Species-specific viability analysis of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus in mixed culture by flow cytometry

BACKGROUND: Bacterial species coexist commonly in mixed communities, for instance those occurring in microbial infections of humans. Interspecies effects contribute to alterations in composition of communities with respect to species and thus, to the course and severity of infection. Therefore, knowledge concerning growth and viability of single species in medically-relevant mixed communities is of high interest to resolve complexity of interspecies dynamics and to support development of treatment strategies. In this study, a flow cytometric method was established to assess the species-specific viability in defined three-species mixed cultures. The method enables the characterization of viability of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus, which are relevant to lung infections of Cystic Fibrosis (CF) patients. The method combines fluorescence detection by antibody and lectin labeling with viability fluorescence staining using SYBR®Green I and propidium iodide. In addition, species-specific cell enumeration analysis using quantitative terminal restriction fragment length polymorphisms (qT-RFLP) was used to monitor the growth dynamics. Finally, to investigate the impact of substrate availability on growth and viability, concentrations of main substrates and metabolites released were determined. RESULTS: For each species, the time course of growth and viability during mixed culture cultivations was obtained by using qT-RFLP analysis in combination with flow cytometry. Comparison between mixed and pure cultures revealed for every species differences in growth properties, e.g. enhanced growth of P. aeruginosa in mixed culture. Differences were also observed for B. cepacia and S. aureus in the time course of viability, e.g. an early and drastic reduction of viability of S. aureus in mixed culture. Overall, P. aeruginosa clearly dominated the mixed culture with regard to obtained cell concentrations. CONCLUSIONS: In combination with qT-RFLP analysis, the methods enabled monitoring of species-specific cell concentrations and viability during co-cultivation of theses strains. Experimental findings suggest that the predominance of P. aeruginosa over B. cepacia and S. aureus in mixed culture under the chosen cultivation conditions is promoted by more efficient substrate consumption of P. aeruginosa, and antagonistic interspecies effects induced by P. aeruginosa

A preliminary phylogeny of Loasaceae subfam. Loasoideae (Angiospermae: Cornales) based on trnL(UAA) sequence data, with consequences for systematics and historical biogeography

AbstractThe phylogeny of Loasaceae subfam. Loasoideae is investigated with sequences of the chloroplast trnL(UAA) intron, all genera and infrageneric entities are included in the analysis. Loasaceae subfam. Loasoideae is monophyletic, and the two most speciose, and monophyletic, clades (which account for approximately 90% of the species total) are Nasa and the so-called Southern Andean Loasas (Blumenbachia, Caiophora, Loasa s.str., Scyphanthus), but the phylogeny of the remainder is not completely resolved. The data underscore a basal position for Chichicaste, Huidobria, Kissenia, and Klaprothieae (Xylopodia, Klaprothia, Plakothira). High bootstrap support values confirm the monophyly both of Klaprothieae and Presliophytum (when expanded to include Loasa ser. Malesherbioideae). Aosa and Blumenbachia are not resolved as monophyletic, but have clear morphological apomorphies. Within Nasa, “N. ser. Saccatae” is paraphyletic, and “N. ser. Carunculatae” is polyphyletic. However, the N. triphylla group in “N. ser. Saccatae” is a well-supported monophyletic group, as is N. ser. Grandiflorae. “Loasa” in its traditional circumscription is paraphyletic, but Loasa s.str. (L. ser. Macrospermae, L. ser. Deserticolae, L. ser. Floribundae) is monophyletic. The remainder of “Loasa” (L. ser. Pinnatae, L. ser. Acaules, L. ser. Volubiles) is probably closely allied to the essentially Patagonian-High Andean group comprising also Scyphanthus and Caiophora. These findings are congruent with morphology and phytogeography. Nasa seems to have undergone its primary radiation at moderate elevations (1500–2500m) in the Andes of northern Peru (Amotape-Huancabamba Zone) and subsequently diversified into high elevations (above 4000m) of the tropical Central Andes. South Andean Loasas appear to have undergone their primary diversification in the southern temperate and mediterranean regions of Chile and Argentina, with a subsequent northwards expansion of Caiophora into the high elevations of the tropical Andes. Hummingbird pollination has evolved independently from melittophily in High Andean clades of Nasa and Caiophora

Mechanical bowel preparation and antimicrobial prophylaxis in elective colorectal surgery in Switzerland—a survey

Background: The aim of this study was to assess the use of mechanical bowel preparation (MBP) and antimicrobial prophylaxis in elective colorectal surgery in Switzerland. Methods: Ninety-eight heads of surgical departments in Switzerland and 42 visceral surgeons in private practice were asked to answer an 18-item questionnaire in October 2008 about arguments in favor of or against MBP. The participants also indicated whether they use MBP and antimicrobial prophylaxis in colorectal surgery, and if so, what agents were used. Of the participants, 117/140 (83%) responded. Additional data were collected pertaining to the respondents' experience and work situation. Results: MBP was used significantly more often for rectal surgery than for left colonic resections (83% vs. 53%; p < 0.001) and more often for left than for right colonic resections (53% vs. 43%; p = 0.001), regardless of the open or laparoscopic approach. Younger surgeons and surgeons with a higher case load in colorectal surgery used MBP significantly less frequently in open right colonic resections. For MBP, cathartics were used in 90% of patients, and enemas were used in 10% of patients. Of the respondents, 37% considered MBP to be useful, even very useful. Based on the literature, because of introduction of fast-track protocols or for considerations of patient comfort, 86% of the respondents had changed the bowel preparation regime during the last 10years in terms of a reduction of the quantity of cathartics or restricted the indications for MBP. Antimicrobial prophylaxis was used by 100% of the respondents, 88% used a single prophylactic dose only, while 70% administered the antibiotics 30-59min before the incision. Most of the surgeons used second-generation cephalosporins in combination with metronidazole, and 24% changed the antibiotic agent or reduced the duration of administration of antibiotics during the last 10years. Conclusions: MBP is often used in open and laparoscopic rectal surgery, but not in right colonic resections. Scientific evidence regarding MBP has yielded a rethinking about rigorous bowel preparation regimes. As of now, surgeons in Switzerland are not yet unanimously ready to abandon MBP in elective colorectal surgery. In Switzerland, surgeons are influenced by the benefit of antimicrobial prophylaxis in colorectal surger

Efficient reconstruction of prior austenite grains in steel from etched light optical micrographs using deep learning and annotations from correlative microscopy

The high-temperature austenite phase is the initial state of practically all technologically relevant hot forming and heat treatment operations in steel processing. The phenomena occurring in austenite, such as recrystallization or grain growth, can have a decisive influence on the subsequent properties of the material. After the hot forming or heat treatment process, however, the austenite transforms into other microstructural constituents and information on the prior austenite morphology are no longer directly accessible. There are established methods available for reconstructing former austenite grain boundaries via metallographic etching or electron backscatter diffraction (EBSD) which both exhibit shortcomings. While etching is often difficult to reproduce and strongly depend on the investigated steel’s alloying concept, EBSD acquisition and reconstruction is rather time-consuming. But in fact, though, light optical micrographs of steels contrasted with conventional Nital etchant also contain information about the former austenite grains. However, relevant features are not directly apparent or accessible with conventional segmentation approaches. This work presents a deep learning (DL) segmentation of prior austenite grains (PAG) from Nital etched light optical micrographs. The basis for successful segmentation is a correlative characterization from EBSD, light and scanning electron microscopy to specify the ground truth required for supervised learning. The DL model shows good and robust segmentation results. While the intersection over union of 70% does not fully reflect the model performance due to the inherent uncertainty in PAG estimation, a mean error of 6.1% in mean grain size derived from the segmentation clearly shows the high quality of the result

Three years of Fermi GBM Earth Occultation Monitoring: Observations of Hard X-ray/Soft Gamma-Ray Sources

The Gamma ray Burst Monitor (GBM) on board Fermi has been providing continuous data to the astronomical community since 2008 August 12. In this paper we present the results of the analysis of the first three years of these continuous data using the Earth occultation technique to monitor a catalog of 209 sources. From this catalog, we detect 99 sources, including 40 low-mass X-ray binary/neutron star systems, 31 high-mass X-ray binary neutron star systems, 12 black hole binaries, 12 active galaxies, 2 other sources, plus the Crab Nebula, and the Sun. Nine of these sources are detected in the 100-300 keV band, including seven black-hole binaries, the active galaxy Cen A, and the Crab. The Crab and Cyg X-1 are also detected in the 300-500 keV band. GBM provides complementary data to other sky-monitors below 100 keV and is the only all-sky monitor above 100 keV. Up-to-date light curves for all of the catalog sources can be found at http://heastro.phys.lsu.edu/gbm/.Comment: 24 pages, 12 figures, accepted for publication in ApJ

Rational Design of Phe‐BODIPY Amino Acids as Fluorogenic Building Blocks for Peptide‐based Detection of Urinary Tract Candida Infections

Fungal infections caused by Candida species are among the most prevalent in hospitalized patients. However, current methods for the detection of Candida fungal cells in clinical samples rely on time‐consuming assays that hamper rapid and reliable diagnosis. Herein, we describe the rational development of new Phe‐BODIPY amino acids as small fluorogenic building blocks and their application to generate fluorescent antimicrobial peptides for rapid labelling of Candida cells in urine. We have used computational methods to analyse the fluorogenic behaviour of BODIPY‐substituted aromatic amino acids and performed bioactivity and confocal microscopy experiments in different strains to confirm the utility and versatility of peptides incorporating Phe‐BODIPYs. Finally, we have designed a simple and sensitive fluorescence‐based assay for the detection of Candida albicans in human urine samples

Time-Resolved Spectroscopy of the 3 Brightest and Hardest Short Gamma-Ray Bursts Observed with the FGST Gamma-Ray Burst Monitor

From July 2008 to October 2009, the Gamma-ray Burst Monitor (GBM) on board the Fermi Gamma-ray Space Telescope (FGST) has detected 320 Gamma-Ray Bursts (GRBs). About 20% of these events are classified as short based on their T90 duration below 2 s. We present here for the first time time-resolved spectroscopy at timescales as short as 2 ms for the three brightest short GRBs observed with GBM. The time-integrated spectra of the events deviate from the Band function, indicating the existence of an additional spectral component, which can be fit by a power-law with index ~-1.5. The time-integrated Epeak values exceed 2 MeV for two of the bursts, and are well above the values observed in the brightest long GRBs. Their Epeak values and their low-energy power-law indices ({\alpha}) confirm that short GRBs are harder than long ones. We find that short GRBs are very similar to long ones, but with light curves contracted in time and with harder spectra stretched towards higher energies. In our time-resolved spectroscopy analysis, we find that the Epeak values range from a few tens of keV up to more than 6 MeV. In general, the hardness evolutions during the bursts follows their flux/intensity variations, similar to long bursts. However, we do not always see the Epeak leading the light-curve rises, and we confirm the zero/short average light-curve spectral lag below 1 MeV, already established for short GRBs. We also find that the time-resolved low-energy power-law indices of the Band function mostly violate the limits imposed by the synchrotron models for both slow and fast electron cooling and may require additional emission processes to explain the data. Finally, we interpreted these observations in the context of the current existing models and emission mechanisms for the prompt emission of GRBs.Comment: 14 pages, 10 figures, 9 tables, Accepted for publication in the Astrophysical Journal September, 23 2010 (Submitted May, 16 2010) Corrections: 1 reference updated, figure 10 captio