Uncovering Suitable Reference Proteins for Expression Studies in Human Adipose Tissue with Relevance to Obesity

Protein expression studies based on the two major intra-abdominal human fat depots, the subcutaneous and the omental fat, can shed light into the mechanisms involved in obesity and its co-morbidities. Here we address, for the first time, the identification and validation of reference proteins for data standardization, which are essential for accurate comparison of protein levels in expression studies based on fat from obese and non-obese individuals.To uncover adipose tissue proteins equally expressed either in omental and subcutaneous fat depots (study 1) or in omental fat from non-obese and obese individuals (study 2), we have reanalyzed our previously published data based on two-dimensional fluorescence difference gel electrophoresis. Twenty-four proteins (12 in study 1 and 12 in study 2) with similar expression levels in all conditions tested were selected and identified by mass spectrometry. Immunoblotting analysis was used to confirm in adipose tissue the expression pattern of the potential reference proteins and three proteins were validated: PARK7, ENOA and FAA. Western Blot analysis was also used to test customary loading control proteins. ENOA, PARK7 and the customary loading control protein Beta-actin showed steady expression profiles in fat from non-obese and obese individuals, whilst FAA maintained steady expression levels across paired omental and subcutaneous fat samples.ENOA, PARK7 and Beta-actin are proper reference standards in obesity studies based on omental fat, whilst FAA is the best loading control for the comparative analysis of omental and subcutaneous adipose tissues either in obese and non-obese subjects. Neither customary loading control proteins GAPDH and TBB5 nor CALX are adequate standards in differential expression studies on adipose tissue. The use of the proposed reference proteins will facilitate the adequate analysis of proteins differentially expressed in the context of obesity, an aim difficult to achieve before this study

Adaptation and Mal-Adaptation to Ambient Hypoxia; Andean, Ethiopian and Himalayan Patterns

The study of the biology of evolution has been confined to laboratories and model organisms. However, controlled laboratory conditions are unlikely to model variations in environments that influence selection in wild populations. Thus, the study of “fitness” for survival and the genetics that influence this are best carried out in the field and in matching environments

Evaluating the Cellular Targets of Anti-4-1BB Agonist Antibody during Immunotherapy of a Pre-Established Tumor in Mice

Manipulation of the immune system represents a promising avenue for cancer therapy. Rational advances in immunotherapy of cancer will require an understanding of the precise correlates of protection. Agonistic antibodies against the tumor necrosis factor receptor family member 4-1BB are emerging as a promising tool in cancer therapy, with evidence that these antibodies expand both T cells as well as innate immune cells. Depletion studies have suggested that several cell types can play a role in these immunotherapeutic regimens, but do not reveal which cells must directly receive the 4-1BB signals for effective therapy.We show that re-activated memory T cells are superior to resting memory T cells in control of an 8-day pre-established E.G7 tumor in mice. We find that ex vivo activation of the memory T cells allows the activated effectors to continue to divide and enter the tumor, regardless of antigen-specificity; however, only antigen-specific reactivated memory T cells show any efficacy in tumor control. When agonistic anti-4-1BB antibody is combined with this optimized adoptive T cell therapy, 80% of mice survive and are fully protected from tumor rechallenge. Using 4-1BB-deficient mice and mixed bone marrow chimeras, we find that it is sufficient to have 4-1BB only on the endogenous host alphabeta T cells or only on the transferred T cells for the effects of anti-4-1BB to be realized. Conversely, although multiple immune cell types express 4-1BB and both T cells and APC expand during anti-4-1BB therapy, 4-1BB on cells other than alphabeta T cells is neither necessary nor sufficient for the effect of anti-4-1BB in this adoptive immunotherapy model.This study establishes alphabeta T cells rather than innate immune cells as the critical target in anti-4-1BB therapy of a pre-established tumor. The study also demonstrates that ex vivo activation of memory T cells prior to infusion allows antigen-specific tumor control without the need for reactivation of the memory T cells in the tumor

Regulation of the V-ATPase along the Endocytic Pathway Occurs through Reversible Subunit Association and Membrane Localization

The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V0 and the cytoplasmic V1. Here we found that the ratio of membrane associated V1/Vo varies along the endocytic pathway, the relative abundance of V1 being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments

Evolution and patterns of global health financing 1995-2014 : development assistance for health, and government, prepaid private, and out-of-pocket health spending in 184 countries

Background An adequate amount of prepaid resources for health is important to ensure access to health services and for the pursuit of universal health coverage. Previous studies on global health financing have described the relationship between economic development and health financing. In this study, we further explore global health financing trends and examine how the sources of funds used, types of services purchased, and development assistance for health disbursed change with economic development. We also identify countries that deviate from the trends. Methods We estimated national health spending by type of care and by source, including development assistance for health, based on a diverse set of data including programme reports, budget data, national estimates, and 964 National Health Accounts. These data represent health spending for 184 countries from 1995 through 2014. We converted these data into a common inflation-adjusted and purchasing power-adjusted currency, and used non-linear regression methods to model the relationship between health financing, time, and economic development. Findings Between 1995 and 2014, economic development was positively associated with total health spending and a shift away from a reliance on development assistance and out-of-pocket (OOP) towards government spending. The largest absolute increase in spending was in high-income countries, which increased to purchasing power-adjusted $5221 per capita based on an annual growth rate of 3.0$914 and $267 per capita in 2014, respectively. Spending in low-income countries grew nearly as fast, at 4.6$51 to $120 per capita. In 2014, 59.2$37.6 billion in 2016. Nonetheless, there is a great deal of variation revolving around these averages. 29 countries spend at least 50% more than expected per capita, based on their level of economic development alone, whereas 11 countries spend less than 50% their expected amount. Interpretation Health spending remains disparate, with low-income and lower-middle-income countries increasing spending in absolute terms the least, and relying heavily on OOP spending and development assistance. Moreover, tremendous variation shows that neither time nor economic development guarantee adequate prepaid health resources, which are vital for the pursuit of universal health coverage.Peer reviewe

Cyclic Nucleotide Phosphodiesterases and Compartmentation in Normal and Diseased Heart

International audienceCyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cAMP and cGMP, thereby regulating multiple aspects of cardiac function. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families which are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP controlling specific cell functions in response to various neurohormonal stimuli. In myocardium, the PDE3 and PDE4 families are predominant to degrade cAMP and thereby regulate cardiac excitation-contraction coupling. PDE3 inhibitors are positive inotropes and vasodilators in human, but their use is limited to acute heart failure and intermittent claudication. PDE5 is particularly important to degrade cGMP in vascular smooth muscle, and PDE5 inhibitors are used to treat erectile dysfunction and pulmonary hypertension. However, these drugs do not seem efficient in heart failure with preserved ejection fraction. There is experimental evidence that these PDEs as well as other PDE families including PDE1, PDE2 and PDE9 may play important roles in cardiac diseases such as hypertrophy and heart failure. After a brief presentation of the cyclic nucleotide pathways in cardiac cells and the major characteristics of the PDE superfamily, this chapter will present their role in cyclic nucleotide compartmentation and the current use of PDE inhibitors in cardiac diseases together with the recent research progresses that could lead to a better exploitation of the therapeutic potential of these enzymes in the future

A review of the systematic biology of fossil and living bony-tongue fishes, Osteoglossomorpha (Actinopterygii: Teleostei)

The bony-tongue fishes, Osteoglossomorpha, have been the focus of a great deal of morphological, systematic, and evolutionary study, due in part to their basal position among extant teleostean fishes. This group includes the mooneyes (Hiodontidae), knifefishes (Notopteridae), the abu (Gymnarchidae), elephantfishes (Mormyridae), arawanas and pirarucu (Osteoglossidae), and the African butterfly fish (Pantodontidae). This morphologically heterogeneous group also has a long and diverse fossil record, including taxa from all continents and both freshwater and marine deposits. The phylogenetic relationships among most extant osteoglossomorph families are widely agreed upon. However, there is still much to discover about the systematic biology of these fishes, particularly with regard to the phylogenetic affinities of several fossil taxa, within Mormyridae, and the position of Pantodon. In this paper we review the state of knowledge for osteoglossomorph fishes. We first provide an overview of the diversity of Osteoglossomorpha, and then discuss studies of the phylogeny of Osteoglossomorpha from both morphological and molecular perspectives, as well as biogeographic analyses of the group. Finally, we offer our perspectives on future needs for research on the systematic biology of Osteoglossomorpha

Identifying associations between diabetes and acute respiratory distress syndrome in patients with acute hypoxemic respiratory failure: an analysis of the LUNG SAFE database

Background: Diabetes mellitus is a common co-existing disease in the critically ill. Diabetes mellitus may reduce the risk of acute respiratory distress syndrome (ARDS), but data from previous studies are conflicting. The objective of this study was to evaluate associations between pre-existing diabetes mellitus and ARDS in critically ill patients with acute hypoxemic respiratory failure (AHRF). Methods: An ancillary analysis of a global, multi-centre prospective observational study (LUNG SAFE) was undertaken. LUNG SAFE evaluated all patients admitted to an intensive care unit (ICU) over a 4-week period, that required mechanical ventilation and met AHRF criteria. Patients who had their AHRF fully explained by cardiac failure were excluded. Important clinical characteristics were included in a stepwise selection approach (forward and backward selection combined with a significance level of 0.05) to identify a set of independent variables associated with having ARDS at any time, developing ARDS (defined as ARDS occurring after day 2 from meeting AHRF criteria) and with hospital mortality. Furthermore, propensity score analysis was undertaken to account for the differences in baseline characteristics between patients with and without diabetes mellitus, and the association between diabetes mellitus and outcomes of interest was assessed on matched samples. Results: Of the 4107 patients with AHRF included in this study, 3022 (73.6%) patients fulfilled ARDS criteria at admission or developed ARDS during their ICU stay. Diabetes mellitus was a pre-existing co-morbidity in 913 patients (22.2% of patients with AHRF). In multivariable analysis, there was no association between diabetes mellitus and having ARDS (OR 0.93 (0.78-1.11); p = 0.39), developing ARDS late (OR 0.79 (0.54-1.15); p = 0.22), or hospital mortality in patients with ARDS (1.15 (0.93-1.42); p = 0.19). In a matched sample of patients, there was no association between diabetes mellitus and outcomes of interest. Conclusions: In a large, global observational study of patients with AHRF, no association was found between diabetes mellitus and having ARDS, developing ARDS, or outcomes from ARDS. Trial registration: NCT02010073. Registered on 12 December 2013