Thermodynamic benchmark study using Biacore technology

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.     

Cofermentation of glucose, xylose, and cellobiose by the beetle-associated yeast Spathaspora passalidarum

Fermentation of cellulosic and hemicellulosic sugars from biomass could resolve food-versus-fuel conflicts inherent in the bioconversion of grains. However, the inability to coferment glucose and xylose is a major challenge to the economical use of lignocellulose as a feedstock. Simultaneous cofermentation of glucose, xylose, and cellobiose is problematic for most microbes because glucose represses utilization of the other saccharides. Surprisingly, the ascomycetous, beetle-associated yeast Spathaspora passalidarum, which ferments xylose and cellobiose natively, can also coferment these two sugars in the presence of 30 g/liter glucose. S. passalidarum simultaneously assimilates glucose and xylose aerobically, it simultaneously coferments glucose, cellobiose, and xylose with an ethanol yield of 0.42 g/g, and it has a specific ethanol production rate on xylose more than 3 times that of the corresponding rate on glucose. Moreover, an adapted strain of S. passalidarum produced 39 g/liter ethanol with a yield of 0.37 g/g sugars from a hardwood hydrolysate. Metabolome analysis of S. passalidarum before onset and during the fermentations of glucose and xylose showed that the flux of glycolytic intermediates is significantly higher on xylose than on glucose. The high affinity of its xylose reductase activities for NADH and xylose combined with allosteric activation of glycolysis probably accounts in part for its unusual capacities. These features make S. passalidarum very attractive for studying regulatory mechanisms enabling bioconversion of lignocellulosic materials by yeasts.     

Bioproduction of benzaldehyde in a solid–liquid two-phase partitioning bioreactor using <Emphasis Type=Italic>Pichia pastoris</Emphasis>

The bioproduction of benzaldehyde from benzyl alcohol using Pichia pastoris was examined in a solid–liquid two-phase partitioning bioreactor (TPPB) to reduce substrate and product inhibition. Rational polymer selection identified Elvax 40W as an effective sequestering phase, possessing partition coefficients for benzyl alcohol and benzaldehyde of 3.5 and 35.4, respectively. The use of Elvax 40W increased the overall mass of benzaldehyde produced by approx. 300% in a 5 l bioreactor, relative to a single phase biotransformation. The two-phase system had a molar yield of 0.99, indicating that only minor losses occurred. These results provide a promising starting point for solid–liquid TPPBs to enhance benzaldehyde production, and suggest that multiple, targeted polymers may provide relief for transformations characterized by multiple inhibitory substrates/product/by-products.     

Screening Mosquito House Entry Points as a Potential Method for Integrated Control of Endophagic Filariasis,Arbovirus and Malaria Vectors

Background

Partial mosquito-proofing of houses with screens and ceilings has the potential to reduce indoor densities of malaria mosquitoes. We wish to measure whether it will also reduce indoor densities of vectors of neglected tropical diseases.

Methodology

The main house entry points preferred by anopheline and culicine vectors were determined through controlled experiments using specially designed experimental huts and village houses in Lupiro village, southern Tanzania. The benefit of screening different entry points (eaves, windows and doors) using PVC-coated fibre glass netting material in terms of reduced indoor densities of mosquitoes was evaluated compared to the control.

Findings

23,027 mosquitoes were caught with CDC light traps; 77.9% (17,929) were Anopheles gambiae sensu lato, of which 66.2% were An. arabiensis and 33.8% An. gambiae sensu stricto. The remainder comprised 0.2% (50) An. funestus, 10.2% (2359) Culex spp. and 11.6% (2664) Mansonia spp. Screening eaves reduced densities of Anopheles gambiae s. l. (Relative ratio (RR)  = 0.91; 95% CI = 0.84, 0.98; P = 0.01); Mansonia africana (RR = 0.43; 95% CI = 0.26, 0.76; P<0.001) and Mansonia uniformis (RR = 0.37; 95% CI = 0.25, 0.56; P<0.001) but not Culex quinquefasciatus, Cx. univittatus or Cx. theileri. Numbers of these species were reduced by screening windows and doors but this was not significant.

Significance

This study confirms that across Africa, screening eaves protects households against important mosquito vectors of filariasis, Rift Valley Fever and O''Nyong nyong as well as malaria. While full house screening is required to exclude Culex species mosquitoes, screening of eaves alone or fitting ceilings has considerable potential for integrated control of other vectors of filariasis, arbovirus and malaria.     

Flexible Mate Guarding Tactics in the Dragonfly Sympelrum Internum (Odonata: Libellulidae)

Mate guarding–a behaviour prevalent in odonates–is a post copulatory association during which males prevent females from re-mating. Some species use two forms of guarding: contact mate guarding, which is energetically costly but highly effective and non-contact mate guarding, which is less costly but less effective. This study aimed to determine if male Sympetrum internum (Odonata:Libellulidae) adjust the duration of contact mate guarding according to environmental, temporal and physiological factors. There was a significant interaction between male density and season on duration of contact mate guarding. Early in the season males increased the duration of contact guarding as the density of rivals increased. Later in the season males guarded mates longer irrespective of male density. Wind and temperature did not detectabiy alter the duration of contact mate guarding, suggesting that the trade-off between current and future reproductive success was more important than were physiological costs.     

Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection

Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis--characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity--show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early infection and suggest novel diagnostic and therapeutic approaches.     

Upregulation ofE. coli 38kDa proteins induced by glutaraldehyde and formaldehyde

Exposure of the W3110 strain ofEscherichia coli K12 to low concentrations of glutaraldehyde or formaldehyde results in an unusual pattern of protein expression, as determined by high-resolution, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). A decline in total protein synthesis is accompanied by the upregulation of three proteins of approximate molecular weight 38 kDa. In the presence of 0.1 mM glutaraldehyde this response occurs within the first 5 min of incubation, and with 0.1 mM formaldehyde, within the first 30 min of incubation. The 38 kDa proteins continue to be expressed at high levels until cell death. Comparison of our 2-D PAGE patterns withE. coli gene-protein and plasmid indexes indicates that one of the proteins may be the major gene product of thepyrC locus. This pattern of protein synthesis may indicate a novelE. coli stress response.     

Developmental partitioning of SYK and ZAP70 prevents autoimmunity and cancer

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