ATPergic signaling disruption in human sepsis as a potential source of biomarkers for clinical use.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthy and ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.

TLR4 expression and functionality are downregulated in glioblastoma cells and in tumor-associated macrophages: A new mechanism of immune evasion?

Glioblastoma (GB) is the most common and aggressive form of primary brain tumor, in which the presence of an inflammatory environment, composed mainly by tumor-associated macrophages (TAMs), is related to its progression and development of chemoresistance. Toll-Like Receptors (TLRs) are key components of the innate immune system and their expression in both tumor and immune-associated cells may impact the cell communication in the tumor microenvironment (TME), further modeling cancer growth and response to therapy. Here, we investigated the participation of TLR4-mediated signaling as a mechanism of induced-immune escape in GB. Initially, bioinformatics analysis of public datasets revealed that TLR4 expression is lower in GB tumors when compared to astrocytomas (AST), and in a subset of TAMs. Further, we confirmed that TLR4 expression is downregulated in chemoresistant GB, as well as in macrophages co-cultured with GB cells. Additionally, TLR4 function is impaired in those cells even following stimulation with LPS, an agonist of TLR4. Finally, experiments performed in a cohort of clinical primary and metastatic brain tumors indicated that the immunostaining of TLR4 and CD45 are inversely proportional, and confirmed the low TLR4 expression in GBs. Interestingly, the cytoplasmic/nuclear pattern of TLR4 staining in cancer tissues suggests additional roles of this receptor in carcinogenesis. Overall, our data suggest the downregulation of TLR4 expression and activity as a strategy for GB-associated immune escape. Additional studies are necessary to better understand TLR4 signaling in TME in order to improve the benefits of immunotherapy based on TLR signaling.

Immortalization of Mesenchymal Stromal Cells by TERT Affects Adenosine Metabolism and Impairs their Immunosuppressive Capacity.

Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.

Polychromatic emission in a wide energy range from InP-InAs-InP multi-shell nanowires.

InP-InAs-InP multi-shell nanowires (NWs) were grown in the wurtzite (WZ) or zincblende (ZB) crystal phase and their photoluminescence (PL) properties were investigated at low temperature (≈6 K) for different measurement geometries. PL emissions from the NWs were carefully studied in a wide energy range from 0.7 to 1.6 eV. The different features observed in the PL spectra for increasing energies are attributed to four distinct emitting domains of these nano-heterostructures: the InAs island (axially grown), the thin InAs capping shell (radially grown), the crystal-phase quantum disks arising from the coexistence of InP ZB and WZ segments in the same NW, and the InP portions of the NW. These results provide a useful frame for the rational implementation of InP-InAs-InP multi-shell NWs containing various quantum confined domains as polychromatic optically active components in nanodevices for quantum information and communication technologies.

Ambulatory blood pressure monitoring phenotypes among individuals with and without diabetes taking antihypertensive medication: the Jackson Heart Study.

Ambulatory blood pressure monitoring (ABPM) can detect phenotypes associated with increased cardiovascular disease (CVD) risk. Diabetes is associated with increased CVD risk but few data are available documenting whether blood pressure (BP) phenotypes, detected by ABPM, differ between individuals with versus without diabetes. We conducted a cross-sectional analysis of 567 participants in the Jackson Heart Study, a population-based study of African Americans, taking antihypertensive medication to evaluate the association between diabetes and ABPM phenotypes. Two clinic BP measurements were taken at baseline following a standardized protocol. ABPM was performed for 24 h following the clinic visit. ABPM phenotypes included daytime, sustained, nocturnal and isolated nocturnal hypertension, a non-dipping BP pattern, and white coat, masked and masked isolated nocturnal hypertension. Diabetes was defined as fasting glucose ⩾126 mg dl-1, haemoglobin A1c ⩾6.5% (48 mmol mol-1) or use of insulin or oral hypoglycaemic medications. Of the included participants (mean age 62.3 years, 71.8% female), 196 (34.6%) had diabetes. After multivariable adjustment, participants with diabetes were more likely to have daytime hypertension (prevalence ratio (PR): 1.32; 95% confidence interval (CI): 1.09-1.60), masked hypertension (PR: 1.46; 95% CI: 1.11-1.93) and masked isolated nocturnal hypertension (PR: 1.39; 95% CI: 1.02-1.89). Although nocturnal hypertension was more common among participants with versus without diabetes, this association was not present after adjustment for daytime systolic BP. Diabetes was not associated with the other ABPM phenotypes investigated. This study highlights the high prevalence of ABPM phenotypes among individuals with diabetes taking antihypertensive medication.

Excitation energy-transfer in functionalized nanoparticles: Going beyond the Förster approach.

We develop a novel approach to treat excitation energy transfer in hybrid nanosystems composed by an organic molecule attached to a semiconductor nanoparticle. Our approach extends the customary Förster theory by considering interaction between transition multipole moments of the nanoparticle at all orders and a point-like transition dipole moment representing the molecule. Optical excitations of the nanoparticle are described through an envelope-function configuration interaction method for a single electron-hole pair. We applied the method to the prototypical case of a core/shell CdSe/ZnS semiconductor quantum dot which shows a complete suppression of the energy transfer for specific transitions which could not be captured by Förster theory.

Identification of valid endogenous control genes for determining gene expression in C6 glioma cell line treated with conditioned medium from adipose-derived stem cell.

INTRODUCTION: There is growing evidence that mesenchymal stem cells (MSCs) can be important players in the tumor microenvironment. They can affect the glioma progression through the modulation of different genes. This modulation can be evaluated through a very useful model, treating the tumor cells with MSC-conditioned medium. However, for an accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is a prerequisite. METHODS: We performed a systematic review in an attempt to find a reference gene to use when analyzing gene expression in C6 glioma cells lines. Considering that we were not able to find a reference gene originated by an appropriate validation, in this study we evaluated candidate genes to be used as reference gene in C6 cells under different treatments with adipose-derived stem cells conditioned medium (CM-ADSCs). ß-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine-guanine phosphoribosyltransferase I (HPRT-1); TATA box binding protein (TBP) and beta-2-microglobulin (B2M) were evaluated by real-time reverse transcription PCR (RT-qPCR). The mean Cq, the maximum fold change (MFC) and NormFinder software were used for reference gene evaluation and selection. RESULTS: The GAPDH and ACTB genes have been the most widely used reference genes to normalize among the different investigated genes in our review, however, controversially these genes underwent a substantial variability among the genes evaluated in the present work. Individually, TBP gene was more stable when compared with other genes analyzed and the combination of TBP and HPRT-1 was even more stable. CONCLUSION: These results evidence the importance of appropriate validation of reference genes before performing qPCR experiments. Besides, our data will contribute with researchers that work analyzing the role of ADSCs in glioma microenvironment through gene expression.

Unintentional high-density p-type modulation doping of a GaAs/AlAs core-multishell nanowire.

Achieving significant doping in GaAs/AlAs core/shell nanowires (NWs) is of considerable technological importance but remains a challenge due to the amphoteric behavior of the dopant atoms. Here we show that placing a narrow GaAs quantum well in the AlAs shell effectively getters residual carbon acceptors leading to an unintentional p-type doping. Magneto-optical studies of such a GaAs/AlAs core-multishell NW reveal quantum confined emission. Theoretical calculations of NW electronic structure confirm quantum confinement of carriers at the core/shell interface due to the presence of ionized carbon acceptors in the 1 nm GaAs layer in the shell. Microphotoluminescence in high magnetic field shows a clear signature of avoided crossings of the n = 0 Landau level emission line with the n = 2 Landau level TO phonon replica. The coupling is caused by the resonant hole-phonon interaction, which points to a large two-dimensional hole density in the structure.

Human platelets express authentic CB1 and CB2 receptors.

In the last decade, the neurovascular effects exerted by endocannabinoids (eCBs) have attracted growing interest, because they hold the promise to open new avenues of therapeutic intervention against major causes of death in Western society. Several actions of eCBs are mediated by type-1 (CB1) or type-2 (CB2) cannabinoid receptors, yet there is no clear evidence of the presence of these proteins in platelets. To demonstrate that CB1 and CB2 are expressed in human platelets, we analyzed their protein level by Western blotting and ELISA, visualized their cellular localization by confocal microscopy, and ascertained their functionality by binding assays. We found that CB1, and to a lesser extent CB2, are expressed in highly purified human platelets. Both receptor subtypes were predominantly localized inside the cell, thus explaining why they might remain undetected in preparations of plasma membranes. The identification of authentic CB1 and CB2 in human platelets supports the potential exploitation of selective agonists or antagonists of these receptors as novel therapeutics to combat neurovascular disorders. It seems remarkable that some of these substances have been already used in humans to treat disease states.

LSD1-mediated demethylation of histone H3 lysine 4 triggers Myc-induced transcription.

Myc is a transcription factor that significantly contributes to cancer progression by modulating the expression of important genes through binding to a DNA sequence, CACGTG, called E-box. We find that on Myc binding to chromatin, the lysine-demethylating enzyme, LSD1, triggers a transient demethylation of lysine 4 in the histone H3. In addition, we demonstrate that Myc binds and recruits LSD1 to the E-box chromatin and the formation of this complex is stimulated by cAMP-PKA. Demethylation by LSD1 produces H(2)O(2), which locally oxidizes guanine and induces the recruitment of 8-oxoguanine-DNA glycosylase (OGG1) and of the nuclease Ape1 on the E-box chromatin. Inhibition of oxidation or silencing of LSD1, OGG1 or Ape1 significantly reduce transcription and inhibit mRNA accumulation of Myc-target genes. Collectively, these data highlight the role of transient LSD1-mediated demethylation of H3K4 leading to local DNA oxidation as driving force in the assembly of the Myc-induced transcription initiation complex.

Physical activity and physiological cardiac remodelling in a community setting: the Multi-Ethnic Study of Atherosclerosis (MESA).

OBJECTIVE: To evaluate the association of physical activity with left ventricular structure and function in the general population in a community setting. DESIGN: Cross-sectional study. SETTING: The Multi-Ethnic Study of Atherosclerosis (MESA), a population-based study of subclinical atherosclerosis. PARTICIPANTS: A multiethnic sample of 4992 participants (aged 45-84 years; 52% female) free of clinically apparent cardiovascular disease. INTERVENTIONS: Physical activity induces beneficial physiological cardiac remodelling in a cross-sectional study of non-athlete individuals. MAIN OUTCOME MEASURES: Left ventricular mass, volumes and function were assessed by cardiac magnetic resonance imaging. Physical activity, defined as intentional exercise and total moderate and vigorous physical activity, was assessed by a standard semiquantitative questionnaire. RESULTS: Left ventricular mass and end-diastolic volume were positively associated with physical activity (eg, 1.4 g/m(2) (women) and 3.1 g/m(2) (men) greater left ventricular mass in the highest category of intentional exercise compared with individuals reporting no intentional exercise; p = 0.05 and p<0.001, respectively). Relationships were non-linear, with stronger positive associations at lower levels of physical activity (test for non-linearity; p = 0.02 and p = 0.03, respectively). Cardiac output and ejection fraction were unchanged with increased physical activity levels. Resting heart rate was lower in women and men with higher physical activity levels (eg, -2.6 beats/minute lower resting heart rate in the highest category of intentional exercise compared with individuals reporting no intentional exercise; p<0.001). CONCLUSIONS: In a community-based population free of clinically apparent cardiovascular disease, higher physical activity levels were associated with proportionally greater left ventricular mass and end-diastolic volume and lower resting heart rate.

Glycosylated hemoglobin and the risk of death and cardiovascular mortality in the elderly.

BACKGROUND AND AIMS: Glycosylated hemoglobin (HbA(1c)) has been associated with incident cardiovascular disease (CVD), but the findings are inconsistent. We tested the hypothesis that HbA(1c) may be associated with an increased risk of death and cardiovascular mortality in older adults. METHODS AND RESULTS: We evaluated the association between HbA(1c) with all-cause and cardiovascular mortality in 810 participants without a history of diabetes in a sub-study of the Cardiovascular Health Study (CHS), a community cohort study of individuals > or =65 years of age. Glycosylated hemoglobin was measured at baseline and all-cause and cardiovascular mortality was assessed during the follow-up period. The relation between baseline HbA(1c) and death was evaluated with multivariate Cox proportional hazards regression models. After a median follow-up of 14.2 years, 416 deaths were observed. The crude incidence rates of all-cause mortality across HbA(1c) groups were: 4.4% per year, 4.3% per year and 4.6% per year for tertile 1 (< or =5.6%), tertile 2 (5.61-6.20%) and tertile 3 (> or =6.21%), respectively. In unadjusted and fully adjusted analyses, baseline HbA(1c) was not associated with all-cause mortality and cardiovascular mortality (hazard ratio: 1.16 [95% confidence interval 0.91-1.47] and hazard ratio: 1.31 [95% confidence interval 0.90-1.93], respectively for the highest HbA(1c) tertile compared with the lowest). CONCLUSION: These results suggest that HbA(1c) does not significantly predict all-cause and cardiovascular mortality in non-diabetic community-dwelling older adults.

Serological evidence of infections and Type 2 diabetes: the MultiEthnic Study of Atherosclerosis.

AIMS: Prospective studies have identified chronic inflammation as a risk factor for Type 2 diabetes. However, it is not known whether infection by specific pathogens or having a greater 'pathogen burden' is associated with diabetes. The aim of this study was to examine the cross-sectional relation of seropositivity to five pathogens (Chlamydia pneumoniae, cytomegalovirus, Helicobacter pylori, hepatitis A virus, herpes simplex virus) and prevalent diabetes. METHODS: Baseline data from a random sample of MultiEthnic Study of Atherosclerosis (MESA) participants (n = 1000; age 45-84 years) were used. Diabetes was defined by American Diabetes Association 2003 criteria, and 'pathogen burden' by the number of pathogens (0-5) for which an individual was seropositive. Logistic regression was used to test differences in diabetes prevalence by seropositivity. Linear regression was used to explore associations between pathogen seropositivity and the inflammation markers C-reactive protein, interleukin-6 and fibrinogen. RESULTS: Diabetes prevalence was 12.7%, whereas seropositivity for C. pnuemoniae was 76%, cytomegalovirus 77%, H. pylori 45%, hepatitis A 58% and herpes simplex virus 85%. Seventy-two percent were seropositive for three or more pathogens. In crude analyses, the prevalence of diabetes was higher among those with a pathogen burden of three or more, and with seropositivity to cytomegalovirus, H. pylori, hepatitis A and herpes simplex virus. After adjustment for demographic covariates (particularly race), all associations became non-significant. Pathogen seropositivity was also not related to inflammation marker levels. CONCLUSIONS: Following demographic adjustments, no associations were observed between infection by several pathogens and diabetes status, suggesting no aetiological role for them in the occurrence of diabetes.

Upgrade of the CATS sample changer on FIP-BM30A at the ESRF: towards a commercialized standard.

An upgraded version of the sample changer ;CATS' (Cryogenic Automated Transfer System) that was developed on the FIP-BM30A beamline at the ESRF is presented. At present, CATS is installed at SLS (three systems), BESSY (one system), DLS (two systems) and APS (four systems for the LSCAT beamline). It consists mainly of an automated Dewar with an assortment of specific grippers designed to obtain a fast and reliable mounting/dismounting rate without jeopardizing the flexibility of the system. The upgraded system has the ability to manage any sample standard stored in any kind of puck.

Validity of the single-particle approach for electron transport in quantum wires assisted by surface acoustic waves.

We study by means of time-dependent numerical simulations the quantum entanglement stemming from the Coulomb interaction between two electrons trapped in the minima of the piezoelectric potential generated by surface acoustic waves. We find that for particles captured in low-energy bound states the quantum correlations turn out to be negligible, thus validating a single-particle approach to the dynamics of such systems. At long times, for high-energy electrons, a substantial entanglement appears, which is an indicator of a mostly correlated dynamics.

HbA 1c as a risk factor for heart failure in persons with diabetes: the Atherosclerosis Risk in Communities (ARIC) study.

AIMS/HYPOTHESIS: Heart failure (HF) incidence in diabetes in both the presence and absence of CHD is rising. Prospective population-based studies can help describe the relationship between HbA(1c), a measure of glycaemia control, and HF risk. METHODS: We studied the incidence of HF hospitalisation or death among 1,827 participants in the Atherosclerosis Risk in Communities (ARIC) study with diabetes and no evidence of HF at baseline. Cox proportional hazard models included age, sex, race, education, health insurance status, alcohol consumption, BMI and WHR, and major CHD risk factors (BP level and medications, LDL- and HDL-cholesterol levels, and smoking). RESULTS: In this population of persons with diabetes, crude HF incidence rates per 1,000 person-years were lower in the absence of CHD (incidence rate 15.5 for CHD-negative vs 56.4 for CHD-positive, p<0.001). The adjusted HR of HF for each 1% higher HbA(1c) was 1.17 (95% CI 1.11-1.25) for the non-CHD group and 1.20 (95% CI 1.04-1.40) for the CHD group. When the analysis was limited to HF cases which occurred in the absence of prevalent or incident CHD (during follow-up) the adjusted HR remained 1.20 (95% CI 1.11-1.29). CONCLUSIONS/INTERPRETATIONS: These data suggest HbA(1c) is an independent risk factor for incident HF in persons with diabetes with and without CHD. Long-term clinical trials of tight glycaemic control should quantify the impact of different treatment regimens on HF risk reduction.

The endocannabinoid 2-arachidonoylglycerol activates human platelets through non-CB1/CB2 receptors.

BACKGROUND: The endocannabinoid 2-arachidonoylglycerol (2-AG) is an endogenous lipid that acts through the activation of G-protein-coupled cannabinoid receptors and plays essential roles in many physiological contexts. In the cardiovascular system 2-AG is generated by both activated endothelial cells and platelets, and participates in the regulation of inflammation and thrombosis. Although human platelets actively metabolize endocannabinoids, 2-AG also binds to platelet surface and leads to cell activation. OBJECTIVE: To investigate the biological consequence of 2-AG interactions with human platelets and to clarify the role of cannabinoid receptors. METHODS: Gel-filtered platelets were stimulated with 2-AG in the presence or absence of various inhibitors. Platelet aggregation and secretion were measured in a lumiaggregometer. Calcium ion movements were measured in FURA-2 loaded platelets. Thromboxane A(2) (TxA(2)) generation was evaluated as Thromboxane B(2) accumulation with a commercial EIA assay. RESULTS: 2-AG induced platelet shape change, aggregation and secretion with a dose-dependent mechanism that required engagement of platelet TxA(2) receptors. 2-AG caused also cytosolic calcium increase; however, it was totally dependent on availability of TxA(2). Indeed 2-AG was able to induce a robust generation of TxA(2) through the cyclooxygenase pathway. Treatment of platelets with inhibitors of monoacylglycerol lipase and fatty acid amide hydrolase did not affect the activation induced by 2-AG. Moreover, neither CB(1) and CB(2) proteins nor CB(1)/CB(2) mRNAs were detected in platelets. CONCLUSIONS: 2-AG can be considered a new physiologic platelet agonist able to induce full platelet activation and aggregation with a non-CB(1)/CB(2) receptor-mediated mechanism.

FIP: a highly automated beamline for multiwavelength anomalous diffraction experiments.

FIP is a French Collaborating Research Group (CRG) beamline at the European Synchrotron Radiation Facility (ESRF) dedicated exclusively to crystallography of biological macromolecules, with a special emphasis on multiwavelength anomalous diffraction data collection in the 0.7-1.81 A wavelength range. The optics, consisting of long cylindrical grazing-angle mirrors associated with a cryocooled double-crystal monochromator, delivers an optimal beam in the corresponding energy range. The high level of automation, which includes automated crystal centring, automated data-collection management and data processing, makes the use of this beamline very easy. This is illustrated by the large number of challenging structures that have been solved since 1999.

Platelet activation by von Willebrand factor requires coordinated signaling through thromboxane A2 and Fc gamma IIA receptor.

Interaction of von Willebrand Factor with glycoprotein Ib-IX-V induces platelet activation through a still poorly defined mechanism. Previous studies have suggested a possible role for the low affinity receptor for immunoglobulin, Fc gamma RIIA, in GPIb-IX-V signaling. Here we show that binding of vWF to platelets induces the tyrosine phosphorylation of Fc gamma RIIA by a Src kinase. Treatment of platelets with the anti-Fc gamma RIIA monoclonal antibody IV.3 specifically inhibits vWF-induced but not thrombin-induced pleckstrin phosphorylation and serotonin secretion. Moreover, vWF fails to induce pleckstrin phosphorylation in mouse platelets, lacking Fc gamma RIIA, and serotonin secretion is impaired. Pleckstrin phosphorylation and serotonin secretion in human platelets stimulated with vWF are blocked by the cyclooxygenase inhibitor acetylsalicylic acid. However, release of arachidonic acid and synthesis of TxA(2) induced by vWF are not affected by the anti-Fc gamma RIIA monoclonal antibody IV.3. Similarly, vWF-induced tyrosine phosphorylation of Fc gamma RIIA, as well as of Syk and PLC gamma 2, occurs normally in aspirinized platelets. Inhibition of the tyrosine kinase Syk by piceatannol does not affect vWF-induced tyrosine phosphorylation of Fc gamma RIIA but prevents phosphorylation of PLC gamma 2. Pleckstrin phosphorylation and platelet secretion induced by vWF, but not by thrombin, are also inhibited by piceatannol. Pleckstrin phosphorylation is also sensitive to the phosphatidylinositol 3-kinase inhibitor wortmannin. These results indicate that PLC gamma 2 plays a central role in platelet activation by vWF and that the stimulation of this enzyme requires coordinated signals through endogenous TxA(2) and Fc gamma RIIA.