High Spatial Resolution Imaging of Endogenous Hydrogen Peroxide in Living Cells by Solid‐State Fluorescence

Herein, we describe selective imaging of hydrogen peroxide using a precipitating dye conjugated to a boronic acid‐based immolative linker. We achieved visualization of endogenous hydrogen peroxide in phagosomes by solid‐state two‐photon fluorescence imaging in living cells with exceptionally high spatial resolution.     

Determination of the Composition of Commercial Tannin Extracts by Liquid Secondary Ion Mass Spectrometry (LSIMS)

The composition of various commercial tannin extracts were determined by liquid secondary ion mass spectrometry (LSIMS). Spectra were obtained directly from tannin extracts without any pre-separation. Eight different tannin powders were analysed: three gallotannins (Chinese, Turkish, tara), three ellagitannins (sweet chestnut, pendunculata oak, sessile oak), one mixed hydrolysable tannin (myrabolans) and one proanthocyanidin (grape seeds). This method enabled the main molecules in these powders to be identified.     

Opioids and Sepsis: Elucidating the Role of the Microbiome and microRNA-146

Sepsis has recently been defined as life-threatening organ dysfunction caused by the dysregulated host response to an ongoing or suspected infection. To date, sepsis continues to be a leading cause of morbidity and mortality amongst hospitalized patients. Many risk factors contribute to development of sepsis, including pain-relieving drugs like opioids, which are frequently prescribed post-operatively. In light of the opioid crisis, understanding the interactions between opioid use and the development of sepsis has become extremely relevant, as opioid use is associated with increased risk of infection. Given that the intestinal tract is a major site of origin of sepsis-causing microbes, there has been an increasing focus on how alterations in the gut microbiome may predispose towards sepsis and mediate immune dysregulation. MicroRNAs, in particular, have emerged as key modulators of the inflammatory response during sepsis by tempering the immune response, thereby mediating the interaction between host and microbiome. In this review, we elucidate contributing roles of microRNA 146 in modulating sepsis pathogenesis and end with a discussion of therapeutic targeting of the gut microbiome in controlling immune dysregulation in sepsis.     

Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.     

Bifunctional DNA Duplexes Permit Efficient Incorporation of pH Probes into Liposomes

Enzyme-mediated proton transport across biological membranes is critical for many vital cellular processes. pH-sensitive fluorescent dyes are an indispensable tool for investigating the molecular mechanism of proton-translocating enzymes. Here, we present a novel strategy to entrap pH-sensitive probes in the lumen of liposomes that has several advantages over the use of soluble or lipid-coupled probes. In our approach, the pH sensor is linked to a DNA oligomer with a sequence complementary to a second oligomer modified with a lipophilic moiety that anchors the DNA conjugate to the inner and outer leaflets of the lipid bilayer. The use of DNA as a scaffold allows subsequent selective enzymatic removal of the probe in the outer bilayer leaflet. The method shows a high yield of insertion and is compatible with reconstitution of membrane proteins by different methods. The usefulness of the conjugate for time-resolved proton pumping measurements was demonstrated by using two large membrane protein complexes.     

Dichotomous feedback: a signal sequestration-based feedback mechanism for biocontroller design

We introduce a new design framework for implementing negative feedback regulation in synthetic biology, which we term ‘dichotomous feedback’. Our approach is different from current methods, in that it sequesters existing fluxes in the process to be controlled, and in this way takes advantage of the process’s architecture to design the control law. This signal sequestration mechanism appears in many natural biological systems and can potentially be easier to realize than ‘molecular sequestration’ and other comparison motifs that are nowadays common in biomolecular feedback control design. The loop is closed by linking the strength of signal sequestration to the process output. Our feedback regulation mechanism is motivated by two-component signalling systems, where a second response regulator could be competing with the natural response regulator thus sequestering kinase activity. Here, dichotomous feedback is established by increasing the concentration of the second response regulator as the level of the output of the natural process increases. Extensive analysis demonstrates how this type of feedback shapes the signal response, attenuates intrinsic noise while increasing robustness and reducing crosstalk.     

Thermal and economic evaluation of heat recovery measures for indoor ice rinks

A comparative study of three ventilation systems supplying air to a typical Canadian indoor ice rink illustrates the power, energy and operating cost savings which can be achieved by using the relatively warm air from the air cooled condensers of the refrigeration system. The direct use of this warm air for ventilation results in a reduction of energy consumption during the winter amounting to 24.2% of the yearly consumption of the heating system. On the other hand, the use of a heat exchanger to heat the ventilation air results in energy consumption reductions throughout the year. Depending on the size of the heat exchanger these gains can be as high as 60.8% of the heating energy consumption by the existing system. Based on actual prices of electricity and the heat exchanger it is established that the cost savings over the life of the equipment are at least three times higher than the cost of the heat exchanger.     

Evaluation and optimisation of five different extraction methods for soy DNA in chocolate and biscuits. Extraction of DNA as a first step in GMO analysis

A method is described to discriminate between genetically modified (GM) and non‐modified foodstuffs by detecting the presence of newly introduced genes at the protein or DNA level. Currently available methods operate almost exclusively at the DNA level and are based on the polymerase chain reaction (PCR). The first and most crucial step in this process is the isolation of DNA. In this study, five different methods for the isolation of DNA from chocolate and biscuits were evaluated, using four commercially available extraction kits and a non‐commercial method for amplification of the soybean‐specific lectin gene. The latter method involves the use of hot‐start Taq polymerase, to prevent the formation of non‐specific amplification products, and an increase in the number of cycles from 35 to 41. The performance of the non‐commercial cetyl trimethylammonium bromide (CTAB)‐based method was the best, taking into consideration the adaptations of the extraction procedure, although this method was more time‐consuming than the others. Chocolate (white, milk and dark) and several biscuits generated positive amplification results using this PCR approach. Copyright © 2004 Society of Chemical Industry     

Determinants of energy demand in the French service sector: A decomposition analysis

This paper analyzes the changes in the energy consumption of the service sector in France over the period 1995–2006, using the logarithmic mean Divisia index I (LMDI I) decomposition method. The analysis is carried out at various disaggregation levels to highlight the specifics of each sub-sector and end-use according to their respective determinants. The results show that in this period the economic growth of the service sector was the main factor that led to the increase in total energy consumption. Structure, productivity, substitution and intensity effects restricted this growth, but with limited effect. By analyzing each end-use, this paper enables a more precise understanding of the impact of these factors. The activity effect was the main determinant of the increase in energy consumption for all end-uses except for air conditioning, for which the equipment rate effect was the main factor. Structural changes in the service sector primarily impacted energy consumption for space heating and cooking. Improvements in productivity limited the growth of energy consumption for all end-uses except for cooking. Finally, energy efficiency improvements mainly affected space-heating energy use.