Visualisation of human rad52 protein and its complexes with hRad51 and DNA.

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.

Association of the Epstein-Barr virus latent membrane protein 1 with lipid rafts is mediated through its N-terminal region.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (Cdelta55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (Ndelta25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, Ndelta25 inhibited WT LMP1-mediated induction of the transcription factors NF-kappaB and AP-1. Morphological data indicate that Ndelta25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.

Angiotensinergic innervation of the kidney: present knowledge and its significance.

Intrarenal neurotransmission implies the co-release of neuropeptides at the neuro-effector junction with direct influence on parameters of kidney function. The presence of an angiotensin (Ang) II-containing phenotype in catecholaminergic postganglionic and sensory fibers of the kidney, based on immunocytological investigations, has only recently been reported. These angiotensinergic fibers display a distinct morphology and intrarenal distribution, suggesting anatomical and functional subspecialization linked to neuronal Ang II-expression. This review discusses the present knowledge concerning these fibers, and their significance for renal physiology and the pathogenesis of hypertension in light of established mechanisms. The data suggest a new role of Ang II as a co-transmitter stimulating renal target cells or modulating nerve traffic from or to the kidney. Neuronal Ang II is likely to be an independent source of intrarenal Ang II. Further physiological experimentation will have to explore the role of the angiotensinergic renal innervation and integrate it into existing concepts.

It’s Not About the Work Ethic

Journal review:Sascha O. Becker and Ludger Woessmann, “Was Weber Wrong? A Human Capital Theory of Protestant Economic History,” The Quarterly Journal of Economics, May 2009.

Dimensionality of the Rosenberg self-esteem scale and its relationships with the three- and five-factor personality models

The authors investigated the dimensionality of the French version of the Rosenberg Self-Esteem Scale (RSES; Rosenberg, 1965) using confirmatory factor analysis. We tested models of 1 or 2 factors. Results suggest the RSES is a 1-dimensional scale with 3 highly correlated items. Comparison with the Revised NEO-Personality Inventory (NEO-PI-R; Costa, McCrae, & Rolland, 1998) demonstrated that Neuroticism correlated strongly and Extraversion and Conscientiousness moderately with the RSES. Depression accounted for 47% of the variance of the RSES. Other NEO-PI-R facets were also moderately related with self-esteem.

Evolution of insulin sensitivity and its variability in out-of-hospital cardiac arrest (OHCA) patients treated with hypothermia.

INTRODUCTION: Therapeutic hypothermia (TH) is often used to treat out-of-hospital cardiac arrest (OHCA) patients who also often simultaneously receive insulin for stress-induced hyperglycaemia. However, the impact of TH on systemic metabolism and insulin resistance in critical illness is unknown. This study analyses the impact of TH on metabolism, including the evolution of insulin sensitivity (SI) and its variability, in patients with coma after OHCA. METHODS: This study uses a clinically validated, model-based measure of SI. Insulin sensitivity was identified hourly using retrospective data from 200 post-cardiac arrest patients (8,522 hours) treated with TH, shortly after admission to the intensive care unit (ICU). Blood glucose and body temperature readings were taken every one to two hours. Data were divided into three periods: 1) cool (T <35°C); 2) an idle period of two hours as normothermia was re-established; and 3) warm (T >37°C). A maximum of 24 hours each for the cool and warm periods was considered. The impact of each condition on SI is analysed per cohort and per patient for both level and hour-to-hour variability, between periods and in six-hour blocks. RESULTS: Cohort and per-patient median SI levels increase consistently by 35% to 70% and 26% to 59% (P <0.001) respectively from cool to warm. Conversely, cohort and per-patient SI variability decreased by 11.1% to 33.6% (P <0.001) for the first 12 hours of treatment. However, SI variability increases between the 18th and 30th hours over the cool to warm transition, before continuing to decrease afterward. CONCLUSIONS: OCHA patients treated with TH have significantly lower and more variable SI during the cool period, compared to the later warm period. As treatment continues, SI level rises, and variability decreases consistently except for a large, significant increase during the cool to warm transition. These results demonstrate increased resistance to insulin during mild induced hypothermia. Our study might have important implications for glycaemic control during targeted temperature management.

Gas6 and its receptors are implicated in sepsis as modulators of innate immunity

Gas6 downregulates the activation state of macrophages and thereby their production of proinflammatory cytokines induced by various stimuli. We aimed to determine whether Gas6 is involved in sepsis. We measured Gas6 plasma levels in 13 healthy subjects, 29 patients with severe sepsis, and 18 patients with non-infectious inflammatory diseases. Gas6 level was higher in septic patients than in control groups (P 0.0001). The sensitivity and specificity of Gas6 levels to predict fatal outcome were 83% and 88%. We next investigated whether Gas6 affects cytokine production and outcome in experimental models of endotoxemia and peritonitis in wild-type (WT) and Gas6-/- mice. Circulating levels of Gas6 after LPS 25mg/kg i.p. peaked at 1 hour (P<0.001). Similarly, TNF- was higher in Gas6-/- than in WT mice 1 hour after LPS (P<0.05). Furthermore, 62 anti- and pro-inflammatory cytokines were quantified in plasma after LPS injection. Their levels were globally higher in Gas6-/- plasma after LPS, 47/62 cytokines being at least 50% higher in Gas6-/- than in WT plasma after 1 hour. Mortality induced by 25mg/kg LPS was 25% in WT versus 87% in Gas6-/- mice (P<0.05). LPS-induced mortality in Gas6 receptors Axl-/-, Tyro3-/- and Merkd was also enhanced when compared to WT mice (P<0.001). In peritonitis models (cecal ligation and puncture, CLP, and i.p. injection of E. coli), Gas6 plasma levels increased and remained elevated at least 24 hours. CLP increased mortality in Gas6-/- mice. Finally, we explored the role of Gas6 in LPS-treated macrophages. We found that Gas6 was released by LPS-stimulated WT macrophages and that Gas6-/- macrophages produced more TNF- and IL-6 than WT macrophages. Cytokine release by Gas6-/- macrophages was higher than by WT macrophages (cytokine array). Adjunction of recombinant Gas6 to the culture medium of Gas6-/- macrophages diminished the cytokine production to WT levels. In LPS-treated Gas6-/- macrophages, Akt and Erk1/2 phosphorylation was reduced whereas p38 and NF B activation was enhanced. Thus, in septic patients, elevated Gas6 levels were associated with fatal outcome. In mice, they raised in experimental endotoxemia and peritonitis models, and correlated also with sepsis severity. However, Gas6-/- mice survival in these models was reduced compared to WT. Gas6 secreted by macrophages in response to LPS activated Akt and restrained p38 and NF B activation, thereby dampening macrophage activation. Altogether these data suggest that, during endotoxemia, Gas6-/- mice phenotype resembles that of mice which have undergone PI3K inhibition, indicating that Gas6 is a major modulator of innate immunity.

Development of mavoglurant and its potential for the treatment of fragile X syndrome.

Introduction: Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability. With no curative treatment available, current therapeutic approaches are aimed at symptom management. FXS is caused by silencing the FMR1 gene, which encodes FMRP; as loss of FMRP leads to the development of symptoms associated with FXS. Areas covered: In this evaluation, the authors examine the role of the metabotropic glutamate receptor 5 (mGluR5) in the pathophysiology of FXS, and its suitability as a target for rescuing the disease state. Furthermore, the authors review the evidence from preclinical studies of pharmacological interventions targeting mGluR5 in FXS. Lastly, the authors assess the findings from clinical studies in FXS, in particular the use of the Aberrant Behavior Checklist-Community Edition (ABC-C) and the recently developed ABC-C for FXS scale, as clinical endpoints to assess disease modification in this patient population. Expert opinion: There is cautious optimism for the successful treatment of the core behavioral and cognitive symptoms of FXS based on preclinical data in animal models and early studies in humans. However, the association between mGluR5-heightened responsiveness and the clinical phenotype in humans remains to be demonstrated. Many questions regarding the optimal treatment and outcome measures of FXS remain unanswered.

Toxoplasma gondii: a rapid method for the isolation of pure tachyzoites: preliminary characterization of its genome

A rapid and simple technique for the purification of Toxoplasma gondii tachyzoites was developed. Highly purified parasites were obtained from the peritoneal exudates of infected mice by means of two consecutive discontinous sucrose gradients run at low speed (10,000xg, 30 min). Parasites obtained by this method conserved its biological activity. Hybridizations tudies with DNA from healthy mice and from purified tachyzoites preparations demonstrated that Toxoplasma gondii tachyzoites DNA could be obtained with better than 90 per cents purity. Preliminary studies with DNA endonucleases showed the presence in the tachyzoites genome of highly repetitives sequences.

New insights into the phylogenetics and biogeography of Arum (Araceae): unravelling its evolutionary history

The heat- and odour-producing genus Arum (Araceae) has interested scientists for centuries. This long-term interest has allowed a deep knowledge of some complex processes, such as the physiology and dynamics of its characteristic lure-and-trap pollination system, to be built up. However, mainly because of its large distributional range and high degree of morphological variation, species' limits and relationships are still under discussion. Today, the genus comprises 28 species subdivided into two subgenera, two sections and six subsections. In this study, the phylogeny of the genus is inferred on the basis of four plastid regions, and the evolution of several morphological characters is investigated. Our phylogenetic hypothesis is not in agreement with the current infrageneric classification of the genus and challenges the monophyly of several species. This demonstrates the need for a new infrageneric classification based on characters reflecting the evolution of this enigmatic genus. To investigate the biogeography of Arum deeply, further spatiotemporal analyses were performed, addressing the importance of the Mediterranean basin in the diversification of Arum. Our results suggest that its centre of origin was the European-Aegean region, and that major diversification happened during the last 10 Myr.

Design and evaluation of a solid sampler for the monitoring of airborne 1,6-hexamethylene diisocyanate (HDI) and its prepolymers in 2-component spray painting

An active, solvent-free solid sampler was developed for the collection of 1,6-hexamethylene diisocyanate (HDI) aerosol and prepolymers. The sampler was made of a filter impregnated with 1-(2-methoxyphenyl)piperazine contained in a filter holder. Interferences with HDI were observed when a set of cellulose acetate filters and a polystyrene filter holder were used; a glass fiber filter and polypropylene filter cassette gave better results. The applicability of the sampling and analytical procedure was validated with a test chamber, constructed for the dynamic generation of HDI aerosol and prepolymers in commercial two-component spray paints (Desmodur(R) N75) used in car refinishing. The particle size distribution, temporal stability, and spatial uniformity of the simulated aerosol were established in order to test the sample. The monitoring of aerosol concentrations was conducted with the solid sampler paired to the reference impinger technique (impinger flasks contained 10 mL of 0.5 mg/mL 1-(2-methoxyphenyl)piperazine in toluene) under a controlled atmosphere in the test chamber. Analyses of derivatized HDI and prepolymers were carried out by using high-performance liquid chromatography and ultraviolet detection. The correlation between the solvent-free and the impinger techniques appeared fairly good (Y = 0.979X - 0.161; R = 0.978), when the tests were conducted in the range of 0.1 to 10 times the threshold limit value (TLV) for HDI monomer and up to 60-mu-g/m3 (3 U.K. TLVs) for total -N = C = O groups.

High-throughput hydrophilic interaction chromatography coupled to tandem mass spectrometry for the optimized quantification of the anti-Gram-negatives antibiotic colistin A/B and its pro-drug colistimethate.

Colistin is a last resort's antibacterial treatment in critically ill patients with multi-drug resistant Gram-negative infections. As appropriate colistin exposure is the key for maximizing efficacy while minimizing toxicity, individualized dosing optimization guided by therapeutic drug monitoring is a top clinical priority. Objective of the present work was to develop a rapid and robust HPLC-MS/MS assay for quantification of colistin plasma concentrations. This novel methodology validated according to international standards simultaneously quantifies the microbiologically active compounds colistin A and B, plus the pro-drug colistin methanesulfonate (colistimethate, CMS). 96-well micro-Elution SPE on Oasis Hydrophilic-Lipophilic-Balanced (HLB) followed by direct analysis by Hydrophilic Interaction Liquid Chromatography (HILIC) with Ethylene Bridged Hybrid - BEH - Amide phase column coupled to tandem mass spectrometry allows a high-throughput with no significant matrix effect. The technique is highly sensitive (limit of quantification 0.014 and 0.006μg/mL for colistin A and B), precise (intra-/inter-assay CV 0.6-8.4%) and accurate (intra-/inter-assay deviation from nominal concentrations -4.4 to +6.3%) over the clinically relevant analytical range 0.05-20μg/mL. Colistin A and B in plasma and whole blood samples are reliably quantified over 48h at room temperature and at +4°C (<6% deviation from nominal values) and after three freeze-thaw cycles. Colistimethate acidic hydrolysis (1M H2SO4) to colistin A and B in plasma was completed in vitro after 15min of sonication while the pro-drug hydrolyzed spontaneously in plasma ex vivo after 4h at room temperature: this information is of utmost importance for interpretation of analytical results. Quantification is precise and accurate when using serum, citrated or EDTA plasma as biological matrix, while use of heparin plasma is not appropriate. This new analytical technique providing optimized quantification in real-life conditions of the microbiologically active compounds colistin A and B offers a highly efficient tool for routine therapeutic drug monitoring aimed at individualizing drug dosing against life-threatening infections.

Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. Copyright © 2014 John Wiley & Sons, Ltd.

The murine model of infection with Leishmania major and its importance for the deciphering of mechanisms underlying differences in Th cell differentiation in mice from different genetic backgrounds.

Mice from the majority of inbred strains are resistant to infection by Leishmania major, an obligate intracellular protozoan parasite of macrophages in the mammalian host. In contrast, mice from BALB strains are unable to control infection and develop progressive disease. In this model of infection, genetically determined resistance and susceptibility have been clearly shown to result from the appearance of parasite-specific CD4+ T helper 1 or T helper 2 cells, respectively. This murine model of infection is considered as one of the best experimental systems for the study of the mechanisms operating in vivo at the initiation of polarised T helper 1 and T helper 2 cell maturation. Among the several factors influencing Th cell development, cytokines themselves critically regulate this process. The results accumulated during the last years have clarified some aspects of the role played by cytokines in Th cell differentiation. They are providing critical information that may ultimately lead to the rational devise of means by which to tailor immune responses to the effector functions that are most efficient in preventing and/or controlling infections with pathogens.

A medium for inducing conversion of Histoplasma capsulatum var. capsulatum into its yeast-like form

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form. Achieving this conversion, however, has been problematical for researchers. The present study tested conversion rates in ten Histoplasma capsulatum var. capsulatum strains using seven culture media, four of wich were conventional and three novel. One of our novel media, MLGema, induced complete conversion, of two strains within five days of incubation at 35 degrees centigrades, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions. MLGema is also inexpensive and easy to produce.