The Toll-Like Receptor Gene Family Is Integrated into Human DNA Damage and p53 Networks

In recent years the functions that the p53 tumor suppressor plays in human biology have been greatly extended beyond “guardian of the genome.” Our studies of promoter response element sequences targeted by the p53 master regulatory transcription factor suggest a general role for this DNA damage and stress-responsive regulator in the control of human Toll-like receptor (TLR) gene expression. The TLR gene family mediates innate immunity to a wide variety of pathogenic threats through recognition of conserved pathogen-associated molecular motifs. Using primary human immune cells, we have examined expression of the entire TLR gene family following exposure to anti-cancer agents that induce the p53 network. Expression of all TLR genes, TLR1 to TLR10, in blood lymphocytes and alveolar macrophages from healthy volunteers can be induced by DNA metabolic stressors. However, there is considerable inter-individual variability. Most of the TLR genes respond to p53 via canonical as well as noncanonical promoter binding sites. Importantly, the integration of the TLR gene family into the p53 network is unique to primates, a recurrent theme raised for other gene families in our previous studies. Furthermore, a polymorphism in a TLR8 response element provides the first human example of a p53 target sequence specifically responsible for endogenous gene induction. These findings—demonstrating that the human innate immune system, including downstream induction of cytokines, can be modulated by DNA metabolic stress—have many implications for health and disease, as well as for understanding the evolution of damage and p53 responsive networks

Discovery of New Compounds Active against Plasmodium falciparum by High Throughput Screening of Microbial Natural Products

Due to the low structural diversity within the set of antimalarial drugs currently available in the clinic and the increasing number of cases of resistance, there is an urgent need to find new compounds with novel modes of action to treat the disease. Microbial natural products are characterized by their large diversity provided in terms of the chemical complexity of the compounds and the novelty of structures. Microbial natural products extracts have been underexplored in the search for new antiparasitic drugs and even more so in the discovery of new antimalarials. Our objective was to find new druggable natural products with antimalarial properties from the MEDINA natural products collection, one of the largest natural product libraries harboring more than 130,000 microbial extracts. In this work, we describe the optimization process and the results of a phenotypic high throughput screen (HTS) based on measurements of Plasmodium lactate dehydrogenase. A subset of more than 20,000 extracts from the MEDINA microbial products collection has been explored, leading to the discovery of 3 new compounds with antimalarial activity. In addition, we report on the novel antiplasmodial activity of 4 previously described natural productsThis work was supported by the Junta de Andalucía [BIO-199, P09-CVI- 5367], the VI Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica 2008-2011, Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET FIS Network: RD12/0018/0017),the Plan Nacional (SAF2013-48999-R), the FEDER funds from the EU and the PARAMET network (FP7-PEOPLE-2011-ITN. GA290080) to DG-P. Research of FV and OG was supported by the Instituto de Salud Carlos III-Subdirección General de Redes y Centros de Investigación Cooperativa-Red de Investigación Cooperativa en Enfermedades Tropicales (RICET FIS Network: RD12/0018/0005) and the FEDER funds from the EU and the PARAMET network (FP7-PEOPLE-2011-ITN. GA290080). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

The Family Physicians in the Film Discourse

Objective: To study the figure of the family physicians in the cinematographic medium. Design: Qualitative study through analysis of documentary content. Location: Primary Care Participants and/or contexts: Selection from cinematographic databases of films in which doctors intervened excluding those in which they performed their functions in the hospital or in areas other than Primary Care or in which their role in the story plot was little relevant Method: Sample selection by purpose by the research team. Individual screening of the selected films and weekly group discussion was done. Each session was recorded in audio extracting sequences and paragraphs of interest that were later codified and classified to obtain relevant categories of meaning. Following the "constant comparative method" each category was evaluated at each step, emerging new hypotheses and topics of research and debate. Results: We selected 21 of the 162 medical films analyzed. We coded the documentary material and obtained 13 categories of meaning, among them: Physician's cinematic image. The family physicians vs other specialists. Professional qualification. Personal versus professional life. Doctor's position before death. Doctor?pacient relationship. Degree of commitment. Professional's position in the health system and its assessment by the community. Conclusions: The family physicians is represented as a polyvalent part of the community, committed to patients, with an unequal level of education and a very personal view of illness and death.Objetivo: estudiar la figura del médico de familia en el medio cinematográfico. Diseño: estudio cualitativo mediante análisis de contenido documental. Emplazamiento: Atención Primaria. Participantes y/o contextos: selección a partir de bases de datos cinematográficas de películas en las que intervenían médicos excluyendo aquellas en las que ejercían sus funciones en el hospital o en ámbitos distintos de la Atención Primaria o en las que su papel en la trama argumental era poco relevante. Método: selección muestral por propósito por parte del equipo investigador. Se procedió al visionado individual de las películas seleccionadas y a la discusión semanal en grupo. Cada sesión se grabó en audio extrayendo secuencias y párrafos de interés que posteriormente fueron codificados y clasificados para obtener categorías de significado relevantes. Siguiendo el “método comparativo constante” cada categoría fue evaluada a cada paso surgiendo nuevas hipótesis y temas de investigación y debate. Resultados: de las 162 películas de médicos analizadas se seleccionaron 21. Codificamos el material documental y obtuvimos 13 categorías de significado, entre ellas: Imagen cinematográfica del médico. El médico de familia vs otros especialistas. Cualificación profesional. Vida personal frente a profesional. Posición del médico ante la muerte. Relación médico-paciente. Grado de compromiso. Posición del profesional en el sistema sanitario y su valoración por parte de la comunidad. Conclusiones: el médico de Familia es representado como parte polivalente de la comunidad, comprometido con los pacientes, con un desigual nivel de formación y con una visión muy personal frente a la enfermedad y la muerte

Determinants of subject visit participation in a prospective cohort study of HTLV infection

<p>Abstract</p> <p>Background</p> <p>Understanding participation in a prospective study is crucial to maintaining and improving retention rates. In 1990–92, following attempted blood donation at five blood centers, we enrolled 155 HTLV-I, 387 HTLV-II and 799 HTLV seronegative persons in a long-term prospective cohort.</p> <p>Methods</p> <p>Health questionnaires and physical exams were administered at enrollment and 2-year intervals through 2004. To examine factors influencing attendance at study visits of the cohort participants we calculated odds ratios (ORs) with generalized estimated equations (GEE) to analyze fixed and time-varying predictors of study visit participation.</p> <p>Results</p> <p>There were significant independent associations between better visit attendance and female gender (OR = 1.31), graduate education (OR = 1.86) and income > $75,000 (OR = 2.68). Participants at two centers (OR = 0.47, 0.67) and of Black race/ethnicity (OR = 0.61) were less likely to continue. Higher subject reimbursement for interview was associated with better visit attendance (OR = 1.84 for$25 vs. $10). None of the health related variables (HTLV status, perceived health status and referral to specialty diagnostic exam for potential adverse health outcomes) significantly affected participation after controlling for demographic variables.</p> <p>Conclusion</p> <p>Increasing and maintaining participation by minority and lower socioeconomic status participants is an ongoing challenge in the study of chronic disease outcomes. Future studies should include methods to evaluate attrition and retention, in addition to primary study outcomes, including qualitative analysis of reasons for participation or withdrawal.</p

Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA

The tumour suppressor p53 is a transcription factor that binds DNA in the vicinity of the genes it controls. The affinity of p53 for specific binding sites relative to other DNA sequences is an inherent driving force for specificity, all other things being equal. We measured the binding affinities of systematically mutated consensus p53 DNA-binding sequences using automated fluorescence anisotropy titrations. Based on measurements of the effects of every possible single base-pair substitution of a consensus sequence, we defined the DNA sequence with the highest affinity for full-length p53 and quantified the effects of deviation from it on the strength of protein–DNA interaction. The contributions of individual nucleotides were to a first approximation independent and additive. But, in some cases we observed significant deviations from additivity. Based on affinity data, we constructed a binding predictor that mirrored the existing p53 consensus sequence definition. We used it to search for high-affinity binding sites in the genome and to predict the effects of single-nucleotide polymorphisms in these sites. Although there was some correlation between the Kd and biological function, the spread of the Kds by itself was not sufficient to explain the activation of different pathways by changes in p53 concentration alone

Probing the Functional Impact of Sequence Variation on p53-DNA Interactions Using a Novel Microsphere Assay for Protein-DNA Binding with Human Cell Extracts

The p53 tumor suppressor regulates its target genes through sequence-specific binding to DNA response elements (REs). Although numerous p53 REs are established, the thousands more identified by bioinformatics are not easily subjected to comparative functional evaluation. To examine the relationship between RE sequence variation—including polymorphisms—and p53 binding, we have developed a multiplex format microsphere assay of protein-DNA binding (MAPD) for p53 in nuclear extracts. Using MAPD we measured sequence-specific p53 binding of doxorubicin-activated or transiently expressed p53 to REs from established p53 target genes and p53 consensus REs. To assess the sensitivity and scalability of the assay, we tested 16 variants of the p21 target sequence and a 62-multiplex set of single nucleotide (nt) variants of the p53 consensus sequence and found many changes in p53 binding that are not captured by current computational binding models. A group of eight single nucleotide polymorphisms (SNPs) was examined and binding profiles closely matched transactivation capability tested in luciferase constructs. The in vitro binding characteristics of p53 in nuclear extracts recapitulated the cellular in vivo transactivation capabilities for eight well-established human REs measured by luciferase assay. Using a set of 26 bona fide REs, we observed distinct binding patterns characteristic of transiently expressed wild type and mutant p53s. This microsphere assay system utilizes biologically meaningful cell extracts in a multiplexed, quantitative, in vitro format that provides a powerful experimental tool for elucidating the functional impact of sequence polymorphism and protein variation on protein/DNA binding in transcriptional networks

Human single-nucleotide polymorphisms alter p53 sequence-specific binding at gene regulatory elements

p53 coordinates the expression of an intricate network of genes in response to stress signals. Sequence-specific DNA binding is essential for p53-mediated tumor suppression. We evaluated the impact of single-nucleotide polymorphisms (SNPs) in p53 response elements (p53RE) on DNA binding and gene expression in response to DNA damage. Using a bioinformatics approach based on incorporating p53 binding strength into a position weight matrix, we selected 32 SNPs in putative and validated p53REs. The microsphere assay for protein–DNA binding (MAPD) and allele-specific expression analysis was employed to assess the impact of SNPs on p53-DNA binding and gene expression, respectively. Comparing activated p53 binding in nuclear extracts from doxorubicin- or ionizing radiation (IR)-treated human cells, we observed little difference in binding profiles. Significant p53 binding was observed for most polymorphic REs and several displayed binding comparable to the p21 RE. SNP alleles predicted to lower p53 binding indeed reduced binding in 25 of the 32 sequences. Chromatin immunoprecipitation-sequencing in lymphoblastoid cells confirmed p53 binding to seven polymorphic p53 REs in response to doxorubicin. In addition, five polymorphisms were associated with altered gene expression following doxorubicin treatment. Our findings demonstrate an effective strategy to identify and evaluate SNPs that may alter p53-mediated stress responses

An emerging role of mTOR in lipid biosynthesis

Lipid biosynthesis is essential for the maintenance of cellular homeostasis. The lipids produced by cells (glycerolipids, fatty acids, phospholipids, cholesterol, and sphingolipids) are used as an energy source/reserve, as building blocks for membrane biosynthesis, as precursor molecules for the synthesis of various cellular products, and as signaling molecules. Defects in lipid synthesis or processing contribute to the development of many diseases, including obesity, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, and cancer. Studies published over the last few years have shown that the target of rapamycin (TOR), a conserved serine/threonine kinase with an important role in regulating cell growth, controls lipid biosynthesis through various mechanisms. Here, we review these findings and briefly discuss their potential relevance for human health and disease