Characterization of an Omnitrap-Orbitrap Platform Equipped with Infrared Multiphoton Dissociation, Ultraviolet Photodissociation, and Electron Capture Dissociation for the Analysis of Peptides and Proteins.

We describe an instrument configuration based on the Orbitrap Exploris 480 mass spectrometer that has been coupled to an Omnitrap platform. The Omnitrap possesses three distinct ion-activation regions that can be used to perform resonant-based collision-induced dissociation, several forms of electron-associated fragmentation, and ultraviolet photodissociation. Each section can also be combined with infrared multiphoton dissociation. In this work, we demonstrate all these modes of operation in a range of peptides and proteins. The results show that this instrument configuration produces similar data to previous implementations of each activation technique and at similar efficiency levels. We demonstrate that this unique instrument configuration is extremely versatile for the investigation of polypeptides.

An Orbitrap/Time-of-Flight Mass Spectrometer for Photofragment Ion Imaging and High-Resolution Mass Analysis of Native Macromolecular Assemblies.

We discuss the design, development, and evaluation of an Orbitrap/time-of-flight (TOF) mass spectrometry (MS)-based instrument with integrated UV photodissociation (UVPD) and time/mass-to-charge ratio (m/z)-resolved imaging for the comprehensive study of the higher-order molecular structure of macromolecular assemblies (MMAs). A bespoke TOF analyzer has been coupled to the higher-energy collisional dissociation cell of an ultrahigh mass range hybrid quadrupole-Orbitrap MS. A 193 nm excimer laser was employed to photofragment MMA ions. A combination of microchannel plates (MCPs)-Timepix (TPX) quad and MCPs-phosphor screen-TPX3CAM assemblies have been used as axial and orthogonal imaging detectors, respectively. The instrument can operate in four different modes, where the UVPD-generated fragment ions from the native MMA ions can be measured with high-mass resolution or imaged in a mass-resolved manner to reveal the relative positions of the UVPD fragments postdissociation. This information is intended to be utilized for retrieving higher-order molecular structural details that include the conformation, subunit stoichiometry, and molecular interactions as well as to understand the dissociation dynamics of the MMAs in the gas phase.

De novo glycan sequencing by electronic excitation dissociation MS2-guided MS3 analysis on an Omnitrap-Orbitrap hybrid instrument.

Comprehensive de novo glycan sequencing remains an elusive goal due to the structural diversity and complexity of glycans. Present strategies employing collision-induced dissociation (CID) and higher energy collisional dissociation (HCD)-based multi-stage tandem mass spectrometry (MSn) or MS/MS combined with sequential exoglycosidase digestions are inherently low-throughput and difficult to automate. Compared to CID and HCD, electron transfer dissociation (ETD) and electron capture dissociation (ECD) each generate more cross-ring cleavages informative about linkage positions, but electronic excitation dissociation (EED) exceeds the information content of all other methods and is also applicable to analysis of singly charged precursors. Although EED can provide extensive glycan structural information in a single stage of MS/MS, its performance has largely been limited to FTICR MS, and thus it has not been widely adopted by the glycoscience research community. Here, the effective performance of EED MS/MS was demonstrated on a hybrid Orbitrap-Omnitrap QE-HF instrument, with high sensitivity, fragmentation efficiency, and analysis speed. In addition, a novel EED MS2-guided MS3 approach was developed for detailed glycan structural analysis. Automated topology reconstruction from MS2 and MS3 spectra could be achieved with a modified GlycoDeNovo software. We showed that the topology and linkage configurations of the Man9GlcNAc2 glycan can be accurately determined from first principles based on one EED MS2 and two CID-EED MS3 analyses, without reliance on biological knowledge, a structure database or a spectral library. The presented approach holds great promise for autonomous, comprehensive and de novo glycan sequencing.

Coherent diffractive imaging of proteins and viral capsids: simulating MS SPIDOC.

MS SPIDOC is a novel sample delivery system designed for single (isolated) particle imaging at X-ray Free-Electron Lasers that is adaptable towards most large-scale facility beamlines. Biological samples can range from small proteins to MDa particles. Following nano-electrospray ionization, ionic samples can be m/z-filtered and structurally separated before being oriented at the interaction zone. Here, we present the simulation package developed alongside this prototype. The first part describes how the front-to-end ion trajectory simulations have been conducted. Highlighted is a quadrant lens; a simple but efficient device that steers the ion beam within the vicinity of the strong DC orientation field in the interaction zone to ensure spatial overlap with the X-rays. The second part focuses on protein orientation and discusses its potential with respect to diffractive imaging methods. Last, coherent diffractive imaging of prototypical T = 1 and T = 3 norovirus capsids is shown. We use realistic experimental parameters from the SPB/SFX instrument at the European XFEL to demonstrate that low-resolution diffractive imaging data (q < 0.3 nm-1) can be collected with only a few X-ray pulses. Such low-resolution data are sufficient to distinguish between both symmetries of the capsids, allowing to probe low abundant species in a beam if MS SPIDOC is used as sample delivery.

High-throughput determination of dry mass of single bacterial cells by ultrathin membrane resonators.

How bacteria are able to maintain their size remains an open question. Techniques that can measure the biomass (dry mass) of single cells with high precision and high-throughput are demanded to elucidate this question. Here, we present a technological approach that combines the transport, guiding and focusing of individual bacteria from solution to the surface of an ultrathin silicon nitride membrane resonator in vacuum. The resonance frequencies of the membrane undergo abrupt variations at the instants where single cells land on the membrane surface. The resonator design displays a quasi-symmetric rectangular shape with an extraordinary capture area of 0.14 mm2, while maintaining a high mass resolution of 0.7 fg (1 fg = 10-15 g) to precisely resolve the dry mass of single cells. The small rectangularity of the membrane provides unprecedented frequency density of vibration modes that enables to retrieve the mass of individual cells with high accuracy by specially developed inverse problem theory. We apply this approach for profiling the dry mass distribution in Staphylococcus epidermidis and Escherichia coli cells. The technique allows the determination of the dry mass of single bacterial cells with an accuracy of about 1% at an unparalleled throughput of 20 cells/min. Finally, we revisit Koch & Schaechter model developed during 60 s to assess the intrinsic sources of stochasticity that originate cell size heterogeneity in steady-state populations. The results reveal the importance of mass resolution to correctly describe these mechanisms.

The Omnitrap Platform: A Versatile Segmented Linear Ion Trap for Multidimensional Multiple-Stage Tandem Mass Spectrometry.

Multidimensional multiple-stage tandem processing of ions is demonstrated successfully in a novel segmented linear ion trap. The enhanced performance is enabled by incorporating the entire range of ion activation methods into a single platform in a highly dynamic fashion. The ion activation network comprises external injection of reagent ions, radical neutral species, photons, electrons, and collisions with neutrals. Axial segmentation of the two-dimensional trapping field provides access to a unique functionality landscape through a system of purpose-designed regions for processing ions with maximum flexibility. Design aspects of the segmented linear ion trap, termed the Omnitrap platform, are highlighted, and motion of ions trapped by rectangular waveforms is investigated experimentally by mapping the stability diagram, tracing secular frequencies, and exploring different isolation techniques. All fragmentation methods incorporated in the Omnitrap platform involving radical chemistry are shown to provide complete sequence coverage for partially unfolded ubiquitin. Three-stage (MS3) tandem mass spectrometry experiments combining collision-induced dissociation of radical ions produced by electron meta-ionization and further involving two intermediate steps of ion isolation and accumulation are performed with high efficiency, producing information rich spectra with signal-to-noise levels comparable to those obtained in a two-stage (MS2) experiment. The advanced capabilities of the Omnitrap platform to provide in-depth top-down MSn characterization of proteins are portrayed. Performance is further enhanced by connecting the Omnitrap platform to an Orbitrap mass analyzer, while successful integration with time-of-flight analyzers has already been demonstrated.

Cost-Effectiveness Analysis of Seven Measures to Reduce Tail Biting Lesions in Fattening Pigs.

Tail biting is an important animal welfare issue in the pig sector. Studies have identified various risk factors which can lead to biting incidents and proposed mitigation measures. This study focused on the following seven key measures which have been identified to affect the risk of tail biting lesions: improvements in straw provision, housing ventilation, genetics, stocking density, herd health, provision of point-source enrichment objects, and adoption of early warning systems. The aim of this study was to examine whether these selected measures to reduce the risk of tail biting lesions in pig fattening are cost-effective. The problem was analyzed by first summarizing the most prospective interventions, their costs and expected impacts on the prevalence of tail biting lesions, second, by using a stochastic bio-economic model to simulate the financial return per pig space unit and per pig at different levels of prevalence of tail biting lesions, and third by looking at how large a reduction in tail biting lesions would be needed at different levels of initial prevalence of lesions to cover the costs of interventions. Tail biting lesions of a severity which would require an action (medication, hospitalization of the pig or other care, or taking preventive measures) by the pig producer were considered in the model. The results provide guidance on the expected benefits and costs of the studied interventions. According to the results, if the average prevalence of tail biting lesions is at a level of 10%, the costs of this damaging behavior can be as high as €2.3 per slaughtered pig (~1.6% of carcass value). Measures which were considered the least expensive to apply, such as provision of point-source enrichment objects, or provided wider production benefits, such as improvements in ventilation and herd health, became profitable at a lower level of efficacy than measures which were considered the most expensive to apply (e.g., straw provision, increased space allowance, automated early warning systems). Measures which were considered most efficient in reducing the risk of tail biting lesions, such as straw provision, can be cost-effective in preventing tail biting, especially when the risk of tail biting is high. At lower risk levels, the provision of point-source objects and other less costly but relatively effective measures can play an important role. However, selection of measures appropriate to the individual farm problem is essential. For instance, if poor health or barren pens are causing the elevated risk of tail biting lesions, then improving health management or enriching the pens may resolve the tail biting problem cost-effectively.

Size-Dependent Hydrogen Atom Attachment to Gas-Phase Hydrogen-Deficient Polypeptide Radical Cations.

Despite significant affinity to carbonyl oxygens, thermal hydrogen atoms attach to unmodified polypeptides at a very low rate, while the hydrogen-hydrogen exchange rate is high. Here, using the novel omnitrap setup, we found that attachment to polypeptides is much more facile when radical site is already present, but the rate decreases for larger radical ions. The likely explanation is the intramolecular hydrogen atom rearrangement in hydrogen-deficient radicals to a more stable or less accessible site.

Identification of PAH Isomeric Structure in Cosmic Dust Analogues: the AROMA setup.

We developed a new analytical experimental setup called AROMA (Astrochemistry Research of Organics with Molecular Analyzer) that combines laser desorption/ionization techniques with ion trap mass spectrometry. We report here on the ability of the apparatus to detect aromatic species in complex materials of astrophysical interests and characterize their structures. A limit of detection of 100 femto-grams has been achieved using pure polycyclic aromatic hydrocarbon (PAH) samples, which corresponds to 2x108 molecules in the case of coronene (C24H12). We detected the PAH distribution in the Murchison meteorite, which is made of a complex mixture of extraterrestrial organic compounds. In addition, collision induced dissociation experiments were performed on selected species detected in Murchison, which led to the first firm identification of pyrene and its methylated derivatives in this sample.

Monitoring particulate matter levels and climate conditions in a Greek sheep and goat livestock building.

Atmospheric pollutants from livestock operations influence air quality inside livestock buildings and the air exhausted from them. The climate that prevails inside the building affects human and animal health and welfare, as well as productivity, while emissions from the building contribute to environmental pollution. The aim of this study was to examine the variation of two climatic parameters (namely temperature and relative humidity) and the levels of particulate matter of different sizes (PM10-PM2.5-PM1), as well as the relationships between them, inside a typical Greek naturally ventilated livestock building that hosts mainly sheep. The concentration of particles was recorded during a 45-day period (27/11-10/1), while temperature and relative humidity were observed during an almost 1-year period. The analysis revealed that the variation of outdoor weather conditions significantly influenced the indoor environment, as temperature and relative humidity inside the building varied in accordance to the outside climate conditions. Temperature remained higher indoors than outdoors during the winter and extremely low values were not recorded inside the building. However, the tolerable relative humidity levels recommended by the International Commission of Agricultural Engineering (CIGR) were fulfilled only in 47% of the hours during the almost 1-year period that was examined. This fact indicates that although temperature was satisfactorily controlled, the control of relative humidity was deficient. The concentration of particulate matter was increased during the cold winter days due to poor ventilation. The maximum daily average value of PM10, PM2.5 and PM1 concentration equaled to 363, 61 and 30 µg/m(3) respectively. The concentration of the coarse particles was strongly influenced by the farming activities that were daily taking place in the building, the dust resuspension being considered as the dominant source. A significant part of the fine particles were secondary, which the production of could be attributed to an increase in relative humidity levels. It is concluded that measures have to be adopted in order to achieve sufficient ventilation and to reduce particulate matter levels.

Temperature, comfort and pollution levels during heat waves and the role of sea breeze.

During the summer of 2007 several Greek regions suffered periods of extreme heat, with midday temperatures of over 40 degrees C on several consecutive days. High temperatures were also recorded on the east coast of central Greece, where a complex sea breeze circulation system frequently develops. The more intense events occurred at the end of June and July. The highest temperatures were observed on 26 June and 25 July, while the sea breeze developed only on 25 July. Meteorological data collected at two sites-a coastal urban location and an inland suburban site that is not reached by the sea breeze flow-as well as pollution data collected at the urban site, were analysed in order to investigate the relationship between sea breeze development and the prevailing environmental conditions during these two heat wave events. The analysis revealed that sea breeze development affects temperature and pollution levels at the shoreline significantly, causing a decrease of approximately 4 degrees C from the maximum temperature value and an increase of approximately 30% in peak PM10 levels. Additionally, several stress indices were calculated in order to assess heat comfort conditions at the two sites. It was found that nocturnal comfort levels are determined mainly by the urban heat island effect, the intensity of which reaches up to 8 degrees C, while the applied indices do not demonstrate any significant daytime thermal stress relief due to sea breeze development.

Simulation of ion motion in FAIMS through combined use of SIMION and modified SDS.

A key application of field asymmetric waveform ion mobility spectrometry (FAIMS) has been in selectively transmitting trace analyte ions that are present in a complex ion mixture to a mass spectrometer (MS) for identification and quantification. The overall sensitivity of FAIMS-MS, however, still needs to be significantly improved through the optimization of ion transmission into FAIMS and at the FAIMS-MS interface. Processes that cause ion losses include diffusion, space charge, separation field in the FAIMS and fringe fields around the edges of the FAIMS electrodes. These were studied here by first developing an algorithm using SIMION as its core structure to compute ion trajectory at different ratios of electric field to buffer gas number density (E/N). The E/N was varied from a few Td to approximately 80 Td by using an asymmetric square waveform. The algorithm was then combined with statistical diffusion simulation (SDS) model, columbic repulsion, and a parabolic gas flow profile to realistically simulate current transmission and peak shape. The algorithm was validated using a FAIMS model identical to the Sionex Corporation SVAC model. Ions modeled included low mass ions with K(o) in the range of 2.17 (m = 55) to 1.39 cm(2) x V(-1) x s(-1) (m = 368). Good agreement was achieved between simulated and experimental CV (peak maxima) values, peak width (fwhm), and transmitted ion current I(output). The model was then used to study fringe fields in a simple arrangement where a 0.5 mm (w) gap was created between the FAIMS exit and a capillary inlet (i.d. = 0.5 mm). At an optimum CV (11.8 V), only approximately 17% (1.3 pA) of the total ion current that correlate to CV = 11.8 V, entered the capillary; bulk of the ion loss was caused by the fringe fields. Current transmission into the capillary was improved, however, by applying a 500 V DC bias across w (0.5 mm).

Study of the eye and lacrimal glands in experimental iron overload in rats in vivo.

A variety of syndromes leading to hemosiderosis in men cause ocular lesions. The purpose of the present study was to determine the distribution of iron and of transferrin in the eyes and lacrimal glands of rats in experimental hemosiderosis, so as to achieve a better understanding of the formation of the ocular lesions observed in patients with advanced hemosiderosis. In order to achieve hemosiderosis the rats were fed 3% (w/w) carbonyl iron or received i.p. or i.v. polymaltose iron. Hemosiderin deposits were detected in macrophages lying in the interlobular connective tissue of lacrimal glands, in the interstitial connective tissue of the choroid, in the ciliary body, in the iris and extracellularly in the sclera in all animals that received iron i.v. Also, scanty hemosiderin laden macrophages were found to a lesser degree in interstitial connective tissue of the choroid and in the interlobular connective tissue of lacrimal glands in animals that received iron i.p. No iron deposits were detected in the eye and lacrimal glands of control rats and in rats that were on an iron enriched diet. No transferrin was detected in the eye and in the lacrimal glands, neither in the control rats nor in the rats that received iron. Experimental iron overload leads to increased iron deposition in tissues of the eye and lacrimal glands, whereas no transferrin could be detected in the aforementioned organs.

Rarity of bacterial and viral meningitis in areas of Western Greece with fewer than 2,000 inhabitants.

The purpose of the present study was to compare the incidence of childhood meningitis in regions with fewer than 2,000 inhabitants (rural regions) and regions with more than 2,000 inhabitants (urban regions) in the prefecture of Achaia in Western Greece during 1991-2005. Included were all 555 children hospitalized for meningitis. The criteria for bacterial meningitis were (i) positive blood/cerebralspinal fluid (CSF) culture, Gram stain, or latex agglutination and/or (ii) increased beta-glucuronidase in CSF. In case of suspected bacterial meningitis, the following findings were considered: compatible clinical and laboratory findings, and whether or not a cure was achieved with antibiotic treatment and finally resulted in negative cultures. In cases of suspected viral meningitis, compatible clinical and laboratory findings were considered, together with observation of a cure without antibiotic treatment. Only 28 of 555 meningitis patients were from rural regions. The incidence per 10,000 children in rural and urban regions, respectively, was as follows: meningitis, 1.13 and 8.99; bacterial meningitis, 0.16 and 2.40; suspected bacterial meningitis, 0.52 and 3.00; and viral meningitis, 0.44 and 3.58. The incidence ratio for bacterial, suspected bacterial, and viral meningitis in urban versus rural regions was 14.85, 5.72, and 8.10, respectively. Only 2 of the 79 cases with a confirmed causative pathogen came from rural regions. In conclusion, compared to those living in urban regions, children living in rural regions are relatively spared from bacterial and viral meningitis.

Use of Doppler ultrasonography in the assessment of bronchodilator response in acute bronchiolitis.

Measurement of the response of acute bronchiolitis (AB) to bronchodilators relies on clinical signs and pulse oximetry. We hypothesized that Doppler ultrasonographic indices of hepatic venous flow may prove to be an objective tool in the assessment of the effect of inhaled salbutamol in infants hospitalized for AB. Previously healthy infants hospitalized for their first episode of AB were prospectively studied. Composite clinical score (CCS, retractions plus wheezing/crackles) and hemoglobin oxygen saturation (SaO(2)) were measured before, and 15-min post-salbutamol nebulization (0.15 mg/kg, minimum 1.5 mg). Peak velocities at the middle hepatic vein (PV-HV) and right renal vein (PV-RV), as well as peripheral-to-middle hepatic vein transit time (TT) of an ultrasound contrast agent were also measured by Doppler ultrasonography pre- and post-nebulization. Nineteen infants were studied. Mean CCS decreased by 0.37 (95% confidence interval [CI]: 0.08-0.66, P = 0.015) and mean SaO(2) increased by 0.68% (95%CI: 0.17-1.19, P = 0.01) post-bronchodilator treatment. Mean TT increased by 9.54 sec (95%CI: 5.95-13.13, P < 0.0001) and PV-HV increased by 16.49 cm/sec (95%CI: 9.07-23.91, P = 0.0002); PV-RV did not change. TT (r = 0.51, P = 0.009), but not PV-HV, correlated negatively with CCS. There was a strong positive correlation between pre- and post-salbutamol TT values (r = 0.92, P < 0.0001). The most likely explanation for these findings is post-salbutamol abolishment of shunting at the pulmonary capillary bed. We conclude that the peripheral-to-middle hepatic vein prolongation of TT measured by Doppler ultrasonography after salbutamol administration in infants with AB can be used as a bedside tool in the objective assessment of clinical response to medication in these patients.

Isolated costal hyperostosis: a rare cause of severe hemothorax.

Chest pain due to hemothorax is rare. However, the possibility of traumatic hemothorax must always be suspected. We report a case of a 15-year-old girl with hemothorax after erosion of the intercostal artery of the left seventh rib by a histological proved solitary costal hyperostosis. She presented with a 2-month history of intermittent left-sided chest pain and 3 episodes of acute chest pain followed by dizziness and loss of consciousness. The chest radiograph revealed a large left pleural effusion and a thin-section chest computed tomography scan that was performed after drainage of the hemothorax indicated a spicule projecting inward into the chest from the left seventh rib. After thoracotomy, a solitary costal hyperostosis causing a traumatic pseudoaneurysm of the intercostal artery was removed by partial resection of the left seventh rib, the intercostals vessels were ligated, and the pseudoaneurysm excised.

First urinary tract infection in neonates, infants and young children: a comparative study.

In an attempt to evaluate first urinary tract infection (UTI) in neonates and infants, we estimated retrospectively in 296 patients (62 neonates and 234 infants) clinical and laboratory findings, occurrence of vesicoureteral reflux (VUR), urinary tract abnormalities and pyelonephritis. First UTI occurred more often in male than female neonates, whereas male and female infants/young children were affected at an equal rate. The pathogens isolated in urine cultures of neonates and infants did not statistically significantly differ (P>0.05); Escherichia coli predominated. Gram-negative bacteria other than E. coli affected boys more often than girls (P=0.0022). Fever was the most frequent symptom. Neonates had lower-grade fever of shorter duration than infants (P<0.05). The incidence of reflux and urinary tract abnormalities did not differ between neonates and infants, male and female neonates and infants (P>0.05). Pyelonephritis affected neonates and infants at an equal rate; it was more prevalent among female patients (P=0.038) and patients with VUR or urinary tract abnormalities other than VUR (P<0.0001). Neonates with reflux were more often affected by Gram-negative bacteria other than E. coli than were neonates without reflux (P=0.0008).

Presentation of childhood brucellosis in Western Greece.

The purpose of this study was to evaluate clinical and laboratory findings, treatment modalities and final outcomes of brucellosis in children and to compare our data with those of other studies performed in Greece. Fifty-two children treated for brucellosis in the Department of Pediatrics during the decade 1995-2004 were analyzed. Of the 52 children, 47 were reexamined during July 2005. Fever, arthritis or arthralgia, hepatomegaly and splenomegaly were the main findings. Young children had positive blood cultures and lower or negative antibody titers statistically significantly more often than did older children. Brucella abortus was isolated in 9 of 18 patients with positive blood cultures. Antibiotic treatment lasted for 28 days on average. There were no complications or relapses, except one, and the final outcomes were excellent.

Low bacterial count urinary tract infections in infants and young children.

UNLABELLED: The purpose of the study was to compare bacterial species, clinical, laboratory and imaging findings ((99m )Tc-dimercaptosuccinic acid renal scan and voiding cystogram) in infants and children with high (>/=10(5) colony forming units (CFU)/ml, group A patients) and low (