Identification of a novel N-acetylmuramic acid (MurNAc) transporter in Tannerella forsythia.

Tannerella forsythia is a Gram-negative periodontal pathogen lacking the ability to undergo de novo synthesis of amino sugars N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) that form the disaccharide-repeating unit of the peptidoglycan backbone. T. forsythia relies on the uptake of these sugars from the environment, which is so far unexplored. Here, we identified a novel transporter system of T. forsythia involved in the uptake of MurNAc across the inner membrane and characterized a homolog of the Escherichia. coli MurQ etherase involved in the conversion of MurNAc-6P to GlcNAc-6P. The genes encoding these components were identified on a three gene cluster spanning Tanf_08375 to Tanf_08385 located downstream from a putative peptidoglycan recycling locus. We show that the three genes, Tanf_08375, Tanf_08380, and Tanf_08385, encoding a MurNAc transporter, a putative sugar kinase, and a MurQ etherase, respectively, are transcriptionally linked. Complementation of the Tanf_08375 and Tanf_08380 genes together in trans, but not individually rescued the inability of an E. coli mutant deficient in the PTS (phosphotransferase system)-dependent MurNAc transporter MurP as well as that of a double mutant deficient in MurP and components of the PTS system to grow on MurNAc. In addition, complementation with this two-gene construct in E. coli caused depletion of MurNAc in the medium, further confirming this observation. Our results show that the products of Tanf_08375 and Tanf_08380 constitute a novel non-PTS MurNAc transporter system that seems to be widespread among bacteria of the Bacteroidetes phylum. To the best of our knowledge, this is the first identification of a PTS-independent MurNAc transporter in bacteria. IMPORTANCE: In this study we report the identification of a novel transporter for peptidoglycan amino-sugar N-acetylmuramic acid (MurNAc) in the periodontal pathogen T. forsythia It has been known since the late 1980s that T. forsythia is a MurNAc auxotroph relying on environmental sources for this essential sugar. Most sugar transporters, and the MurNAc transporter MurP in particular require a PTS phosho-relay to drive the uptake and concurrent phosphorylation of the sugar through the inner membrane in Gram-negative bacteria. Our study uncovered a novel type of PTS-independent MurNAc transporter, and although so far unique to T. forsythia, may be present in a range of bacteria both of the oral cavity and gut especially of the phylum Bacteroidetes

EFFECTS OF SHORT COURSE VIRTUAL REALITY OR VIDEO BOX TRAINING FOR LAPAROSCOPIC BASIC SKILLS AND SIMULATED SURGICAL PERFORMANCE IN VETERINARY STUDENTS

Objective: To compare novices’ performance of simulated laparoscopic tasks on a box trainer and VR trainer before and after training on one system, and to compare performance between groups following training. Study Design: Randomized, prospective study Population: Veterinary students without prior hands-on laparoscopic experience were randomly assigned to the box or VR training group. Methods: Performance of basic laparoscopic tasks on box and VR trainers and a simulated surgical task were scored before and after completion of a designated training program. Scores for each task were compared within and between groups. Results: The box group’s post-training scores were higher than pre-training scores for all box tasks. The VR group’s post-training scores were higher than pre-training scores for most VR tasks. Median post-training scores were higher in the box group compared to the VR group for all box tasks. Median post-training scores were higher for the VR group compared to the box group only for VR intracorporeal suturing. Neither program led to significant improvement in simulated surgical task performance. Conclusion: In this study the box trainer seemed advantageous over the VR trainer for training novices to perform most basic tasks. The lack of significant improvement of simulated surgical task performance is likely related to study limitations including small sample size and short training timeline. Impact: Both training systems increase laparoscopic skills in novices. Simulation training of laparoscopic skills may be applicable within veterinary school curricula. Additional studies are warranted to assess these systems with different laparoscopic tasks and trainee skill levels

Photochemie und Verteilung von Hydroperoxiden im Bereich der Arabischen Halbinsel : Messungen während OMO und AQABA

Zusammenfassung Diese Arbeit behandelt Messungen und Analysen von Hydroperoxiden (ROOH) aus zwei Forschungsexpeditionen namens OMO und AQABA. Während OMO wurden Spurengase an Bord des HALO (High Altitude LOng range) Forschungsflugzeugs im Sommer 2015 gemessen um die Auswirkungen des Asiatischen Monsuns und dessen Einfluss auf die Asiatische Monsun Antizyklone (AMA) in der oberen Troposphäre (UT) über der Arabischen Halbinsel und dem Mittelmeer (MM) zu untersuchen. Bei dieser Studie liegt der Fokus auf Wasserstoffperoxid (H2O2) und organischen Hydroperoxiden (OrgHP), sowie deren Vorläufer und Verlustprozesse. Die gemessenen Daten wurden mit Berechnungen über das photostationäre Gleichgewicht (PSS) für H2O2, Methylhydroperoxid (MHP) und sich daraus ergebenden unbekannten Hydroperoxiden (UHP), sowie dem globalen Zirkulations-modell EMAC verglichen. Dabei sind erhöhte Mischungsverhältnisse für H2O2, MHP und UHP in der AMA im Vergleich zum nordhemisphärischen Hintergrund zu beobachten. Die höchsten Mischungsverhältnisse für H2O2 und MHP werden im südhemisphärischen Hintergrund erhalten, während für UHP maximale Werte in der AMA vorzufinden sind. Insgesamt liegen die gemessenen Mischungs-verhältnisse höher als in EMAC und den PSS-Berechnungen. Besonders in der AMA unterschätzt EMAC H2O2 und MHP. Ein Gradient entlang des Längengrads weist auf ein Reservoir von Hydroperoxiden in Richtung des AMA-Zentrums hin, welches vermutlich durch konvektive Zufuhr über Indien entsteht. Das lässt auf einen hohen Beitrag von atmosphärischem Transport zu dem lokalen ROOH Budget entlang der Flugroute schließen, welcher die große Abweichung zum lokalen PSS Modell erklärt. Die Diskrepanz zwischen Messung und EMAC kann mit Hilfe einer Sensitivitätsstudie auf Unsicherheiten der einzelnen ROOH Auswasch-effizienzen während der Konvektion zurückgeführt werden. Vermutlich handelt es sich bei UHP damit um MHP, welches aus der Grenzschicht über Südostasien in die UT gelangt und mit der AMA in Richtung Westen transportiert wird. AQABA fand im Sommer 2017 auf dem Schiff Kommandor Iona während einer Fahrt von Frankreich um die Arabische Halbinsel herum bis nach Kuwait und zurück statt. Dabei wurde der Einfluss von Luftverschmutzung auf die menschliche Gesundheit, das Klima und die natürliche Umgebung erforscht. Die befahrenen Regionen reichen von sauberer maritimer Luft über der Arabischen See (AS) über unbeeinflusste aber staubige Luft mit afrikanischer Herkunft über dem Roten Meer (RM) und leicht beeinflussten Luftmassen über dem MM im Abwind großer Städte bis hin zu stark durch Schiffsabgase und Ölförderung beeinflussten Luftmassen im Persischen Golf. Erhöhte ROOH Mischungsverhältnisse und leichte Tagesgänge treten immer dann auf, wenn der Einfluss aus der planetaren Grenzschicht (PBL) erhöht ist, während in unbeeinflussten Luftmassen besonders OrgHP konstant niedrige Werte aufweist. Berechnungen der nächtlichen Depositionsrate kdep sowie der Produktionsrate von H2O2 liegen im Bereich früherer Studien, während besonders bei Sonnenhöchststand im MM deutlich erhöhte kdep vorliegen. Thermisch induzierte Turbulenz und damit erhöhter Transport zur Oberfläche verbunden mit steigender Deposition am Tag kann den schwachen Tagesgang erklären, obwohl keine direkte (Anti-)Korrelation mit Staubpartikeln besteht. Der Vergleich mit EMAC zeigt trotz einer leichten Überschätzung in der AS gute qualitative und quantitative Übereinstimmung für OrgHP. Im Gegensatz dazu wird H2O2 mit einer signifikant höheren Löslichkeit durch unterschätzte Verluste in Form von Deposition am Tag deutlich überschätzt. Darüber hinaus zeigt der Abgleich der Grenzschichthöhe mit ERA5 Daten einen deutlich zu großen Beitrag der PBL in EMAC, welcher besonders in den Morgenstunden einen starken Anstieg durch eingetragenes H2O2 aus der freien Troposphäre zur Folge hat.Abstract This thesis deals with the observations and analyses of two research expeditions called OMO and AQABA. During OMO, trace gas measurements were performed onboard the HALO (High Altitude LOng range) research aircraft in summer 2015 in order to investigate the outflow of the South Asian summer monsoon and its influence on the composition of the Asian Monsoon Anticyclone (AMA) in the upper troposphere (UT) over the eastern Mediterranean and the Arabian Peninsula. This study focuses on in-situ observations of hydrogen peroxide (H2O2) and organic hydroperoxides (OrgHP) and their precursors and loss processes. Observations are compared to steady-state calculations of H2O2, methyl hydroperoxide (MHP) and inferred unidentified hydroperoxide (UHP) mixing ratios as well as to simulations with the general circulation ECHAM/MESSy for Atmospheric Chemistry (EMAC) model. Enhanced mixing ratios of H2O2, MHP and UHP were observed in the AMA relative to the northern hemispheric background. The highest mixing ratios for H2O2 and MHP were found in the southern hemisphere outside the AMA, while for UHP, the highest concentrations were encountered within the AMA. In general, the observed concentrations are higher than steady-state calculations and EMAC simulations. Especially in the AMA, EMAC underestimates H2O2 and OrgHP. Longitudinal gradients indicate a pool of hydroperoxides towards the center of the AMA, which is most likely associated with upwind convection over India. This indicates main contributions of atmospheric transport to the local budgets of hydroperoxides along the flight track, explaining strong deviations to the steady-state calculation, which only accounts for local photochemistry. Deviations to EMAC simulations are most likely due to uncertainties in the scavenging efficiencies for individual hydroperoxides in deep convective transport to the UT, corroborated by a sensitivity study. It seems that the observed excess UHP is excess MHP transported to the west from an upper tropospheric source related to convection in the summer monsoon over South-East Asia. AQABA took place onboard the vessel Kommandor Iona. Measurements were performed during the shipping from France around the Arabian Peninsula to Kuwait and back. Thereby the influence of pollution on human health, the climate and the natural environment were investigated. During the trip air masses from clean marine conditions above the Arabian Sea (AS) over clean but dusty air masses originating over Africa and moderate polluted air downwind of megacities to polluted regions with ship exhaust and petrochemical industry in the Persian Gulf were encountered. Enhanced mixing ratios and slight diel variations for H2O2 and OrgHP occurred in areas with larger influence of the planetary boundary layer (PBL) while for clean conditions especially OrgHP mixing ratios were constantly low. Calculations of the production and nighttime deposition rates kdep for H2O2 are in the range of previous studies while especially at noon in the Mediterranean significantly higher values for kdep were observed. Thermally induced turbulences and thus increased transport to the surface together with higher daytime deposition rates explain the weak diel variations although no (anti)correlation with dust particles was found. The comparison to EMAC shows good qualitative and quantitative agreement for OrgHP even though there is a slight overestimation in the AS. In contrast, for the highly soluble H2O2 there is a large overestimation due to underestimated kdep during daytime. Additionally, the comparison to the ERA5 boundary layer height shows a substantial influence of the PBL in EMAC which causes a large contribution of H2O2 from the free troposphere and thus a steep increase of mixing ratios in the morning

The performance of stochastic designs in wellbore drilling operations

© 2018, The Author(s). Wellbore drilling operations frequently entail the combination of a wide range of variables. This is underpinned by the numerous factors that must be considered in order to ensure safety and productivity. The heterogeneity and sometimes unpredictable behaviour of underground systems increases the sensitivity of drilling activities. Quite often the operating parameters are set to certify effective and efficient working processes. However, failings in the management of drilling and operating conditions sometimes result in catastrophes such as well collapse or fluid loss. This study investigates the hypothesis that optimising drilling parameters, for instance mud pressure, is crucial if the margin of safe operating conditions is to be properly defined. This was conducted via two main stages: first a deterministic analysis—where the operating conditions are predicted by conventional modelling procedures—and then a probabilistic analysis via stochastic simulations—where a window of optimised operation conditions can be obtained. The outcome of additional stochastic analyses can be used to improve results derived from deterministic models. The incorporation of stochastic techniques in the evaluation of wellbore instability indicates that margins of the safe mud weight window are adjustable and can be extended considerably beyond the limits of deterministic predictions. The safe mud window is influenced and hence can also be amended based on the degree of uncertainty and the permissible level of confidence. The refinement of results from deterministic analyses by additional stochastic simulations is vital if a more accurate and reliable representation of safe in situ and operating conditions is to be obtained during wellbore operations.Published versio

N-Acetylmuramic Acid (MurNAc) Auxotrophy of the Oral Pathogen Tannerella forsythia: Characterization of a MurNAc Kinase and Analysis of Its Role in Cell Wall Metabolism

Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104 - fold higher catalytic efficiency (kcat/Km) for MurNAc than for N-acetylglucosamine (GlcNAc) with kcat values of 10.5 s−1 and 0.1 s−1 and Km values of 200 µM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia, a kinase deletion mutant (1Tf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the 1Tf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 µg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 µg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis

Impact of the South Asian monsoon outflow on atmospheric hydroperoxides in the upper troposphere

During the OMO (Oxidation Mechanism Observation) mission, trace gas measurements were performed on board the HALO (High Altitude Long Range) research aircraft in summer 2015 in order to investigate the outflow of the South Asian summer monsoon and its influence on the composition of the Asian monsoon anticyclone (AMA) in the upper troposphere over the eastern Mediterranean and the Arabian Peninsula. This study focuses on in situ observations of hydrogen peroxide (H$_{2}$O$_{2}$$^{obs}$) and organic hydroperoxides (ROOH$^{obs}$) as well as their precursors and loss processes. Observations are compared to photostationary-state (PSS) calculations of H$_{2}$O$_{2}$$^{PSS}$ and extended by a separation of ROOH$^{obs}$ into methyl hydroperoxide (MHP$^{PSS}$) and inferred unidentified hydroperoxide (UHP$^{PSS}$) mixing ratios using PSS calculations. Measurements are also contrasted to simulations with the general circulation ECHAM–MESSy for Atmospheric Chemistry (EMAC) model. We observed enhanced mixing ratios of H$_{2}$O$_{2}$$^{obs}$ (45 %), MHP$^{PSS}$ (9 %), and UHPPSS (136 %) in the AMA relative to the northern hemispheric background. Highest concentrations for H$_{2}$O$_{2}$$^{obs}$ and MHP$^{PSS}$ of 211 and 152 ppb$_{V}$, respectively, were found in the tropics outside the AMA, while for UHP$^{PSS}$, with 208 ppt$_{V}$, highest concentrations were found within the AMA. In general, the observed concentrations are higher than steady-state calculations and EMAC simulations by a factor of 3 and 2, respectively. Especially in the AMA, EMAC underestimates the H$_{2}$O$_{2}$$^{EMAC}$ (medians: 71 ppt$_{V}$ vs. 164 ppt$_{V}$) and ROOHEMAC (medians: 25 ppt$_{V}$ vs. 278 ppt$_{V}$) mixing ratios. Longitudinal gradients indicate a pool of hydroperoxides towards the center of the AMA, most likely associated with upwind convection over India. This indicates main contributions of atmospheric transport to the local budgets of hydroperoxides along the flight track, explaining strong deviations from steady-state calculations which only account for local photochemistry. Underestimation of H$_{2}$O$_{2}$$^{EMAC}$ by approximately a factor of 2 in the Northern Hemisphere (NH) and the AMA and overestimation in the Southern Hemisphere (SH; factor 1.3) are most likely due to uncertainties in the scavenging efficiencies for individual hydroperoxides in deep convective transport to the upper troposphere, corroborated by a sensitivity study. It seems that the observed excess UHPPSS is excess MHP transported to the west from an upper tropospheric source related to convection in the summer monsoon over Southeast Asia

The exo-β-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases

Endo-β-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the β-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-β-N-acetylmuramidases, which catalyze an exo-lytic cleavage of β-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-β-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl β-MurNAc and the peptidoglycan-derived disaccharide MurNAc-β-1,4-GlcNAc. Together with the exo-β-N-acetylglucosaminidase NagZ and the exo-muramoyl-L-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www. cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria

Kaiserwette(r)

Der Band "Kaiserwette(r) – Engelbert Humperdinck in seiner Zeit", geht auf eine Tagung zurück, die Ende September 2021 in Siegburg stattfand, am berufenen Ort, der Geburtsstadt des Komponisten. Sein Ziel ist es allerdings nicht, Humperdinck zu sezieren, indem man ihn sozusagen in einen ‚Guten‘ und einen ‚Schlechten‘ zerlegt, also hier weiß- und dort schwarzmalt. Die Intention ist es vielmehr auf jene differenzierenden Grautöne gerichtet, die für das historische Verständnis unabdingbare Voraussetzung sind

A Photoelectron Spectroscopic Investigation of Vinyl Fluoride (C2H3F). The HeI, Threshold and CIS Photoelectron Spectroscopy.

The threshold photoelectron spectrum (TPES) and the constant ion state (CIS) spectra of the individual ionic states of C2H3F have been recorded using synchrotron radiation. For comparison a well resolved HeI photoelectron spectrum (HeI-PES) has also been measured and analyzed in detail. The TPES has been measured between 9.5 eV and 35 eV photon energy. Numerous vibrational structures, reported for the first time, observed in the ground state and the six excited states of the cation are analyzed. Quantum chemical calculations have been performed and provide strong support to the assignments. State selected CIS spectra highlighted the major importance of autoionization for the production of almost all ionized states of C2H3F observed in this work