Production of the biocontrol agent Pantoea agglomerans PBC-1 in a stirred tank reactor by batch and fed-batch cultures

Concerns about food safety as well as the development of resistance to many fungicides by major postharvest pathogens have increased recently. Biological control, using microorganisms antagonistic to the fungal plant pathogens, appears to be promising as an alternative to fungicides. The microbial biocontrol agent has to be produced on an industrial scale, maintaining its biocontrol efficacy. The purpose of the current study was to optimize the conditions for microbial biomass production of the biocontrol agent Pantoea agglomerans PBC-1 in a 2-l mechanically stirred reactor (STR), defining mixing and mass transfer technological parameters and the growth kinetics for different saccharides. In the batch mode, different impellers and spargers were tested. Despite the oxygen mass transfer improvement achieved with marine propeller combined with porous sparger, the biomass did not increase, if compared with the use of a Rushton turbine and L-sparger, pointing out the relevance of a radial flux for better broth homogenization. Different carbon sources were used: sucrose, glucose and fructose; each of which led to viable populations 3.9 × 10⁹, 1.4 × 10⁹, 3.9 × 10⁹ c.f.u/ml, respectively, after 20 h of incubation. Fed-batch technology allows the maintenance of high cell viability for longer periods of time in the stationary growth phase, which can be crucial for the scale-up of biocontrol agent production process that is achieved together with a reduction of 85% on the incidence caused by the pathogens, brought about by fresh microbial biomass preparation on artificially wounded apples or oranges, stored for 7 days at 25°C against Penicillium expansum and Penicillium digitatum.     

Association between GSTP1, GSTM1 and GSTT1 polymorphisms involved in xenobiotic metabolism and head and neck cancer development

Polymorphisms in the glutathione S-transferase superfamily genes that encodes enzymes involved in the phase II xenobiotic metabolism may lead head and neck cancer development. In this study we investigate the association of A313G and C341T GSTP1 polymorphisms, GSTM1 and GSTT1 null genotypes in the head and neck cancer development, interactions between these polymorphisms,the tumor histopathologic parameters and risk factors (smoking and drinking) were also evaluated in the case–control study. 775 individuals (261 patients/514 controls) were included in the study. Molecular analyzes were performed by PCR and PCR–RFLP; and statistical analyzes by Chi square and multiple logistic regression. Chi square test showed that only the genotype frequencies for GSTM1 and GSTT1 were in Hardy–Weinberg disequilibrium in both groups. Significant results with p ≤ 0.05 showed that age ≥ 48 years (OR = 11.87; 7.55–18.65), smoking (OR = 4.25; 2.70–6.69), drinking (OR = 1.59; 1.02–2.46) were possible predictors for the head and neck cancer development and the presence of A313G GSTP1 polymorphism (OR = 0.62; 0.42–0.92) decreased the risk for this disease. Individuals with the 313AG/GG GSTP1 and age ≥ 48 years (OR = 0.59; 0.38–0.91), male gender (OR = 0.54; 0.35–0.83), smokers (OR = 0.63; 0.40–0.99) and drinkers (OR = 0.57; 0.35–0.95); the GSTM1 null genotype and age < 48 years (OR = 2.46; 1.09–5.55); the GSTT1 null genotype and primary anatomical sites of pharynx (OR = 0.37; 0.17–0.79) and larynx (OR = 3.60; 1.93–6.72), can modulate the risk for the disease development. The variables age ≥ 48 years, smoking and drinking can be predictors for head and neck cancer development; moreover, A313G GSTP1 polymorphism, GSTM1 and GSTT1 null genotypes can modulate the risk for this disease.     

Cells respond to mechanical stress by rapid disassembly of caveolae

The functions of caveolae, the characteristic plasma membrane invaginations, remain debated. Their abundance in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by osmotic swelling or by uniaxial stretching results in a rapid disappearance of caveolae, in a reduced caveolin/Cavin1 interaction, and in an increase of free caveolins at the plasma membrane. Tether-pulling force measurements in cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin- and ATP-independent cell response that buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin- and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that quickly accommodates sudden and acute mechanical stresses.     

Co‐operational PCR Coupled with Dot Blot Hybridization for the Detection of Phaeomoniella chlamydospora on Infected Grapevine Wood

The technique consisting of the co‐operational PCR coupled with dot blot hybridization and posterior colorimetric visualization was developed for the detection of Phaeomoniella chlamydospora, one of the major pathogenic fungi involved in the Petri disease of grapevine. A partial region of the fungal rDNA including the internal transcribed spacer (ITS) region was amplified through co‐operational PCR for P. chlamydospora and 17 additional grapevine‐associated fungi included in the genera Botryosphaeria, Cryptovalsa, Cylindrocarpon, Dematophora, Diplodia, Dothiorella, Eutypa, Fomitiporia, Lasiodiplodia, Neofusicoccum, Phaeoacremonium, Phomopsis and Stereum, by using the primer pairs NSA3/NLC2 (external pair) and NSI1/NLB4 (inner pair). A specific probe (Pch2D) targeting the ITS2 region in the rDNA was developed for the detection of P. chlamydospora. Dot blot hybridizations carried out with the PCR products showed the specificity of the probe. Results indicated that Pch2D only hybridized with DNA amplicons of P. chlamydospora isolates, thus proving the specific detection of this fungus, while the 17 remaining species tested for the Pch2D probe resulted in negative results. Sensitivity of the technique was established below 0.1 pg of genomic DNA. This technique was further validated using artificially inoculated grapevine cuttings with P. chlamydospora. The efficacy of detection was established at 80% after two independent blind assays.     

Nine new polymorphic microsatellite loci for the amplification of archived otolith DNA of Atlantic cod,Gadus morhua L.

Nine out of 22 microsatellite primers tested were successfully amplified on three samples of cod Gadus morhua L. (two contemporary and one archived otolith samples). All loci were polymorphic (5–23 alleles/locus). The average observed heterozygosity across loci and samples was 0.625, ranging from 0.294 to 0.895 at each locus. All loci were under Hardy–Weinberg equilibrium, except PGmo56 that showed significant excess of heterozygotes in all studied samples. The isolated loci were suitable for degraded DNA and therefore useful for conducting a long‐term temporal study with DNA obtained from archived otoliths of cod.     

Seed weevils living on the edge: pressures and conflicts over body size in the endoparasitic Curculio larvae

Abstract 1. Body size in parasitic insects can be subjected to contrasting selective pressures, especially if they complete their development within a single host. On the one hand, a larger body size is associated with a higher fitness. On the other hand, the host offers a discrete amount of resources, thus constraining the evolution of a disproportionate body size. 2. The present study used the weevil Curculio elephas as a study model. Larvae develop within a single acorn, feeding on its cotyledons, and larval body size is strongly related to individual fitness. 3. The relationship between larval and acorn size was negatively exponential. Larval growth was constrained in small acorns, which did not provide enough food for the weevils to attain their potential size. Larval size increased and levelled off in acorns over a certain size (inflexion point), in which cotyledons were rarely depleted. When there were more than one larva per acorn, a larger acorn was necessary to avoid food depletion. 4. The results show that C. elephas larvae are sometimes endoparasitic, living on the edge of host holding capacity. If they were smaller they could avoid food depletion more easily, but the fitness benefits linked to a larger size have probably promoted body size increase. The strong negative effects of conspecific competition may have possibly influenced female strategy of laying a single egg per seed. 5. Being larger and fitter, but always within the limits of the available host sizes, may be one main evolutionary dilemma in endoparasites.     

Deficient Production of Reactive Oxygen Species Leads to Severe Chronic DSS-Induced Colitis in Ncf1/p47phox-Mutant Mice

Background

Colitis is a common clinical complication in chronic granulomatous disease (CGD), a primary immunodeficiency caused by impaired oxidative burst. Existing experimental data from NADPH-oxidase knockout mice propose contradictory roles for the involvement of reactive oxygen species in colitis chronicity and severity. Since genetically controlled mice with a point-mutation in the Ncf1 gene are susceptible to chronic inflammation and autoimmunity, we tested whether they presented increased predisposition to develop chronic colitis.

Methods

Colitis was induced in Ncf1-mutant and wild-_type mice by a 1^st 7-days cycle of dextran sulfate sodium (DSS), intercalated by a 7-days resting period followed by a 2^nd 7-days DSS-cycle. Cytokines were quantified locally in the colon inflammatory infiltrates and in the serum. Leukocyte infiltration and morphological alterations of the colon mucosa were assessed by immunohistochemistry.

Results

Clinical scores demonstrated a more severe colitis in Ncf1-mutant mice than controls, with no recovery during the resting period and a severe chronic colitis after the 2^nd cycle, confirmed by histopathology and presence of infiltrating neutrophils, macrophages, plasmocytes and lymphocytes in the colon. Severe colitis was mediated by increased local expression of cytokines (IL-6, IL-10, TNF-α, IFN-γ and IL-17A) and phosphorylation of Leucine-rich repeat kinase 2 (LRRK2). Serological cytokine titers of those inflammatory cytokines were more elevated in Ncf1-mutant than control mice, and were accompanied by systemic changes in functional subsets of monocytes, CD4⁺T and B cells.

Conclusion

This suggests that an ineffective oxidative burst leads to severe chronic colitis through local accumulation of peroxynitrites, pro-inflammatory cytokines and lymphocytes and systemic immune deregulation similar to CGD.     

How landscape heterogeneity frames optimal diffusivity in searching processes

Theoretical and empirical investigations of search strategies typically have failed to distinguish the distinct roles played by density versus patchiness of resources. It is well known that motility and diffusivity of organisms often increase in environments with low density of resources, but thus far there has been little progress in understanding the specific role of landscape heterogeneity and disorder on random, non-oriented motility. Here we address the general question of how the landscape heterogeneity affects the efficiency of encounter interactions under global constant density of scarce resources. We unveil the key mechanism coupling the landscape structure with optimal search diffusivity. In particular, our main result leads to an empirically testable prediction: enhanced diffusivity (including superdiffusive searches), with shift in the diffusion exponent, favors the success of target encounters in heterogeneous landscapes.