Reduced ribosomal thermal denaturation in Listeria monocytogenes following osmotic and heat shocks

Abstract Increased thermotolerance of Listeria monocytogenes induced by exposure to a high NaCl concentration or a sublethal heat shock was concurrent with increased thermal stability of the 30S ribosomal subunit as measured by differential scanning calorimetry. It is proposed that protection of the 30S subunit is a critical mechanism for increased thermotolerance.     

Catechol production by O-demethylation of 2-methoxyphenol using the obligate anaerobe, Acetobacterium woodii

Acetobacterium woodii produced catechol (up to 7.84 mM) by demethylating 2-methoxyphenol during growth in the presence or absence of fructose. The highest product concentrations were obtained when 2-methoxyphenol was the sole energy source but the highest substrate conversion (97%) was obtained in fructose-limited chemostat culture. Growing cells were the most suitable form of the biocatalyst since the catalytic activity was 5-fold higher than in harvested cells.     

The expression of a synthetic rainbow trout metallothionein gene in E. coli

A synthetic gene for rainbow trout metallothionein was constructed and inserted into a dual origin plasmid where expression was induced by a temperature shift in a proteinase-deficient strain of Escherichia coli. The recombinant protein was purified to homogeneity, and a partial amino acid sequence was determined to confirm its identity. Its immunochemical characteristics were similar to those of native metallothionein from rainbow trout. The amounts of recombinant metallothionein produced were quantified in soluble cell extracts by ELISA. Low concentrations were detected when growth was performed either in L-broth or defined (GMM-II) medium. Supplementation of the medium with zinc or copper had no effect on the amount of metallothionein produced. By contrast, when cadmium was included in either L-broth or GMM-II medium, much higher concentrations of the protein within the cells (approx. 13 micrograms/mg soluble cell protein) were detected. This stabilisation of the protein by metal reconstitution in vivo is considered in relation to the selective uptake/exclusion of metals by the cells and its significance for the scavenging of certain precious or toxic heavy metals is discussed.     

An optimized procedure for the capture,fractionation and proteomic analysis of proteins using hydrogel nanoparticles

We have developed an optimized procedure using dual size exclusion/affinity hydrogel nanoparticles to capture and comparatively analyze low molecular mass proteins directly from biological samples. The method described facilitates charge‐ and size‐dependent protein binding, direct analysis by MS or other means and is highly reproducible. A comparative analysis of the low molecular mass proteome of plasma following freeze–thaw immediately after venipuncture is used to illustrate proof‐of‐concept. The technique described is rapid and may be easily reproduced in any laboratory.     

A Comparison of Supervised Classification Methods for the Prediction of Substrate Type Using Multibeam Acoustic and Legacy Grain-Size Data

Detailed seabed substrate maps are increasingly in demand for effective planning and management of marine ecosystems and resources. It has become common to use remotely sensed multibeam echosounder data in the form of bathymetry and acoustic backscatter in conjunction with ground-truth sampling data to inform the mapping of seabed substrates. Whilst, until recently, such data sets have typically been classified by expert interpretation, it is now obvious that more objective, faster and repeatable methods of seabed classification are required. This study compares the performances of a range of supervised classification techniques for predicting substrate type from multibeam echosounder data. The study area is located in the North Sea, off the north-east coast of England. A total of 258 ground-truth samples were classified into four substrate classes. Multibeam bathymetry and backscatter data, and a range of secondary features derived from these datasets were used in this study. Six supervised classification techniques were tested: Classification Trees, Support Vector Machines, k-Nearest Neighbour, Neural Networks, Random Forest and Naive Bayes. Each classifier was trained multiple times using different input features, including i) the two primary features of bathymetry and backscatter, ii) a subset of the features chosen by a feature selection process and iii) all of the input features. The predictive performances of the models were validated using a separate test set of ground-truth samples. The statistical significance of model performances relative to a simple baseline model (Nearest Neighbour predictions on bathymetry and backscatter) were tested to assess the benefits of using more sophisticated approaches. The best performing models were tree based methods and Naive Bayes which achieved accuracies of around 0.8 and kappa coefficients of up to 0.5 on the test set. The models that used all input features didn''t generally perform well, highlighting the need for some means of feature selection.     

CD36 and Fyn Kinase Mediate Malaria-Induced Lung Endothelial Barrier Dysfunction in Mice Infected with Plasmodium berghei

Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36−/− mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σ_alb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36−/− mice had near normal fluid conductance but a similar reduction in σ_alb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36−/− mice were exposed to H₂O₂. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H₂O₂ indicating loss of barrier function, CD36−/− MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36−/− endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36−/− lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn−/− mice, which were also protected from H₂O₂- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.