Rainfall,geology and landscape position generate large‐scale spatiotemporal fire pattern heterogeneity in an African savanna

Fire is considered a critical management tool in fire prone landscapes. Often studies and policies relating to fire focus on why and how the fire regime should be managed, often neglecting to subsequently evaluate management's ability to achieve these objectives over long temporal and large spatial scales. This study explores to what extent the long‐term spatio‐temporal fire patterns recorded in the Kruger National Park, South Africa has been influenced by management policies and to what extent it was dictated by underlying variability in the abiotic template. This was done using a spatially explicit fire‐scar database from 1941 to 2006 across the 2 million ha Park. Fire extent (ha burnt per annum) 1) is correlated with rainfall cycles 2) exhibits no long‐term trend and 3) is largely non‐responsive to prevailing fire management policies. Rainfall, geology and distance from the closest perennial river and the interactions between these variables influence large‐scale fire pattern heterogeneity: areas with higher rainfall, on basaltic substrates and far from rivers are more fire prone and have less heterogeneous fire regimes than areas with lower rainfall, on granitic substrates and closer to rivers. This study is the first to illustrate that under a range of rainfall and geological conditions, perennial rivers influence long‐term, landscape‐scale fire patterns well beyond the riparian zone (typically up to 15 km from the river). It was concluded that despite fire management policies which historically aimed for largely homogeneous fire return regimes, spatially and temporally heterogeneous patterns have emerged. This is primarily because of differences in rainfall, geology and distance from perennial rivers. We postulate that large‐scale spatio‐temporal fire pattern heterogeneity is implicit to heterogeneous savannas, even under largely homogenizing fire policies. Management should be informed by these patterns, embracing the natural heterogeneity‐producing template. We therefore suggest that management actions will be better directed when operating at appropriate scales, nested within the broader implicit landscape patterns, and when focusing on fire regime parameters over which they have more influence (e.g. fire season).     

Influence of some physicochemical factors on the viscosity of aqueous levan solutions of Zymomonas mobilis

Zymomonas mobilis strain 113 “S” produces levan – an extracellular, viscous, biologically active, non-toxic fructose polymer with a unique structure and extraordinary properties. This polysaccharide was isolated at two different degrees of purity by alcohol precipitation from aqueous solutions and was characterized with respect to some rheological properties and stability of viscous solutions. The effects of temperature, pH and salt concentration on the viscosity of 1–3% levan solutions were examined. The viscosity of levan solutions was found to be quite stable and reversible at room temperature over a wide range of pH from 4 to 11. The viscosity was slightly affected by increased salt concentration. Levan solutions were rather stable at high temperatures (up to 70°C, 1 h, pH 6), where the viscosity could be almost completerly restored (up to 80–100%). Therefore, the degradation of the polymer structure under these conditions is probably insignificant. Temperatures of 70–100°C with a pH of less than 3.5 caused irreversible degradation of the levan structure. The above-mentioned properties of levan, obtained from Zymomonas mobilis 113 “S”, demonstrated the potential for the development of various therapeutic forms of pharmacologically-active levan and their application in medicine as well as in the food and other industries.     

Increased sequence diversity coverage improves detection of HIV-specific T cell responses

The accurate identification of HIV-specific T cell responses is important for determining the relationship between immune response, viral control, and disease progression. HIV-specific immune responses are usually measured using peptide sets based on consensus sequences, which frequently miss responses to regions where test set and infecting virus differ. In this study, we report the design of a peptide test set with significantly increased coverage of HIV sequence diversity by including alternative amino acids at variable positions during the peptide synthesis step. In an IFN-gamma ELISpot assay, these "toggled" peptides detected HIV-specific CD4(+) and CD8(+) T cell responses of significantly higher breadth and magnitude than matched consensus peptides. The observed increases were explained by a closer match of the toggled peptides to the autologous viral sequence. Toggled peptides therefore afford a cost-effective and significantly more complete view of the host immune response to HIV and are directly applicable to other variable pathogens.     

Characterisation of preYvaY export reveals differences in the substrate specificities of Bacillus subtilis and Escherichia coli leader peptidases

Translocation, processing and secretion of YvaY, a Bacillus subtilis protein of unknown function, were characterised both in B. subtilis and in Escherichia coli. In its natural host B. subtilis, YvaY was transiently synthesised at the end of the exponential growth phase. It was efficiently secreted into the culture supernatant in spite of a calculated membrane spanning domain in the mature part of the protein. In E. coli, despite the high conservation of Sec-dependent transport components, processing of preYvaY was strongly impaired. To uncover which elements of E. coli and B. subtilis translocation systems are responsible for the observed substrate specificity, components of the B. subtilis Sec-system were co-expressed besides yvaY in E. coli. Expression of B. subtilis secA or secYEG genes did not affect processing, but expression of B. subtilis signal peptidase genes significantly enhanced processing of preYvaY in E. coli. While the major signal peptidases SipS or SipT had a strong stimulatory effect on preYvaY processing, the minor signal peptidases SipU, SipV or SipW had a far less stimulatory effect in E. coli. These results reveal that targeting and translocation of preYvaY is mediated by the E. coli Sec proteins but processing of preYvaY is not performed by E. coli signal peptidase LepB. Thus, differences in substrate specificities of E. coli LepB and the B. subtilis Sip proteins provide the bottleneck for export of YvaY in E. coli. Significant slower processing of preYvaY in absence of SecB indicated that SecB mediates targeting of the B. subtilis precursor.     

Integrating macrophages into organotypic co-cultures: a 3D in vitro model to study tumor-associated macrophages

Tumor progression is controlled by signals from cellular and extra-cellular microenvironment including stromal cells and the extracellular matrix. Consequently, three-dimensional in vitro tumor models are essential to study the interaction of tumor cells with their microenvironment appropriately in a biologically relevant manner. We have previously used organotypic co-cultures to analyze the malignant growth of human squamous cell carcinoma (SCC) cell lines on a stromal equivalent in vitro. In this model, SCC cell lines are grown on a collagen-I gel containing fibroblasts. Since macrophages play a critical role in the progression of many tumor types, we now have expanded this model by integrating macrophages into the collagen gel of these organotypic tumor co-cultures. This model was established as a murine and a human system of skin SCCs. The effect of macrophages on tumor progression depends on their polarization. We demonstrate that macrophage polarization in organotypic co-cultures can be modulated towards and M1 or an M2 phenotype by adding recombinant IFN-γ and LPS or IL-4 respectively to the growth medium. IL-4 stimulation of macrophage-containing cultures resulted in enhanced tumor cell invasion evidenced by degradation of the basement membrane, enhanced collagenolytic activity and increased MMP-2 and MMP-9. Interestingly, extended co-culture with tumor cells for three weeks resulted in spontaneous M2 polarization of macrophages without IL-4 treatment. Thus, we demonstrate that macrophages can be successfully integrated into organotypic co-cultures of murine or human skin SCCs and that this model can be exploited to analyze macrophage activation towards a tumor supporting phenotype.     

Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin

A cDNA encoding the multifunctional cytochrome P450, CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin from Sorghum bicolor (L.) Moench was isolated. A PCR approach based on three consensus sequences of A-type cytochromes P450 – (V/I)KEX(L/F)R, FXPERF, and PFGXGRRXCXG – was applied. Three novel cytochromes P450 (CYP71E1, CYP98, and CYP99) in addition to a PCR fragment encoding sorghum cinnamic acid 4-hydroxylase were obtained.Reconstitution experiments with recombinant CYP71E1 heterologously expressed in Escherichia coli and sorghum NADPH–cytochrome P450–reductase in L--dilaurylphosphatidyl choline micelles identified CYP71E1 as the cytochrome P450 that catalyses the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis. In accordance to the proposed pathway for dhurrin biosynthesis CYP71E1 catalyses the dehydration of the oxime to the corresponding nitrile, followed by a C-hydroxylation of the nitrile to produce p-hydroxymandelonitrile. In vivo administration of oxime to E. coli cells results in the accumulation of the nitrile, which indicates that the flavodoxin/flavodoxin reductase system in E. coli is only able to support CYP71E1 in the dehydration reaction, and not in the subsequent C-hydroxylation reaction.CYP79 catalyses the conversion of tyrosine to p-hydroxyphenylacetaldoxime, the first committed step in the biosynthesis of the cyanogenic glucoside dhurrin. Reconstitution of both CYP79 and CYP71E1 in combination with sorghum NADPH-cytochrome P450–reductase resulted in the conversion of tyrosine to p-hydroxymandelonitrile, i.e. the membranous part of the biosynthetic pathway of the cyanogenic glucoside dhurrin. Isolation of the cDNA for CYP71E1 together with the previously isolated cDNA for CYP79 provide important tools necessary for tissue-specific regulation of cyanogenic glucoside levels in plants to optimize food safety and pest resistance.