The role of monocyte/macrophages as vehicles of dissemination of Simkania negevensis: an in vitro simulation model

Exposure to Simkania negevensis (Sn), an intracellular microorganism that has been associated with respiratory tract infections in infants and adults, is prevalent. Sn can multiply within free-living amoebae and has been detected in domestic water supplies, which may constitute a source of infection with the organism. Its path of transport from its portal of entry to the body to its target organs is unknown. In this study, the possibility that monocytes/macrophages may serve as vehicles of transmission was examined. In vitro cocultivation of Sn-infected Acanthamoeba polyphaga with the monocyte/macrophage cell line U937 resulted in the death of the amoebae and infection of the U937 cells. Sn entered and multiplied in U937 cells within short periods of time, and the microorganism could be transferred from U937 cells to cell cultures of various origins. Uninfected monocyte/macrophages could become infected when in contact with either actively or persistently Sn-infected cell cultures. Persistently infected cultures in contact with uninfected U937 cells became actively infected. The results of this study provide a basis for determination of the molecular mechanisms of monocyte/macrophage-cell interactions in transfer of infection and may contribute to a better understanding of the pathogenesis of Sn infections in vivo.     

CpG drives human transitional B cells to terminal differentiation and production of natural antibodies

The receptor TLR9, recognizing unmethylated bacterial DNA (CpG), is expressed by B cells and plays a role in the maintenance of serological memory. Little is known about the response of B cells stimulated with CpG alone, without additional cytokines. In this study, we show for the first time the phenotypic modification, changes in gene expression, and functional events downstream to TLR9 stimulation in human B cell subsets. In addition, we demonstrate that upon CpG stimulation, IgM memory B cells differentiate into plasma cells producing IgM Abs directed against the capsular polysaccharides of Streptococcus pneumoniae. This novel finding proves that IgM memory is the B cell compartment responsible for the defense against encapsulated bacteria. We also show that cord blood transitional B cells, corresponding to new bone marrow emigrants, respond to CpG. Upon TLR9 engagement, they de novo express AID and Blimp-1, genes necessary for hypersomatic mutation, class-switch recombination, and plasma cell differentiation and produce Abs with anti-pneumococcal specificity. Transitional B cells, isolated from cord blood, have not been exposed to pneumococcus in vivo. In addition, it is known that Ag binding through the BCR causes apoptotic cell death at this stage of development. Therefore, the ability of transitional B cells to sense bacterial DNA through TLR9 represents a tool to rapidly build up the repertoire of natural Abs necessary for our first-line defense at birth.     

Picosecond dynamics of T and R forms of aspartate transcarbamylase: a neutron scattering study

E. coli aspartate transcarbamylase (ATCase) is a 310 kDa allosteric enzyme which catalyses the first committed step in pyrimidine biosynthesis. The binding of its substrates, carbamylphosphate and aspartate, induces significant conformational changes. This enzyme shows homotropic cooperative interactions between the catalytic sites for the binding of aspartate. This property is explained by a quaternary structure transition from T state (aspartate low affinity) to R state (aspartate high affinity) accompanied by a 5% increase of radius of gyration of ATCase. The same quaternary structure change is observed upon binding of the bisubstrate analogue PALA (N-(phosphonacetyl)-L-aspartate. Owing to the large incoherent neutron scattering cross-section of the hydrogen atom and the abundance of this element in proteins, inelastic neutron scattering gives a global view of protein dynamics as sensed via the individual motions of its hydrogen atoms. We present neutron scattering results of the local dynamics (few angstroms), at short time (few tens of picoseconds), of ATCase in T and R forms. Compared to the T form, we observe an increased mobility of the protein in the R form that we associate to an increase of accessible surface area to the solvent. Beyond this specific result, this highlights the key role of the accessible surface area (ASA) in dynamic contribution to inelastic neutron data in the picosecond time scale. In particular, we want to stress out (i) that a difference at the picosecond time scale does not allow to conclude to a difference in the dynamics at a longer time scale and to address whether the T state is looser than the R state (ii) how challenging is, any comparison in terms of general dynamics (tense or relaxed) between dynamic values deduced from experimental neutron data on proteins with different sequences and therefore ASA. This caveat holds particularly when comparing dynamics of a mesophile with the corresponding extremophile.     

A Simple,Realistic Stochastic Model of Gastric Emptying

Several models of Gastric Emptying (GE) have been employed in the past to represent the rate of delivery of stomach contents to the duodenum and jejunum. These models have all used a deterministic form (algebraic equations or ordinary differential equations), considering GE as a continuous, smooth process in time. However, GE is known to occur as a sequence of spurts, irregular both in size and in timing. Hence, we formulate a simple stochastic process model, able to represent the irregular decrements of gastric contents after a meal. The model is calibrated on existing literature data and provides consistent predictions of the observed variability in the emptying trajectories. This approach may be useful in metabolic modeling, since it describes well and explains the apparently heterogeneous GE experimental results in situations where common gastric mechanics across subjects would be expected.     

Proposals for the catalytic mechanism of glycogen phosphorylase b prompted by crystallographic studies on glucose 1-phosphate binding

The crystal structure of glycogen phosphorylase b in the presence of the weak activator 2 mm-inosine 5′-phosphate has been solved at 3 Å resolution. The binding interactions of the substrate, glucose 1-phosphate, at the catalytic site are described. The nearby presence (6 Å) of the essential co-factor, pyridoxal phosphate, is consistent with biochemical studies but an analysis of the way in which this group might act in catalysis leads to results that are inconsistent with solution studies. Moreover it is difficult to accommodate a glycogen substrate with its terminal glucose in the position defined by glucose 1-phosphate. Model-building studies show that an alternative binding mode for glucose 1-phosphate is possible and that this alternative mode allows a glycogen substrate to be fitted with ease. The alternative binding site leads directly to proposals for the mechanism in which the phosphate group of pyridoxal phosphate acts as a nucleophile and the imidazole of histidine 376 functions as a general acid. It is suggested that these are the essential features of the catalytic mechanism and that, in the absence of the second substrate, glycogen, and in the absence of AMP, the enzyme binds glucose 1-phosphate in a non-productive mode. Conversion of the enzyme to the active conformation through association with AMP may result in conformational changes that direct the binding to the productive mode.     

Inhibition of net nitrification activity in a Mediterranean woodland: possible role of chemicals produced by Arbutus unedo

Nitrification is a key biological process for the control of soil NO₃ ^? availability and N losses from terrestrial ecosystems. The study investigates the causes for the absence of net nitrification activity in the soil of a Mediterranean monospecific woodland of Arbutus unedo, focusing in particular on the possible role of chemicals produced by this plant. The mineral N pool, net rates of mineralization and nitrification were measured in the soil top 10 cm over 18 months. Raw extracts of leaves and roots of Arbutus unedo and soil underneath Arbutus plant canopy were purified using chromatographic techniques and the structure of chemicals was defined using spectroscopic and spectrometric methods. Leaf extracts (raw, aqueous and organic fractions) were tested for their toxicity on net nitrification, using a test soil. Field and laboratory incubations showed soil NO₃ ^? concentration below the detection limit over the whole study period, despite the significant NH₄ ⁺ availability. Toxicity tests indicated that more than 400 μg of extract g^?1 dry soil were needed to have more than 50% reduction of net NO₃ ^? production. Gallocatechin and catechin were among the most abundant chemicals in the extracts of leaves, roots and soil. Their soil concentration was significantly higher than the annual calculated input via leaf litter, and it was in the range of toxic concentrations, as deduced from the dose-response curve of the toxicity test. Data support the hypothesis that plant produced chemicals might be involved in the limited net nitrate production in this Mediterranean woodland.

     

Substrate preference of Bifidobacterium adolescentis MB 239: compared growth on single and mixed carbohydrates

The utilization of mono-, di-, and oligosaccharides by Bifidobacterium adolescentis MB 239 was investigated. Raffinose, fructooligosaccharides (FOS), lactose, and the monomeric moieties glucose and fructose were used. To establish a hierarchy of sugars preference, the kinetics of growth and sugar consumption were determined on individual and mixed carbohydrates. On single carbon sources, higher specific growth rates and cell yields were attained on di- and oligosaccharides compared to monosaccharides. Analysis of the carbohydrates in steady-state chemostat cultures, growing at the same dilution rate on FOS, lactose, or raffinose, showed that monomeric units and hydrolysis products were present. In chemostat cultures on individual carbohydrates, B. adolescentis MB 239 simultaneously displayed α-galactosidase, β-galactosidase, and β-fructofuranosidase activities on all the sugars, including monosaccharides. Glycosyl hydrolytic activities were found in cytosol, cell surface, and growth medium. Batch experiments on mixtures of carbohydrates showed that they were co-metabolized by B. adolescentis MB 239, even if different disappearance kinetics were registered. When mono-, di-, and oligosaccharides were simultaneously present in the medium, no precedence for monosaccharides utilization was observed, and di- and oligosaccharides were consumed before their constitutive moieties.     

Thermal Manipulation of Gold Nanocomposites for Microfluidic Platform Optimization

Plasmonics - Surface gold–polymer nanocomposites are prepared by using the thermal convection method for the deposition of gold colloids onto the surface of four polymer films: poly(-vinyl...