Electrospray tandem mass spectrometry of mixed-sequence RNA/DNA oligonucleotides

The fragmentation of electrospray-generated multiply deprotonated RNA and mixed-sequence RNA/DNA pentanucleotides upon low-energy collision-induced dissociation (CID) in a hybrid quadrupole time-of-flight mass spectrometer was investigated. The goal of unambiguous sequence identification of mixed-sequence RNA/DNA oligonucleotides requires detailed understanding of the gas-phase dissociation of this class of compounds. The two major dissociation events, base loss and backbone fragmentation, are discussed and the unique fragmentation behavior of oligoribonucleotides is demonstrated. Backbone fragmentation of the all-RNA pentanucleotides is characterized by abundant c-ions and their complementary y-ions as the major sequence-defining fragment ion series. In contrast to the dissociation of oligodeoxyribonucleotides, where backbone fragmentation is initiated by the loss of a nucleobase which subsequently leads to the formation of the w- and [a-base]-ions, backbone dissociation of oligoribonucleotides is essentially decoupled from base loss. The different behavior of RNA and DNA oligonucleotides is related to the presence of the 2'-hydroxyl substituent, which is the only structural alteration between the DNA and RNA pentanucleotides studied. CID of mixed-sequence RNA/DNA pentanucleotides results in a combination of the nucleotide-typical backbone fragmentation products, with abundant w-fragment ions generated by cleavage of the phosphodiester backbone adjacent to the deoxy building blocks, whereas backbone cleavage adjacent to ribonucleotides induces the formation of c- and y-ions. (C) 2002 American Society for Mass Spectrometry.

Metabolomics reveals herbivore-induced metabolites of resistance and susceptibility in maize leaves and roots

Plants respond to herbivory by reprogramming their metabolism. Most research in this context has focused on locally induced compounds that function as toxins or feeding deterrents. We developed an ultra-high-pressure liquid chromatography time-of-flight mass spectrometry (UHPLC-TOF-MS)-based metabolomics approach to evaluate local and systemic herbivore-induced changes in maize leaves, sap, roots and root exudates without any prior assumptions about their function. Thirty-two differentially regulated compounds were identified from Spodoptera littoralis-infested maize seedlings and isolated for structure assignment by microflow nuclear magnetic resonance (CapNMR). Nine compounds were quantified by a high throughput direct nano-infusion tandem mass spectrometry/mass spectrometry (MS/MS) method. Leaf infestation led to a marked local increase of 1,3-benzoxazin-4-ones, phospholipids, N-hydroxycinnamoyltyramines, azealic acid and tryptophan. Only few changes were found in the root metabolome, but 1,3-benzoxazin-4-ones increased in the vascular sap and root exudates. The role of N-hydroxycinnamoyltyramines in plant–herbivore interactions is unknown, and we therefore tested the effect of the dominating p-coumaroyltyramine on S. littoralis. Unexpectedly, p-coumaroyltyramine was metabolized by the larvae and increased larval growth, possibly by providing additional nitrogen to the insect. Taken together, this study illustrates that herbivore attack leads to the induction of metabolites that can have contrasting effects on herbivore resistance in the leaves and roots.

Thrombophilia and risk of VTE recurrence according to the age at the time of first VTE manifestation

BACKGROUND Whether screening for thrombophilia is useful for patients after a first episode of venous thromboembolism (VTE) is a controversial issue. However, the impact of thrombophilia on the risk of recurrence may vary depending on the patient's age at the time of the first VTE. PATIENTS AND METHODS Of 1221 VTE patients (42 % males) registered in the MAISTHRO (MAin-ISar-THROmbosis) registry, 261 experienced VTE recurrence during a 5-year follow-up after the discontinuation of anticoagulant therapy. RESULTS Thrombophilia was more common among patients with VTE recurrence than those without (58.6 % vs. 50.3 %; p = 0.017). Stratifying patients by the age at the time of their initial VTE, Cox proportional hazards analyses adjusted for age, sex and the presence or absence of established risk factors revealed a heterozygous prothrombin (PT) G20210A mutation (hazard ratio (HR) 2.65; 95 %-confidence interval (CI) 1.71 - 4.12; p < 0.001), homozygosity/double heterozygosity for the factor V Leiden and/or PT mutation (HR 2.35; 95 %-CI 1.09 - 5.07, p = 0.030), and an antithrombin deficiency (HR 2.12; 95 %-CI 1.12 - 4.10; p = 0.021) to predict recurrent VTE in patients aged 40 years or older, whereas lupus anticoagulants (HR 3.05; 95%-CI 1.40 - 6.66; p = 0.005) increased the risk of recurrence in younger patients. Subgroup analyses revealed an increased risk of recurrence for a heterozygous factor V Leiden mutation only in young females without hormonal treatment whereas the predictive value of a heterozygous PT mutation was restricted to males over the age of 40 years. CONCLUSIONS Our data do not support a preference of younger patients for thrombophilia testing after a first venous thromboembolic event.

Density-dependent dynamics of a dominant rain forest tree change with juvenile stage and time of masting

Although negative density dependence (NDD) can facilitate tree species coexistence in forests, the underlying mechanisms can differ, and rarely are the dynamics of seedlings and saplings studied together. Herein we present and discuss a novel mechanism based on our investigation of NDD predictions for the large, grove-forming ectomycorrhizal mast fruiting tree, Microberlinia bisulcata (Caesalpiniaceae), in an 82.5-ha plot at Korup, Cameroon. We tested whether juvenile density, size, growth and survival decreases with increasing conspecific adult basal area for 3245 ‘new’ seedlings and 540 ‘old’ seedlings (< 75-cm tall) during an approximately 4-year study period (2008–2012) and for 234 ‘saplings’ (≥ 75-cm tall) during an approximately 6-year study period (2008–2014). We found that the respective densities of new seedlings, old seedlings and saplings were positively, not and negatively related to increasing BA. Maximum leaf numbers and heights of old seedlings were negatively correlated with increasing basal areas, as were sapling heights and stem diameters. Whereas survivorship of new seedlings decreased by more than one-half with increasing basal area over its range in 2010–2012, that of old seedlings decreased by almost two-thirds, but only in 2008–2010, and was generally unrelated to conspecific seedling density. In 2010–2012 relative growth rates in new seedlings’ heights decreased with increasing basal area, as well as with increasing seedling density, together with increasing leaf numbers, whereas old seedlings’ growth was unrelated to either conspecific density or basal area. Saplings of below-average height had reduced survivorship with increasing basal area (probability decreasing from approx. 0.4 to 0.05 over the basal area range tested), but only sapling growth in terms of leaf numbers decreased with increasing basal area. These static and dynamic results indicate that NDD is operating within this system, possibly stabilizing the M. bisulcata population. However, these NDD patterns are unlikely to be caused by symmetric competition or by consumers. Instead, an alternative mechanism for conspecific adult–juvenile negative feedback is proposed, one which involves the interaction between tree phenology and ectomycorrhizal linkages.

Preparatory studies for a high-precision Penning-trap measurement of the 163Ho electron capture Q-value

The ECHo Collaboration (Electron Capture 163Ho aims to investigate the calorimetric spectrum following the electron capture decay of 163Ho to determine the mass of the electron neutrino. The size of the neutrino mass is reflected in the endpoint region of the spectrum, i.e., the last few eV below the transition energy. To check for systematic uncertainties, an independent determination of this transition energy, the Q-value, is mandatory. Using the TRIGA-TRAP setup, we demonstrate the feasibility of performing this measurement by Penning-trap mass spectrometry. With the currently available, purified 163Ho sample and an improved laser ablation mini-RFQ ion source, we were able to perform direct mass measurements of 163Ho and 163Dy with a sample size of less than 1017 atoms. The measurements were carried out by determining the ratio of the cyclotron frequencies of the two isotopes to those of carbon cluster ions using the time-of-flight ion cyclotron resonance method. The obtained mass excess values are ME(163Ho)= −66379.3(9) keV and ME(163Dy)= −66381.7(8) keV. In addition, the Q-value was measured for the first time by Penning-trap mass spectrometry to be Q = 2.5(7) keV.

Chronological Order of Appearance of Extraintestinal Manifestations Relative to the Time of IBD Diagnosis in the Swiss Inflammatory Bowel Disease Cohort.

BACKGROUND Data evaluating the chronological order of appearance of extraintestinal manifestations (EIMs) relative to the time of inflammatory bowel disease (IBD) diagnosis is currently lacking. We aimed to assess the type, frequency, and chronological order of appearance of EIMs in patients with IBD. METHODS Data from the Swiss Inflammatory Bowel Disease Cohort Study were analyzed. RESULTS The data on 1249 patients were analyzed (49.8% female, median age: 40 [interquartile range, 30-51 yr], 735 [58.8%] with Crohn's disease, 483 [38.7%] with ulcerative colitis, and 31 [2.5%] with indeterminate colitis). A total of 366 patients presented with EIMs (29.3%). Of those, 63.4% presented with 1, 26.5% with 2, 4.9% with 3, 2.5% with 4, and 2.7% with 5 EIMs during their lifetime. Patients presented with the following diseases as first EIMs: peripheral arthritis 70.0%, aphthous stomatitis 21.6%, axial arthropathy/ankylosing spondylitis 16.4%, uveitis 13.7%, erythema nodosum 12.6%, primary sclerosing cholangitis 6.6%, pyoderma gangrenosum 4.9%, and psoriasis 2.7%. In 25.8% of cases, patients presented with their first EIM before IBD was diagnosed (median time 5 mo before IBD diagnosis: range, 0-25 mo), and in 74.2% of cases, the first EIM manifested itself after IBD diagnosis (median: 92 mo; range, 29-183 mo). CONCLUSIONS In one quarter of patients with IBD, EIMs appeared before the time of IBD diagnosis. Occurrence of EIMs should prompt physicians to look for potential underlying IBD.

Posterior Vertical Bite Reconstructions of Erosively Worn Dentitions and the "Stamp Technique" - A Case Series with a Mean Observation Time of 40 Months

PURPOSE In the present case series, the authors report on seven cases of erosively worn dentitions (98 posterior teeth) which were treated with direct resin composite. MATERIALS AND METHODS In all cases, both arches were restored by using the so-called stamp technique. All patients were treated with standardized materials and protocols. Prior to treatment, a waxup was made on die-cast models to build up the loss of occlusion as well as ensure the optimal future anatomy and function of the eroded teeth to be restored. During treatment, teeth were restored by using templates of silicone (ie, two "stamps," one on the vestibular, one on the oral aspect of each tooth), which were filled with resin composite in order to transfer the planned, future restoration (ie, in the shape of the waxup) from the extra- to the intraoral situation. Baseline examinations were performed in all patients after treatment, and photographs as well as radiographs were taken. To evaluate the outcome, the modified United States Public Health Service criteria (USPHS) were used. RESULTS The patients were re-assessed after a mean observation time of 40 months (40.8 ± 7.2 months). The overall outcome of the restorations was good, and almost exclusively "Alpha" scores were given. Only the marginal integrity and the anatomical form received a "Charlie" score (10.2%) in two cases. CONCLUSION Direct resin composite restorations made with the stamp technique are a valuable treatment option for restoring erosively worn dentitions.

Chemical Composition of Micrometer-Sized Filaments in an Aragonite Host by a Miniature Laser Ablation/Ionization Mass Spectrometer

Detection of extraterrestrial life is an ongoing goal in space exploration, and there is a need for advanced instruments and methods for the detection of signatures of life based on chemical and isotopic composition. Here, we present the first investigation of chemical composition of putative microfossils in natural samples using a miniature laser ablation/ionization time-of-flight mass spectrometer (LMS). The studies were conducted with high lateral (similar to 15 mu m) and vertical (similar to 20-200 nm) resolution. The primary aim of the study was to investigate the instrument performance on micrometer-sized samples both in terms of isotope abundance and element composition. The following objectives had to be achieved: (1) Consider the detection and calculation of single stable isotope ratios in natural rock samples with techniques compatible with their employment of space instrumentation for biomarker detection in future planetary missions. (2) Achieve a highly accurate chemical compositional map of rock samples with embedded structures at the micrometer scale in which the rock matrix is easily distinguished from the micrometer structures. Our results indicate that chemical mapping of strongly heterogeneous rock samples can be obtained with a high accuracy, whereas the requirements for isotope ratios need to be improved to reach sufficiently large signal-to-noise ratio (SNR). Key Words: Biogenicity-Biomarkers-Biosignatures-Filaments-Fossilization. Astrobiology 15, 669-682.

Prototype of the gas chromatograph-mass spectrometer to investigate volatile species in the lunar soil for the Luna-Resurs mission

In preparation for the Russian Luna-Resurs mission we combined our compact time-of-flight mass spectrometer (TOF-MS) with a chemical pre-separation of the species by gas chromatography (GC). Coupled measurements with both instruments were successfully performed with the prototype of the mass spectrometer and a flight-like gas chromatograph. The system was tested with two test gas mixtures, a mixture of hydrocarbons and a mixture of noble gases. Due to its capability to record mass spectra over the full mass range at once with high sensitivity and a dynamic range of up to 10(6) within 1 s, the TOF-MS system is a valuable extension of the GC analytical system. Based on the measurements with calibration gases performed with the combined GC-MS prototype and under assumption of mean characteristics for the Moon's regolith, the detection limit for volatile species in a soil sample is estimated to 2.10(-10) by mass for hydrocarbons and 2.10(-9) by mass for noble gases. (C) 2015 Elsevier Ltd. All rights reserved.

On the prospective detection of polyoxymethylene in comet 67P/Churyumov–Gerasimenko with the COSIMA instrument onboard Rosetta

The presence of polyoxymethylene (POM) in cometary grains has been debated years ago. Although never proven, its presence can not be excluded. Rosetta, the ESA mission to comet 67P/Churyumov–Gerasimenko, may answer this question. On board the spacecraft, COSIMA (COmetary Secondary Ion Mass Analyzer) will analyze the grains ejected from the nucleus using a Time Of Flight Secondary Ion Mass Spectrometer (TOF-SIMS). In this paper we report the extent to which COSIMA will be able to detect POM if this compound is present on cometary grains. We have analyzed two kinds of POM polymers with a laboratory model of COSIMA. Positive mass spectra display alternating sequence of peaks with a separation of 30.011 Da between 1 and 600 Da related to formaldehyde and its oligomers but also to the fragmentation of these oligomers. The separation of 30.011 Da of numbers peaks, corresponding to the fragmentation into H2CO is characteristic of POM and we show that it could be highlight by mathematical treatment. POM lifetime on COSIMA targets have also been studied as POM is thermally instable. It can be concluded that the cometary grains analysis have to be planned not too long after their collection in order to maximize the chances to detect POM. This work was supported by the Centre National d'Etudes Spatiales (CNES).

Short communication: Role of Streptococcus pluranimalium in Mediterranean buffaloes (Bubalus bubalis) with different udder health statuses

The aims of the current study were to describe presence and clinical role over time of Streptococcus pluranimalium isolated in milk samples of Mediterranean buffalo (MB). Two hundred composite milk samples originating from 40 primiparous MB were collected at 10, 30, 60, 90, and 150d in milk (DIM) and from 20 pluriparous MB at 77 to 120 DIM. Milk samples were used for analysis of somatic cell counts, bacteriological cultures, and identification (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). Nine of 200 (4.5%) samples of primiparous MB and 3 of 20 (15%) samples of pluriparous MB were positive for Strep. pluranimalium. The prevalence of the bacterium in primipari was 0% (0/40) at 10, 30, and 150 DIM, whereas it was 5 (2/40) and 17.5% (7/40) at 60 and 90 DIM, respectively. Eight primipari were positive only once, whereas 1 was positive at 2 different samplings. Mono-infection was not detected in any of the age categories or udder health status. Infections were transient in primipari. Clinical mastitis was observed in primipari once at 90 DIM, subclinical mastitis detected twice in the same animals at 60 and 90 DIM, and intramammary infections were diagnosed 1 and 5 times at 60 and 90 DIM in primipari, respectively, whereas 3 infections were diagnosed in pluripari. The clinical reflections demonstrate for the first time the presence of Strep. pluranimalium in MB and its association with different udder health status. Nevertheless, it cannot be excluded that the bacterium may simply follow a pattern of commensal or opportunistic behavior, taking advantage of a preexisting bacterial udder infection.