The identification, characterization, and expression of truncated hemoglobin genes in the nitrogen-fixing actinorhizal symbiont Frankia

Using a molecular approach (PCR), a group II truncated hemoglobin (trHb) gene was identified in several diverse isolates of Frankia. An analysis of three draft genome sequences for Frankia isolates EAN1pec, Ccl3, and ACN14a also revealed the presence of second trHb, homologous to group I trHbs. Phylogenetic analysis suggested that the Frankia trHb genes were grouped based on their respective genotype and clustered closest to Mycobacterium trHb genes. Frankia strain Ccl3 was grown under a variety of environmental stimuli to evaluate the expression of trHbN and trHbO genes. Nitrogen status did not affect expression of either gene, while oxidative stress caused a decrease in expression levels for both genes. The expression of trHbO increased under low oxygen environments, suggesting a role in increasing respiration rates. The expression of trHbN increased in response to spontaneously generated nitric oxide, suggesting a role in the protection from reactive nitrogen species

Causes of prehospital misinterpretations of ST elevation myocardial infarction

Objectives: To determine the causes of software misinterpretation of ST elevation myocardial infarction (STEMI) compared to clinically identified STEMI to identify opportunities to improve prehospital STEMI identification. Methods: We compared ECGs acquired from July 2011 through June 2012 using the LIFEPAK 15 on adult patients transported by the Los Angeles Fire Department. Cases included patients ≥18 years who received a prehospital ECG. Software interpretation of the ECG (STEMI or not) was compared with data in the regional EMS registry to classify the interpretation as true positive (TP), true negative (TN), false positive (FP), or false negative (FN). For cases where classification was not possible using registry data, 3 blinded cardiologists interpreted the ECG. Each discordance was subsequently reviewed to determine the likely cause of misclassification. The cardiologists independently reviewed a sample of these discordant ECGs and the causes of misclassification were updated in an iterative fashion. Results: Of 44,611 cases, 50% were male (median age 65; inter-quartile range 52–80). Cases were classified as 482 (1.1%) TP, 711 (1.6%) FP, 43371 (97.2%) TN, and 47 (0.11%) FN. Of the 711 classified as FP, 126 (18%) were considered appropriate for, though did not undergo, emergent coronary angiography, because the ECG showed definite (52 cases) or borderline (65 cases) ischemic ST elevation, a STEMI equivalent (5 cases) or ST-elevation due to vasospasm (4 cases). The sensitivity was 92.8% [95% CI 90.6, 94.7%] and the specificity 98.7% [95% CI 98.6, 98.8%]. The leading causes of FP were ECG artifact (20%), early repolarization (16%), probable pericarditis/myocarditis (13%), indeterminate (12%), left ventricular hypertrophy (8%), and right bundle branch block (5%). There were 18 additional reasons for FP interpretation (<4% each). The leading causes of FN were borderline ST-segment elevations less than the algorithm threshold (40%) and tall T waves reducing the ST/T ratio below threshold (15%). There were 11 additional reasons for FN interpretation occurring ≤3 times each. Conclusion: The leading causes of FP automated interpretation of STEMI were ECG artifact and non-ischemic causes of ST-segment elevation. FN were rare and were related to ST-segment elevation or ST/T ratio that did not meet the software algorithm threshold

Factors in perioperative care that determine blood loss in liver surgery

AbstractObjectivesExcessive blood loss during liver surgery contributes to postoperative morbidity and mortality and the minimizing of blood loss improves outcomes. This study examines pre- and intraoperative factors contributing to blood loss and identifies areas for improvement.MethodsAll patients who underwent elective hepatic resection between June 2007 and June 2009 were identified. Detailed information on the pre- and perioperative clinical course was analysed. Univariate and multivariate analyses were used to identify factors associated with intraoperative blood loss.ResultsA total of 175 patients were studied, of whom 95 (54%) underwent resection of three or more segments. Median blood loss was 782ml. Greater blood loss occurred during major resections and prolonged surgery and was associated with an increase in postoperative complications (P= 0.026). Peak central venous pressure (CVP) of >10cm H2O was associated with increased blood loss (P= 0.01). Although no differences in case mix were identified, blood loss varied significantly among anaesthetists, as did intraoperative volumes of i.v. fluids and transfusion practices.ConclusionsThis study confirms a relationship between CVP and blood loss in hepatic resection. Intraoperative CVP values were higher than those described in other studies. There was variation in the intraoperative management of patients. Collaboration between surgical and anaesthesia teams is required to minimize blood loss and the standardization of intraoperative anaesthesia practice may improve outcomes following liver surgery

Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

BACKGROUND: Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe. RESULTS: As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs. CONCLUSIONS: Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different species

Fluxes and fate of dissolved methane released at the seafloor at the landward limit of the gas hydrate stability zone offshore western Svalbard

Widespread seepage of methane from seafloor sediments offshore Svalbard close to the landward limit of the gas hydrate stability zone (GHSZ) may, in part, be driven by hydrate destabilization due to bottom water warming. To assess whether this methane reaches the atmosphere where it may contribute to further warming, we have undertaken comprehensive surveys of methane in seawater and air on the upper slope and shelf region. Near the GHSZ limit at ?400 m water depth, methane concentrations are highest close to the seabed, reaching 825 nM. A simple box model of dissolved methane removal from bottom waters by horizontal and vertical mixing and microbially mediated oxidation indicates that ?60% of methane released at the seafloor is oxidized at depth before it mixes with overlying surface waters. Deep waters are therefore not a significant source of methane to intermediate and surface waters; rather, relatively high methane concentrations in these waters (up to 50 nM) are attributed to isopycnal turbulent mixing with shelf waters. On the shelf, extensive seafloor seepage at <100 m water depth produces methane concentrations of up to 615 nM. The diffusive flux of methane from sea to air in the vicinity of the landward limit of the GHSZ is ?4–20 ?mol m?2 d?1, which is small relative to other Arctic sources. In support of this, analyses of mole fractions and the carbon isotope signature of atmospheric methane above the seeps do not indicate a significant local contribution from the seafloor source

Silence on Shangri-La: attenuation of Huygens acoustic signals suggests surface volatiles

Objective: Characterize and understand acoustic instrument performance on the surface of Titan. Methods: The Huygens probe measured the speed of sound in Titan's atmosphere with a 1 MHz pulse time-of-flight transducer pair near the bottom of the vehicle. We examine the fraction of pulses correctly received as af unction of time. Results: This system returned good data from about 11 km altitude, where the atmosphere became thick enough to effectively transmit the sound, down to the surface just before landing: these data have been analyzed previously. After an initial transient at landing, the instrument operated nominally for about 10 min, recording pulses much as during descent. The fraction of pulses detected then declined and the transmitted sound ceased to be detected altogether, despite no indication of instrument or probe configuration changes. Conclusions: The most likely explanation appears to be absorption of the signal by polyatomic gases with relaxation losses at the instrument frequency, such as ethane, acetylene and carbon dioxide. These vapors, detected independently by the GCMS instrument, were evolved from the surface material by the warmth leaking from the probe, and confirm the nature of the surface materials as ‘damp’ with a cocktail of volatile compounds. Some suggestions for future missions are considered. Practice implications: None

Measurement and modelling of dark current decay transients in perovskite solar cells

The current decay in response to a sudden change of applied bias up to 1 V has been measured on a methylammonium lead triiodide perovskite solar cell with titania and spiro-OMeTAD transport layers, for temperatures between 258 and 308 K. These measurements are highly reproducible, in contrast to most other techniques used to investigate perovskite cells. A drift-diffusion model that accounts for slow moving ions as well as electrons and holes acting as charge carriers was used to predict the current transients. The close fit of the model predictions to the measurements shows that mobile ions in the perovskite layer influence transient behaviour on timescales of up to 50 s. An activation energy of 0.55 eV is inferred from fitting simulations to measurements made at room temperature

Bedaquiline and clofazimine resistance in Mycobacterium tuberculosis: an in-vitro and in-silico data analysis

Background: Bedaquiline is a core drug for the treatment of multidrug-resistant tuberculosis; however, the understanding of resistance mechanisms is poor, which is hampering rapid molecular diagnostics. Some bedaquiline-resistant mutants are also cross-resistant to clofazimine. To decipher bedaquiline and clofazimine resistance determinants, we combined experimental evolution, protein modelling, genome sequencing, and phenotypic data. Methods: For this in-vitro and in-silico data analysis, we used a novel in-vitro evolutionary model using subinhibitory drug concentrations to select bedaquiline-resistant and clofazimine-resistant mutants. We determined bedaquiline and clofazimine minimum inhibitory concentrations and did Illumina and PacBio sequencing to characterise selected mutants and establish a mutation catalogue. This catalogue also includes phenotypic and genotypic data of a global collection of more than 14 000 clinical Mycobacterium tuberculosis complex isolates, and publicly available data. We investigated variants implicated in bedaquiline resistance by protein modelling and dynamic simulations. Findings: We discerned 265 genomic variants implicated in bedaquiline resistance, with 250 (94%) variants affecting the transcriptional repressor (Rv0678) of the MmpS5–MmpL5 efflux system. We identified 40 new variants in vitro, and a new bedaquiline resistance mechanism caused by a large-scale genomic rearrangement. Additionally, we identified in vitro 15 (7%) of 208 mutations found in clinical bedaquiline-resistant isolates. From our in-vitro work, we detected 14 (16%) of 88 mutations so far identified as being associated with clofazimine resistance and also seen in clinically resistant strains, and catalogued 35 new mutations. Structural modelling of Rv0678 showed four major mechanisms of bedaquiline resistance: impaired DNA binding, reduction in protein stability, disruption of protein dimerisation, and alteration in affinity for its fatty acid ligand. Interpretation: Our findings advance the understanding of drug resistance mechanisms in M tuberculosis complex strains. We have established an extended mutation catalogue, comprising variants implicated in resistance and susceptibility to bedaquiline and clofazimine. Our data emphasise that genotypic testing can delineate clinical isolates with borderline phenotypes, which is essential for the design of effective treatments. Funding: Leibniz ScienceCampus Evolutionary Medicine of the Lung, Deutsche Forschungsgemeinschaft, Research Training Group 2501 TransEvo, Rhodes Trust, Stanford University Medical Scientist Training Program, National Institute for Health and Care Research Oxford Biomedical Research Centre, Oxford University Hospitals NHS Foundation Trust, Bill & Melinda Gates Foundation, Wellcome Trust, and Marie Skłodowska-Curie Actions