sarcomas, which result in approximately 10,700 diagnoses and 3,800 deaths per year in the United States, show remarkable histologic diversity, with more than 50 recognized subtypes. However, knowledge of their genomic alterations is limited. We describe an integrative analysis of DNA sequence, copy number and mRNA expression in 207 samples encompassing seven major subtypes. Frequently mutated genes included TP53 (17% of pleomorphic liposarcomas), NF1 (10.5% of myxofibrosarcomas and 8% of pleomorphic liposarcomas) and PIK3CA (18% of myxoid/round-cell liposarcomas, or MRCs). PIK3CA mutations in MRCs were associated with Akt activation and poor clinical outcomes. In myxofibrosarcomas and pleomorphic liposarcomas, we found both point mutations and genomic deletions affecting the tumor suppressor NF1. Finally, we found that short hairpin RNA (shRNA)-based knockdown of several genes amplified in dedifferentiated liposarcoma, including CDK4 and YEATS4, decreased cell proliferation. Our study yields a detailed map of molecular alterations across diverse sarcoma subtypes and suggests potential subtype-specific targets for therapy.

Targeted quantitation of overexpressed and endogenous cystic fibrosis transmembrane conductance regulator using multiple reaction monitoring tandem mass spectrometry and oxygen stable isotope dilution.

Cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ion channel in the apical plasma membrane of epithelial cells. Mutations in the gene coding for CFTR cause cystic fibrosis (CF). A major cellular dysfunction is insufficient apical plasma membrane expression of the protein. Its correction is important for developing new CF therapeutics and treatments, which requires a sensitive and precise method for quantifying apical plasma membrane CFTR. We report the first method of liquid chromatography-tandem mass spectrometry for quantifying endogenous and overexpressed CFTR in HT29 and BHK cells. For low level of endogenous CFTR from HT29, the target protein in the cell lysate was enriched by immunoprecipitation using anti-CFTR antibody MAB3484 or M3A7. For overexpressed CFTR from BHK, the cell lysate prepared by differential detergent fractionation or surface biotinylation was used directly without immunoprecipitation. Proteins in the enriched CFTR preparations or cell lysates were digested with proteases, and a surrogate marker peptide designated as CFTR01 (NSILTETLHR) was successfully quantified using the method of multiple reaction monitoring and stable isotope dilution with an (18)O-labeled reference peptide (CFTR01-(18)O(4)) as the internal standard. CFTR quantified in this work ranged from a few tens of picograms to low nanograms per million of cells.

Averagine-scaling analysis and fragment ion mass defect labeling in peptide mass spectrometry.

A method termed as the averagine-scaling analysis (ASA) is proposed for predictive design and selection of chemical reagents for modifying peptides, as well as for facile mass spectral analysis of peptide fragment ions with increased mass defects. The ASA method scales mass spectral data using the mass of the hypothetical averagine residue as reference. The scaling analysis is used in conjunction with a strategy of fragment ion mass defect labeling (FIMDL) for effectively using the broad, unoccupied mass zones in the low m/ z region of mass spectra. The FIMDL approach involves the solution modification of peptide termini with chemical reagents of large mass defects and the gas-phase generation of peptide terminal fragment ions that carry the FIMDL groups. The scaling analysis reveals that iodine has the highest FIMDL efficiency among halogens. Iodine-containing reagents, 4-iodophenylisocyanate and 4-iodophenylisothiocyanate, are used to label primary amines on peptides to demonstrate the scaling analysis. The ASA method successfully distinguishes peptide fragment ions with and without an FIMDL group and specifically and efficiently reduces the data complexity of peptide tandem mass spectra. The combination of ASA with FIMDL extends the instrument suitability for the mass defect analysis from mass spectrometers of ultrahigh mass resolution and accuracy to those of medium ones. This combination is expected to have a profound impact on peptide tandem mass spectrometry.

Discerning the ancestry of European Americans in genetic association studies.

European Americans are often treated as a homogeneous group, but in fact form a structured population due to historical immigration of diverse source populations. Discerning the ancestry of European Americans genotyped in association studies is important in order to prevent false-positive or false-negative associations due to population stratification and to identify genetic variants whose contribution to disease risk differs across European ancestries. Here, we investigate empirical patterns of population structure in European Americans, analyzing 4,198 samples from four genome-wide association studies to show that components roughly corresponding to northwest European, southeast European, and Ashkenazi Jewish ancestry are the main sources of European American population structure. Building on this insight, we constructed a panel of 300 validated markers that are highly informative for distinguishing these ancestries. We demonstrate that this panel of markers can be used to correct for stratification in association studies that do not generate dense genotype data.