The Aeromonas salmonicida subsp. salmonicida exoproteome: determination of the complete repertoire of Type-Three Secretion System effectors and identification of other virulence factors

BACKGROUND Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen of fisheries worldwide. Several virulence factors have been described, but the type-three secretion system (T3SS) is recognized as having a major effect on virulence by injecting effectors directly into fish cells. In this study we used high-throughput proteomics to display the differences between in vitro secretome of A. salmonicida wild-type (wt, hypervirulent, JF2267) and T3SS-deficient (isogenic ΔascV, extremely low-virulent, JF2747) strains in exponential and stationary phases of growth. RESULTS Results confirmed the secretion of effectors AopH, AexT, AopP and AopO via T3SS, and for the first time demonstrated the impact of T3SS in secretion of Ati2, AopN and ExsE that are known as effectors in other pathogens. Translocators, needle subunits, Ati1, and AscX were also secreted in supernatants (SNs) dependent on T3SS. AopH, Ati2, AexT, AopB and AopD were in the top seven most abundant excreted proteins. EF-G, EF-Tu, DnaK, HtpG, PNPase, PepN and MdeA were moderately secreted in wt SNs and predicted to be putative T3 effectors by bioinformatics. Pta and ASA_P5G088 were increased in wt SNs and T3-associated in other bacteria. Ten conserved cytoplasmic proteins were more abundant in wt SNs than in the ΔascV mutant, but without any clear association to a secretion system. T1-secreted proteins were predominantly found in wt SNs: OmpAI, OmpK40, DegQ, insulinase ASA_0716, hypothetical ASA_0852 and ASA_3619. Presence of T3SS components in pellets was clearly decreased by ascV deletion, while no impact was observed on T1- and T2SS. Our results demonstrated that the ΔascV mutant strain excreted well-described (VapA, AerA, AerB, GCAT, Pla1, PlaC, TagA, Ahe2, GbpA and enolase) and yet uncharacterized potential toxins, adhesins and enzymes as much as or even more than the wt strain. Other putative important virulence factors were not detected. CONCLUSIONS We demonstrated the whole in vitro secretome and T3SS repertoire of hypervirulent A. salmonicida. Several toxins, adhesins and enzymes that are not part of the T3SS secretome were secreted to a higher extent in the extremely low-virulent ΔascV mutant. All together, our results show the high importance of an intact T3SS to initiate the furunculosis and offer new information about the pathogenesis.

Determination of genetic transferrin variants in human serum by high-resolution capillary zone electrophoresis(†)

High-resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate-deficient transferrin in human serum. The assay comprises mixing of human serum with a Fe(III) -containing solution prior to analysis of the iron-saturated mixture in a dynamically double-coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, one BD and three CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high-resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate-deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.

Automatic estimation of noise parameters in Fourier-domain optical coherence tomography cross sectional images using statistical information

We present an application and sample independent method for the automatic discrimination of noise and signal in optical coherence tomography Bscans. The proposed algorithm models the observed noise probabilistically and allows for a dynamic determination of image noise parameters and the choice of appropriate image rendering parameters. This overcomes the observer variability and the need for a priori information about the content of sample images, both of which are challenging to estimate systematically with current systems. As such, our approach has the advantage of automatically determining crucial parameters for evaluating rendered image quality in a systematic and task independent way. We tested our algorithm on data from four different biological and nonbiological samples (index finger, lemon slices, sticky tape, and detector cards) acquired with three different experimental spectral domain optical coherence tomography (OCT) measurement systems including a swept source OCT. The results are compared to parameters determined manually by four experienced OCT users. Overall, our algorithm works reliably regardless of which system and sample are used and estimates noise parameters in all cases within the confidence interval of those found by observers.

The impact of common versus separate estimation of orbit parameters on GRACE gravity field solutions

Gravity field parameters are usually determined from observations of the GRACE satellite mission together with arc-specific parameters in a generalized orbit determination process. When separating the estimation of gravity field parameters from the determination of the satellites’ orbits, correlations between orbit parameters and gravity field coefficients are ignored and the latter parameters are biased towards the a priori force model. We are thus confronted with a kind of hidden regularization. To decipher the underlying mechanisms, the Celestial Mechanics Approach is complemented by tools to modify the impact of the pseudo-stochastic arc-specific parameters on the normal equations level and to efficiently generate ensembles of solutions. By introducing a time variable a priori model and solving for hourly pseudo-stochastic accelerations, a significant reduction of noisy striping in the monthly solutions can be achieved. Setting up more frequent pseudo-stochastic parameters results in a further reduction of the noise, but also in a notable damping of the observed geophysical signals. To quantify the effect of the a priori model on the monthly solutions, the process of fixing the orbit parameters is replaced by an equivalent introduction of special pseudo-observations, i.e., by explicit regularization. The contribution of the thereby introduced a priori information is determined by a contribution analysis. The presented mechanism is valid universally. It may be used to separate any subset of parameters by pseudo-observations of a special design and to quantify the damage imposed on the solution.

Determination of vertebral range of motion using inertial measurement units in 27 Franches-Montagnes stallions and comparison between conditions and with a mixed population

REASONS FOR PERFORMING STUDY: The diagnosis of equine back disorders is challenging. Objectively determining movement of the vertebral column may therefore be of value in a clinical setting. OBJECTIVES: To establish whether surface-mounted inertial measurement units (IMUs) can be used to establish normal values for range of motion (ROM) of the vertebral column in a uniform population of horses trotting under different conditions. STUDY DESIGN: Vertebral ROM was established in Franches-Montagnes stallions and a general population of horses and the variability in measurements compared between the two groups. Repeatability and the influence of specific exercise condition (on ROM) were assessed. Finally, attempts were made to explain the findings of the study through the evaluation of factors that might influence ROM. METHODS: Dorsoventral (DV) and mediolateral (ML) vertebral ROM was measured at a trot under different exercise conditions in 27 Franches-Montagnes stallions and six general population horses using IMUs distributed over the vertebral column. RESULTS: Variability in the ROM measurements was significantly higher for general population horses than for Franches-Montagnes stallions (both DV and ML ROM). Repeatability was strong to very strong for DV measurements and moderate for ML measurements. Trotting under saddle significantly reduced the ROM, with sitting trot resulting in a significantly lower ROM than rising trot. Age is unlikely to explain the low variability in vertebral ROM recorded in the Franches-Montagnes horses, while this may be associated with conformational factors. CONCLUSIONS: It was possible to establish a normal vertebral ROM for a group of Franches-Montagnes stallions. While within-breed variation was low in this population, further studies are necessary to determine variation in vertebral ROM for other breeds and to assess their utility for diagnosis of equine back disorders.

Doppler Orbit Determination of Deep Space Probes by the Bernese GNSS Software: First Results of the Combined Orbit Determination from DSN and Inter-Satellite Ka-Band Data from the Grail Mission

Navigation of deep space probes is most commonly operated using the spacecraft Doppler tracking technique. Orbital parameters are determined from a series of repeated measurements of the frequency shift of a microwave carrier over a given integration time. Currently, both ESA and NASA operate antennas at several sites around the world to ensure the tracking of deep space probes. Just a small number of software packages are nowadays used to process Doppler observations. The Astronomical Institute of the University of Bern (AIUB) has recently started the development of Doppler data processing capabilities within the Bernese GNSS Software. This software has been extensively used for Precise Orbit Determination of Earth orbiting satellites using GPS data collected by on-board receivers and for subsequent determination of the Earth gravity field. In this paper, we present the currently achieved status of the Doppler data modeling and orbit determination capabilities in the Bernese GNSS Software using GRAIL data. In particular we will focus on the implemented orbit determination procedure used for the combined analysis of Doppler and intersatellite Ka-band data. We show that even at this earlier stage of the development we can achieve an accuracy of few mHz on two-way S-band Doppler observation and of 2 µm/s on KBRR data from the GRAIL primary mission phase.

Radiochemical determination of 129I and 36Cl in MEGAPIE, a proton irradiated lead-bismuth eutectic spallation target

The concentrations of the long-lived nuclear reaction products 129I and 36Cl have been measured in samples from the MEGAPIE liquid metal spallation target. Samples from the bulk target material (lead-bismuth eutectic, LBE), from the interface of the metal free surface with the cover gas, from LBE/steel interfaces and from noble metal absorber foils installed in the cover gas system were analysed using Accelerator Mass Spectrometry at the Laboratory of Ion beam Physics at ETH Zürich. The major part of 129I and 36Cl was found accumulated on the interfaces, particularly at the interface of LBE and the steel walls of the target container, while bulk LBE samples contain only a minor fraction of these nuclides. Both nuclides were also detected on the absorber foils to a certain extent (≪ 1% of the total amount). The latter number is negligible concerning the radio-hazard of the irradiated target material; however it indicates a certain affinity of the absorber foils for halogens, thus proving the principle of using noble metal foils for catching these volatile radionuclides. The total amounts of 129I and 36Cl in the target were estimated from the analytical data by averaging within the different groups of samples and summing up these averages over the total target. This estimation could account for about half of the amount of 129I and 36Cl predicted to be produced using nuclear physics modelling codes for both nuclides. The significance of the results and the associated uncertainties are discussed.