Post-transcriptional modification mapping in the Clostridium acetobutylicum 16S rRNA by mass spectrometry and reverse transcriptase assays

Post-transcriptional modifications in ribosomal RNA are believed to fine-tune the RNA functions. The present study describes the characterization of the post-transcriptional modifications in Clostridium acetobutylicum 16S rRNA, using high-pressure liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry and reverse transcriptase assays. The combination of these techniques allowed the identification of eleven modified nucleosides, which were mapped onto the rRNA sequence. The C. acetobutylicum modification map is similar to that of Escherichia coli, with the majority of the modifications near functionally important sites in the rRNA. Although, in general, the number of modifications in rRNA is smaller than in tRNA, the conservation of the modification sites seems to indicate that the post-transcriptional modifications in 16S rRNA provide a necessary prerequisite for the ribosomal function

Air Pollution–Related Prothrombotic Changes in Persons with Diabetes

Background: Population studies suggest that persons with diabetes are more sensitive to the effects of particulate matter (PM) air pollution. However, the biological mechanisms of a possible prothrombotic effect underlying this enhanced susceptibility remain largely unknown.Objective: We hypothesized that exposure to PM causes prothrombotic changes in persons with diabetes, possibly via systemic inflammation.Methods: Our study included 137 nonsmoking adults with diabetes who were outpatients at the University Hospital Leuven. Recent exposure (2 hr before examination) to ambient PM was measured at the entrance of the hospital. Individual chronic exposure to PM was assessed by measuring the area occupied by carbon in airway macrophages obtained by sputum induction. Platelet function was measured ex vivo with the PFA-100 platelet function analyzer, which simulates a damaged blood vessel; we analyzed the function of platelets in primary hemostasis under high shear conditions. Total and differential blood leukocytes were counted.Results: Independent of antiplatelet medication, an interquartile range (IQR) increase of 39.2 microg/m3 in PM10 (PM with aerodynamic diameter </= 10 microm) concentration measured 2 hr before the clinical examination (recent exposure) was associated with a decrease of 21.1 sec [95% confidence interval (CI), 35.3 to 6.8] in the PFA-100 closure time (i.e., increased platelet activation) and an increase in blood leukocytes of 512 per microliter of blood (95% CI, 45.2979). Each area increase of 0.25 microm2 (IQR) in carbon load of airway macrophages (chronic exposure) was associated with an increase of 687 leukocytes per microliter of blood (95% CI, 2241,150).Conclusions: A relevant increase in recent PM exposure was associated with a change in platelet function toward a greater prothrombotic tendency. The magnitude of the change was about two-thirds (in the opposite direction) of the average effect of antiplatelet medication. Diabetic patients showed evidence of proinflammatory response to both recent and chronic exposure to PM air pollution. Editor's SummaryDiabetics are at increased risk of cardiovascular diseases, and the association between particulate matter (PM) air pollution and cardiovascular outcomes may be stronger among diabetics than among nondiabetics. Jacobs et al. (p. 191) hypothesized that susceptibility to adverse cardiovascular outcomes among diabetics might be related to prothrombotic and inflammatory effects of PM. The authors estimated associations between PM exposures and measures of platelet function (estimated using the PFA-100 platelet function analyzer) and systemic inflammation (total and differential white blood cell counts) among 63 well-controlled diabetics (29 type I, 34 type II). Exposures included modeled estimates of average ambient residential PM10 (PM with aerodynamic diameter </= 10 microm), recent PM10 and PM2.5 (aerodynamic diameter </= 2.5 microm) exposures (at the study hospital), and a proxy measure of chronic carbon load (median area occupied by carbon in 50 airway macrophages from an induced sputum sample.) The authors report that recent PM10 exposure was associated with increased platelet activation, both before and after adjustment for type of diabetes and use of medications that inhibit platelet aggregation, and that carbon load was positively associated with platelet and white blood cell counts. The authors conclude that findings are consistent with proinflammatory responses to PM air pollution among diabetics.status: publishe

The Belgian Virtual Tumorbank: A Tool for Translational Cancer Research

Background: Biobanks play a critical role in cancer research by providing high quality biological samples for research. However, the availability of tumor samples in single research institutions is often limited, especially for rare cancers. In order to facilitate the search for samples scattered among different Belgian institutions, a nationwide virtual tumorbank project was launched and is operational since February 2012. The Belgian Virtual Tumorbank (BVT) network encompasses the tumor biobanks from eleven Belgian university hospitals that collect and store residual human tumor samples locally and is coordinated by the Belgian Cancer Registry.Materials and Methods: A web application was developed and consists of two modules. The registration module (BVTr) centralizes the tumor sample data from the local partner biobanks. The catalog module (BVTc) allows researchers to trace the tumor samples in the 11 tumor biobanks. The BVTc contains patient, medical and technical data, but excludes identifying information to ensure privacy of individuals. Automatic and manual controls guarantee high quality data on the samples requested by scientists for research purposes in oncology. A major advantage of the BVT network is that the available data can be linked to the data of the Belgian Cancer Registry for quality control purposes.Results: Currently, more than 92,000 registrations are available in the catalog. Twenty-seven percent of the residual primary tumor samples originate from breast tissue, but also less frequent localisations such as head and neck (4%), male genital organs (1.7%), and urinary tract (1%) are available. In addition to the residual tumor tissue samples, also other available material can be stored and registered by the local biobanks. The most common type is corresponding normal tissue (19%).Other frequently available materials are plasma, blood, serum, DNA, and buffy coat. Even PBMCs, RNA, cytology, and urine are available in some cases.Discussion and Conclusion: The BVT catalog is a valuable source of information for oncology research and the ultimate goal is to promote multidisciplinary cancer research (i.e., pathogenesis, disease prediction, prevention, diagnosis, treatment, and prognosis) for the benefit of all cancer patients

Improved circulating microparticle analysis in acid-citrate dextrose (ACD) anticoagulant tube.

INTRODUCTION: Recently extracellular vesicles (exosomes, microparticles also referred to as microvesicles and apoptotic bodies) have attracted substantial interest as potential biomarkers and therapeutic vehicles. However, analysis of microparticles in biological fluids is confounded by many factors such as the activation of cells in the blood collection tube that leads to in vitro vesiculation. In this study we aimed at identifying an anticoagulant that prevents in vitro vesiculation in blood plasma samples. MATERIALS AND METHODS: We compared the levels of platelet microparticles and non-platelet-derived microparticles in platelet-free plasma samples of healthy donors. Platelet-free plasma samples were isolated using different anticoagulant tubes, and were analyzed by flow cytometry and Zymuphen assay. The extent of in vitro vesiculation was compared in citrate and acid-citrate-dextrose (ACD) tubes. RESULTS: Agitation and storage of blood samples at 37 degrees C for 1hour induced a strong release of both platelet microparticles and non-platelet-derived microparticles. Strikingly, in vitro vesiculation related to blood sample handling and storage was prevented in samples in ACD tubes. Importantly, microparticle levels elevated in vivo remained detectable in ACD tubes. CONCLUSIONS: We propose the general use of the ACD tube instead of other conventional anticoagulant tubes for the assessment of plasma microparticles since it gives a more realistic picture of the in vivo levels of circulating microparticles and does not interfere with downstream protein or RNA analyses

Traffic Air Pollution and Oxidized LDL

BACKGROUND: Epidemiologic studies indirectly suggest that air pollution accelerates atherosclerosis. We hypothesized that individual exposure to particulate matter (PM) derived from fossil fuel would correlate with plasma concentrations of oxidized low-density lipoprotein (LDL), taken as a marker of atherosclerosis. We tested this hypothesis in patients with diabetes, who are at high risk for atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: In a cross-sectional study of non-smoking adult outpatients with diabetes we assessed individual chronic exposure to PM by measuring the area occupied by carbon in airway macrophages, collected by sputum induction and by determining the distance from the patient's residence to a major road, through geocoding. These exposure indices were regressed against plasma concentrations of oxidized LDL, von Willebrand factor and plasminogen activator inhibitor 1 (PAI-1). We could assess the carbon load of airway macrophages in 79 subjects (58 percent). Each doubling in the distance of residence from major roads was associated with a 0.027 µm(2) decrease (95% confidence interval (CI): -0.048 to -0.0051) in the carbon load of airway macrophages. Independently from other covariates, we found that each increase of 0.25 µm(2) [interquartile range (IQR)] in carbon load was associated with an increase of 7.3 U/L (95% CI: 1.3 to 13.3) in plasma oxidized LDL. Each doubling in distance of residence from major roads was associated with a decrease of -2.9 U/L (95% CI: -5.2 to -0.72) in oxidized LDL. Neither the carbon load of macrophages nor the distance from residence to major roads, were associated with plasma von Willebrand factor or PAI-1. CONCLUSIONS: The observed positive association, in a susceptible group of the general population, between plasma oxidized LDL levels and either the carbon load of airway macrophages or the proximity of the subject's residence to busy roads suggests a proatherogenic effect of traffic air pollution

An analytical platform for mass spectrometry-based identification and chemical analysis of RNA in ribonucleoprotein complexes

We describe here a mass spectrometry (MS)-based analytical platform of RNA, which combines direct nano-flow reversed-phase liquid chromatography (RPLC) on a spray tip column and a high-resolution LTQ-Orbitrap mass spectrometer. Operating RPLC under a very low flow rate with volatile solvents and MS in the negative mode, we could estimate highly accurate mass values sufficient to predict the nucleotide composition of a ∼21-nucleotide small interfering RNA, detect post-transcriptional modifications in yeast tRNA, and perform collision-induced dissociation/tandem MS-based structural analysis of nucleolytic fragments of RNA at a sub-femtomole level. Importantly, the method allowed the identification and chemical analysis of small RNAs in ribonucleoprotein (RNP) complex, such as the pre-spliceosomal RNP complex, which was pulled down from cultured cells with a tagged protein cofactor as bait. We have recently developed a unique genome-oriented database search engine, Ariadne, which allows tandem MS-based identification of RNAs in biological samples. Thus, the method presented here has broad potential for automated analysis of RNA; it complements conventional molecular biology-based techniques and is particularly suited for simultaneous analysis of the composition, structure, interaction, and dynamics of RNA and protein components in various cellular RNP complexes

Ambient air pollution and thrombosis

Abstract Air pollution is a growing public health concern of global significance. Acute and chronic exposure is known to impair cardiovascular function, exacerbate disease and increase cardiovascular mortality. Several plausible biological mechanisms have been proposed for these associations, however, at present, the pathways are incomplete. A seminal review by the American Heart Association (2010) concluded that the thrombotic effects of particulate air pollution likely contributed to their effects on cardiovascular mortality and morbidity. The aim of the current review is to appraise the newly accumulated scientific evidence (2009–2016) on contribution of haemostasis and thrombosis towards cardiovascular disease induced by exposure to both particulate and gaseous pollutants. Seventy four publications were reviewed in-depth. The weight of evidence suggests that acute exposure to fine particulate matter (PM2.5) induces a shift in the haemostatic balance towards a pro-thrombotic/pro-coagulative state. Insufficient data was available to ascertain if a similar relationship exists for gaseous pollutants, and very few studies have addressed long-term exposure to ambient air pollution. Platelet activation, oxidative stress, interplay between interleukin-6 and tissue factor, all appear to be potentially important mechanisms in pollution-mediated thrombosis, together with an emerging role for circulating microvesicles and epigenetic changes. Overall, the recent literature supports, and arguably strengthens, the contention that air pollution contributes to cardiovascular morbidity by promoting haemostasis. The volume and diversity of the evidence highlights the complexity of the pathophysiologic mechanisms by which air pollution promotes thrombosis; multiple pathways are plausible and it is most likely they act in concert. Future research should address the role gaseous pollutants play in the cardiovascular effects of air pollution mixture and direct comparison of potentially susceptible groups to healthy individuals