Guidelines for the use and interpretation of assays for monitoring autophagy.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Common variants associated with plasma triglycerides and risk for coronary artery disease.

Triglycerides are transported in plasma by specific triglyceride-rich lipoproteins; in epidemiological studies, increased triglyceride levels correlate with higher risk for coronary artery disease (CAD). However, it is unclear whether this association reflects causal processes. We used 185 common variants recently mapped for plasma lipids (P < 5 × 10(-8) for each) to examine the role of triglycerides in risk for CAD. First, we highlight loci associated with both low-density lipoprotein cholesterol (LDL-C) and triglyceride levels, and we show that the direction and magnitude of the associations with both traits are factors in determining CAD risk. Second, we consider loci with only a strong association with triglycerides and show that these loci are also associated with CAD. Finally, in a model accounting for effects on LDL-C and/or high-density lipoprotein cholesterol (HDL-C) levels, the strength of a polymorphism's effect on triglyceride levels is correlated with the magnitude of its effect on CAD risk. These results suggest that triglyceride-rich lipoproteins causally influence risk for CAD.

Prognostic effect of epithelial cell adhesion molecule overexpression in untreated node-negative breast cancer.

PURPOSE: Epithelial cell adhesion molecule (Ep-CAM) recently received increased attention not only as a prognostic factor in breast cancer but also as a potential target for immunotherapy. We examined Ep-CAM expression in 402 consecutive node-negative breast cancer patients with long-term follow-up not treated in the adjuvant setting. EXPERIMENTAL DESIGN: Ep-CAM expression was evaluated by immunostaining. Its prognostic effect was estimated relative to overexpression/amplification of HER-2, histologic grade, tumor size, age, and hormone receptor expression. RESULTS: Ep-CAM status was positive in 106 (26.4%) patients. In multivariate analysis, Ep-CAM status was associated with disease-free survival independent of age, pT stage, histologic grade, estrogen receptor (ER), progesterone receptor (PR), as well as HER2 status (P = 0.028; hazard ratio, 1.60; 95% confidence interval, 1.05-2.44). Recently, so-called triple-negative (HER-2, ER, and PR) breast cancer has received increased attention. We noticed a similar association of Ep-CAM with disease-free survival in the triple-negative group as for the entire cohort. CONCLUSION: In this study of untreated breast cancer patients, Ep-CAM overexpression was associated with poor survival in the entire cohort and in the subgroup of triple-negative breast cancer. This suggests that Ep-CAM may be a well-suited target for specific therapies particularly in HER-2-, ER-, and PR-negative tumors.

Hypolipidemic therapeutics and muscle metabolism

Summary : Lipid metabolism disorders, leading to obesity and cardiovascular diseases, are a major public health issue worldwide. These diseases have been treated by drugs and surgery, leading to tremendous costs and secondary morbidity. The aim of this thesis work is to investigate the mechanisms of actions of a new, micronutrition-based, approach to prevent obesity and cardiovascular diseases. This specific combination of micronutrients, Lipistase, incorporated into any dietary ail can be used in the daily food. Micronutrients are substances used by the living organism in small quantities to maintain physiological homeostasis. However, the human body is not able to produce them and has to obtain them from dietary sources. The combination of micronutrients investigated here, is composed of 26 compounds including trace elements, vitamins, minerals, ails and plant extracts, known to have individually a beneficial effect on lipid metabolism regulation. These specific micronutrients are used for the first time in a combinatorial mode targeting several metabolic pathways for better homeostasis control as opposed to a single target treatment, either chemical or natural. Short and long term studies, in different mouse strains, showed a significant decrease in plasma triglycerides, body weight gain and body fat mass in animals that were fed with a standard diet containing Lipistase. Additionally, a greatly reduced fat accumulation was observed in adipose tissue and liver of Lipistase-treated animals, while lipid and glucose utilization by skeletal muscle was enhanced. Moreover, the size of atherosclerotic plaques was significantly reduced in mice whose masher was treated during pregnancy and suckling, without showing any adverse effect. Finally, Lipistase has been shown to increase longevity by 20%. The control mice that did not receive Lipistase in their diet did not show all these beneficial effects. These micronutrients are used at the lowest dosage ever reported for treating Lipid disorders, resulting in far much lower costs as well as probably a higher safety. This is the first approach being very suitable for an effective large scale prevention policy for obesity and cardiovascular diseases, like iodine in dietary salt has been for goiter. Résumé : Les dysrégulations du métabolisme des lipids, à l'origine d'obésité et de maladies cardiovasculaires, sont un problème de santé publique majeur et mondial. Ces maladies impliquent des traitements médicamenteux et chirurgicaux dont le coût la morbidité secondaire sont très important. Le but de ce travail de thèse est d'étudier les mécanismes d'action d'une nouvelle approche préventive, basée sur la micronutrition. Cette combinaison spécifique de micronutriments, Lipistase, peut être incorporée dans n'importe quelle huile alimentaire et utilisée dans l'alimentation quotidienne. Les micronutrirnents sont des substances essentielles, à très faibles doses, pour le maintien de l'homéostasie physiologique des organismes vivants. Cependant, étant incapable de les synthétiser, le corps humain est dépendant en cela de l'apport alimentaire. La combinaison de micronutriments que nous avons étudié contient 26 composants, incluant des extraits de plantes, des huiles, des vitamines, des métaux et des minéraux, tous connus pour avoir individuellement des effets bénéfiques sur la régulation du métabolisme des lipides. Ces micronutriments spécifiques sont utilisés pour la première fois en mode combinatoire, ciblant ainsi plusieurs voies métaboliques pour un meilleur control de l'homéostasie, par opposition monothérapies chimiques ou naturelles. Des expériences de court et long terme, avec divers modèles de souris, ont montré une diminution significative des taux de triglycérides plasmatiques, de la prise de poids et de la masse graisseuse corporelle chez les animaux qui ont reçu Lipistase dans la nourriture standard. Une accumulation significativement moins importante des graisses a été observée dans le tissu adipeux et hépatique des souris traitées, alors que l'utilisation des lipides et glucose a été favorisée dans le muscle. En outre, la taille des plaques d'athérosclérose aété significativement réduite chez les souris dont la mère a été traitée pendant la grossesse et l'allaitement, sans montrer aucun effet indésirable. Enfin, les souris traitées par Lipistase ont vécu 20% plus longtèmps. Les souris contrôles qui n'ont pas reçu Lipistase dans la nourriture n'ont montré aucun de ces effets bénéfiques. Ces micronutriments sont utilisés au dosage le plus faible jamais rapporté pour le traitement des maladies du métabolisme lipidique, permettant ainsi un coût plus faible et surtout une meilleure sécurité. C'est une approche adéquate pour une politique de prévention de santé publique à large échelle de l'obésité et des maladies cardiovasculaires. C'est en cela et sous bien d'autres aspects, une première dans la prise en charge des maladies du métabolisme lipidique et pourrait même être pour ces dernières ce que l'iode du sel de cuisine a été pour le goitre.

New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk.

Levels of circulating glucose are tightly regulated. To identify new loci influencing glycemic traits, we performed meta-analyses of 21 genome-wide association studies informative for fasting glucose, fasting insulin and indices of beta-cell function (HOMA-B) and insulin resistance (HOMA-IR) in up to 46,186 nondiabetic participants. Follow-up of 25 loci in up to 76,558 additional subjects identified 16 loci associated with fasting glucose and HOMA-B and two loci associated with fasting insulin and HOMA-IR. These include nine loci newly associated with fasting glucose (in or near ADCY5, MADD, ADRA2A, CRY2, FADS1, GLIS3, SLC2A2, PROX1 and C2CD4B) and one influencing fasting insulin and HOMA-IR (near IGF1). We also demonstrated association of ADCY5, PROX1, GCK, GCKR and DGKB-TMEM195 with type 2 diabetes. Within these loci, likely biological candidate genes influence signal transduction, cell proliferation, development, glucose-sensing and circadian regulation. Our results demonstrate that genetic studies of glycemic traits can identify type 2 diabetes risk loci, as well as loci containing gene variants that are associated with a modest elevation in glucose levels but are not associated with overt diabetes.

Estudio de reconocimiento en el Norte del Perù Salaverry – Puerto Pizarro

Breve recuento de observaciones efectuadas e impresiones registradas durante un estudio de reconocimiento de la actividad pesquera en la Región del Norte del Perú, hecho durante la primera quincena de abril de 1962. El viaje se efectuó en camioneta, duró trece días y fueron cubiertos cerca de 3,500 kilómetros desde Lima hasta la frontera con el Ecuador. En total, se visitaron 17 comunidades pesqueras y 4 ciudades y centros de comercio de pescado desde el norte de Chimbote

Estudio de reconocimiento en el Sur del Perú Callao - Ilo

El informe abarca, en forma descriptiva, los datos recopilados durante un viaje realizado por la Región sur del Perú, en el mes de octubre de 1962. Refiere sobre la naturaleza de la pesca y su organización en la región

TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-kappaB dependent induction of cFLIPL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a "death ligand"-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-kappaB and JNK signalling pathways. To determine the role of TGF-beta-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-kappaB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-kappaB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-kappaB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-kappaB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1-/- MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-kappaB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

Appropriateness of colonoscopy in Europe (EPAGE II). Chronic diarrhea and known inflammatory bowel disease.

BACKGROUND AND STUDY AIMS: To summarize the published literature on assessment of appropriateness of colonoscopy for investigation of chronic diarrhea, management of patients with known inflammatory bowel disease (IBD), and for colorectal cancer (CRC) surveillance in such patients, and to report report appropriateness criteria developed by an expert panel, the 2008 European Panel on the Appropriateness of Gastrointestinal Endoscopy, EPAGE II. METHODS: A systematic search of guidelines, systematic reviews, and primary studies regarding the evaluation of chronic diarrhea, the management of IBD, and colorectal cancer surveillance in IBD was performed. The RAND/UCLA Appropriateness Method was applied to develop appropriateness criteria for colonoscopy for these conditions. RESULTS: According to the literature, colonoscopic evaluation may be justified for patients aged > 50 years with recent-onset chronic diarrhea or with alarm symptoms. Surveillance colonoscopy for CRC should be offered to all patients with extensive ulcerative colitis or colonic Crohn's disease of 8 years' duration, and to all patients with less extensive disease of 15 years' duration. Intervals for surveillance colonoscopy depend on duration of evolution, initial diagnosis, and histological findings. The EPAGE II criteria also confirmed the appropriateness of diagnostic colonoscopy for diarrhea of > 4 weeks' duration. They also suggest that, in addition to assessing extent of IBD by colonoscopy, further colonoscopic examination is appropriate in the face of persistent or worsening symptoms. Surveillance colonoscopy in IBD patients was generally appropriate after a lapse of 2 years. In the presence of dysplasia at previous colonoscopy, it was not only appropriate but necessary. CONCLUSIONS: Despite or perhaps because of the limitations of the available published studies, the panel-based EPAGE II (http://www.epage.ch) criteria can help guide appropriate colonoscopy use in the absence of strong evidence from the literature.

Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis

Background: Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. Results: The study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ¿5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. Conclusion: The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy.

The immune space: a concept and template for rationalizing vaccine development.

Abstract Empirical testing of candidate vaccines has led to the successful development of a number of lifesaving vaccines. The advent of new tools to manipulate antigens and new methods and vectors for vaccine delivery has led to a veritable explosion of potential vaccine designs. As a result, selection of candidate vaccines suitable for large-scale efficacy testing has become more challenging. This is especially true for diseases such as dengue, HIV, and tuberculosis where there is no validated animal model or correlate of immune protection. Establishing guidelines for the selection of vaccine candidates for advanced testing has become a necessity. A number of factors could be considered in making these decisions, including, for example, safety in animal and human studies, immune profile, protection in animal studies, production processes with product quality and stability, availability of resources, and estimated cost of goods. The "immune space template" proposed here provides a standardized approach by which the quality, level, and durability of immune responses elicited in early human trials by a candidate vaccine can be described. The immune response profile will demonstrate if and how the candidate is unique relative to other candidates, especially those that have preceded it into efficacy testing and, thus, what new information concerning potential immune correlates could be learned from an efficacy trial. A thorough characterization of immune responses should also provide insight into a developer's rationale for the vaccine's proposed mechanism of action. HIV vaccine researchers plan to include this general approach in up-selecting candidates for the next large efficacy trial. This "immune space" approach may also be applicable to other vaccine development endeavors where correlates of vaccine-induced immune protection remain unknown.

Separability and distillability of multiparticle quantum systems

We present a family of 3-qubit states to which any arbitrary state can be depolarized. We fully classify those states with respect to their separability and distillability properties. This provides a sufficient condition for nonseparability and distillability for arbitrary states. We generalize our results to N-particle states.

Expansion and tissue infiltration of an allospecific CD4+CD25+CD45RO+IL-7Ralphahigh cell population in solid organ transplant recipients.

It has been recently shown (Seddiki, N., B. Santner-Nanan, J. Martinson, J. Zaunders, S. Sasson, A. Landay, M. Solomon, W. Selby, S.I. Alexander, R. Nanan, et al. 2006. J. Exp. Med. 203:1693-1700.) that the expression of interleukin (IL) 7 receptor (R) alpha discriminates between two distinct CD4 T cell populations, both characterized by the expression of CD25, i.e. CD4 regulatory T (T reg) cells and activated CD4 T cells. T reg cells express low levels of IL-7Ralpha, whereas activated CD4 T cells are characterized by the expression of IL-7Ralpha(high). We have investigated the distribution of these two CD4 T cell populations in 36 subjects after liver and kidney transplantation and in 45 healthy subjects. According to a previous study (Demirkiran, A., A. Kok, J. Kwekkeboom, H.J. Metselaar, H.W. Tilanus, and L.J. van der Laan. 2005. Transplant. Proc. 37:1194-1196.), we observed that the T reg CD25(+)CD45RO(+)IL-7Ralpha(low) cell population was reduced in transplant recipients (P < 0.00001). Interestingly, the CD4(+)CD25(+)CD45RO(+)IL-7Ralpha(high) cell population was significantly increased in stable transplant recipients compared with healthy subjects (P < 0.00001), and the expansion of this cell population was even greater in patients with documented humoral chronic rejection compared with stable transplant recipients (P < 0.0001). The expanded CD4(+)CD25(+)CD45RO(+)IL-7Ralpha(high) cell population contained allospecific CD4 T cells and secreted effector cytokines such as tumor necrosis factor alpha and interferon gamma, thus potentially contributing to the mechanisms of chronic rejection. More importantly, CD4(+)IL-7Ralpha(+)and CD25(+)IL-7Ralpha(+) cells were part of the T cell population infiltrating the allograft of patients with a documented diagnosis of chronic humoral rejection. These results indicate that the CD4(+)CD25(+)IL-7Ralpha(+) cell population may represent a valuable, sensitive, and specific marker to monitor allospecific CD4 T cell responses both in blood and in tissues after organ transplantation.

Seventy-five genetic loci influencing the human red blood cell.

Anaemia is a chief determinant of global ill health, contributing to cognitive impairment, growth retardation and impaired physical capacity. To understand further the genetic factors influencing red blood cells, we carried out a genome-wide association study of haemoglobin concentration and related parameters in up to 135,367 individuals. Here we identify 75 independent genetic loci associated with one or more red blood cell phenotypes at P < 10(-8), which together explain 4-9% of the phenotypic variance per trait. Using expression quantitative trait loci and bioinformatic strategies, we identify 121 candidate genes enriched in functions relevant to red blood cell biology. The candidate genes are expressed preferentially in red blood cell precursors, and 43 have haematopoietic phenotypes in Mus musculus or Drosophila melanogaster. Through open-chromatin and coding-variant analyses we identify potential causal genetic variants at 41 loci. Our findings provide extensive new insights into genetic mechanisms and biological pathways controlling red blood cell formation and function.

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.