Mapping ephemeral wetlands: manual digitisation and logistic regression modelling in Nelson Mandela Bay Municipality,South Africa

Until recently, little research has been conducted on the distribution and structure of ephemeral systems in semi-arid areas. This information is critical for appropriate wetland management and conservation. The Nelson Mandela Bay Municipality is a semi-arid area along the south-eastern coastline of the Eastern Cape Province of South Africa. The Municipality encapsulates a wide range of geological and geomorphological features as well as vegetation types within an area of some 1950 km², providing an ideal area for such research. The distribution and abundance of wetlands were defined, and a logistic regression (LR) model was used to establish whether this modelling technique is viable in semi-arid areas with highly variable rainfall patterns. Wetlands were delineated manually using geographical information systems, high-resolution aerial photographs and environmental data. More than 1700 wetland polygons were identified, with 80% of the systems being categorised as depressions, seeps and wetland flats. Unchannelled (8%) and channelled (7%) valley bottom wetlands and floodplain wetlands (5%) were also identified. The wetland database was then used to create a wetland occurrence probability model. There were 19 environmental variables used to develop the LR model, with eight variables used in the final model output. The predictive capacity of the model was good, with an area under curve value of 0.68 and an overall accuracy of 66%. This indicates that probabilistic wetland models are useful in highly variable environments with high numbers of small (<1 ha) wetlands. Such predictive models provide a tool to assist in improving the accuracy of land cover datasets in semi-arid areas, and can be used to inform management decisions on flood risk areas and key conservation zones. In addition, abiotic variables that are significant in the model output provide an indication of the factors influencing wetland functioning in the region.     

Effector and memory CD8+ T cells can be generated in response to alloantigen independently of CD4+ T cell help

There is now considerable evidence suggesting that CD8(+) T cells are able to generate effector but not functional memory T cells following pathogenic infections in the absence of CD4(+) T cells. We show that following transplantation of allogeneic skin, in the absence of CD4(+) T cells, CD8(+) T cells become activated, proliferate, and expand exclusively in the draining lymph nodes and are able to infiltrate and reject skin allografts. CD44(+)CD8(+) T cells isolated 100 days after transplantation rapidly produce IFN-gamma following restimulation with alloantigen in vitro. In vivo CD44(+)CD8(+) T cells rejected donor-type skin allografts more rapidly than naive CD8(+) T cells demonstrating the ability of these putative memory T cells to mount an effective recall response in vivo. These data form the first direct demonstration that CD8(+) T cells are able to generate memory as well as effector cells in response to alloantigen during rejection in the complete absence of CD4(+) T cells. These data have important implications for the design of therapies to combat rejection and serve to reinforce the view that CD8(+) T cell responses to allografts require manipulation in addition to CD4(+) T cell responses to completely prevent the rejection of foreign organ transplants.     

Maternal transfer of strain-specific immunity in an invertebrate

The most celebrated component of the vertebrate immune system is the acquired response in which memory cells established during primary infection enhance the proliferation of antibodies during secondary infection. Additionally, the strength of vertebrate acquired immune responses varies dramatically depending on the infecting pathogen species or on the pathogen genotype within species. Because invertebrates lack the T-cell receptors and Major Histocompatibility Complex (MHC) molecules that mediate vertebrate adaptive immune responses, they are thought to lack adaptive immunity and be relatively unspecific in their interactions with pathogens. With only innate immunity, invertebrate hosts are believed to be nai;ve at each new encounter with pathogens. Nevertheless, some forms of facultative immunity appear to be important in insects; some individuals have enhanced immunity due to population density, and some social insects benefit when their nest-mates have been exposed to a pathogen or pathogen mimic (; see for a predation example.) Here we provide evidence for acquired strain-specific immunity in the crustacean Daphnia magna infected with the pathogenic bacteria Pasteuria ramosa. Specifically, the fitness of hosts was enhanced when challenged with a bacterial strain their mother had experienced relative to cases when mother and offspring were challenged with different strains.     

Annexin-mediated secretory vesicle aggregation in plants

The mechanism by which membranes fuse during vesicle-mediated secretion is of considerable importance for plant cell growth, but remains unknown. We have identified Ca²⁺-dependent phospholipid-binding proteins (annexins) from maize ( Zea mays ), that may play a part in this process. An assay for Ca²⁺-dependent binding of annexins to liposomes, revealed that the maize proteins (p23, p33 and p35) and annexins from bovine lung, bind over a similar range of Ca²⁺ concentrations. Turbidity assays further revealed that both maize and bovine annexins induced liposome aggregation and that the plant annexins were also effective at aggregating plant secretory vesicles. This aggregation occurred at levels of free Ca²⁺ similar to that required for the binding of annexins p33 and p35. We discuss the significance of these results for the plant secretory apparatus.     

Transmissible spongiform encephalopathy strain-associated diversity of N-terminal proteinase K cleavage sites of PrP(Sc) from scrapie-infected and bovine spongiform encephalopathy-infected mice.

Assessment of the different conformational states of the abnormal prion protein (PrP(Sc)) in the CNS provides an established basis for distinguishing transmissible spongiform encephalopathy (TSE) strains. PrP(Sc) conformers are variably resistant to N-terminal proteinase K (PK) digestion, and analysis of the consensus products (PrP(res)) by immunoassay enables effective, but relatively low-resolution differentiation. Determination of the precise N-terminal amino acid profile (N-TAAP) of PrP(res) presents a potential high-resolution means of TSE-strain typing, and thus of differential disease diagnosis. This approach was evaluated using individual mice affected by model scrapie (22A, ME7, 87V and 79A) and bovine spongiform encephalopathy (BSE) (301V) strains. Nano liquid chromatography-mass spectrometry (LC-MS) was used to determine PrP(res) N-terminal tryptic digestion products. Four major N-terminal tryptic peptides were generated from all mouse TSE strains investigated, corresponding with predominant N-termination of PrP(res) at G(81), G(85), G(89) and G(91). Both the mass spectrometric abundance of the individual peptides and the ratios of pairs of these peptides were evaluated as markers of conformation in relation to their potential for strain discrimination. The yield of peptides was significantly greater for BSE than scrapie strains and the relative quantities of particular peptide pairs differed between strains. Thus, whereas peptide G(91)-K(105) was a dominant peptide from 301V, this was not the case for other strains and, significantly, the ratio of peptides G(91)-K(105):G(89)-K(105) was substantially higher for BSE-infected compared with scrapie-infected mice. These data support the potential of the N-TAAP approach for high-resolution TSE strain typing and differential diagnosis.     

Combining evolutionary and structural information for local protein structure prediction

We study the effects of various factors in representing and combining evolutionary and structural information for local protein structural prediction based on fragment selection. We prepare databases of fragments from a set of non-redundant protein domains. For each fragment, evolutionary information is derived from homologous sequences and represented as estimated effective counts and frequencies of amino acids (evolutionary frequencies) at each position. Position-specific amino acid preferences called structural frequencies are derived from statistical analysis of discrete local structural environments in database structures. Our method for local structure prediction is based on ranking and selecting database fragments that are most similar to a target fragment. Using secondary structure type as a local structural property, we test our method in a number of settings. The major findings are: (1) the COMPASS-type scoring function for fragment similarity comparison gives better prediction accuracy than three other tested scoring functions for profile-profile comparison. We show that the COMPASS-type scoring function can be derived both in the probabilistic framework and in the framework of statistical potentials. (2) Using the evolutionary frequencies of database fragments gives better prediction accuracy than using structural frequencies. (3) Finer definition of local environments, such as including more side-chain solvent accessibility classes and considering the backbone conformations of neighboring residues, gives increasingly better prediction accuracy using structural frequencies. (4) Combining evolutionary and structural frequencies of database fragments, either in a linear fashion or using a pseudocount mixture formula, results in improvement of prediction accuracy. Combination at the log-odds score level is not as effective as combination at the frequency level. This suggests that there might be better ways of combining sequence and structural information than the commonly used linear combination of log-odds scores. Our method of fragment selection and frequency combination gives reasonable results of secondary structure prediction tested on 56 CASP5 targets (average SOV score 0.77), suggesting that it is a valid method for local protein structure prediction. Mixture of predicted structural frequencies and evolutionary frequencies improve the quality of local profile-to-profile alignment by COMPASS.     

Flow cytometry and FACS applied to filamentous fungi

Flow cytometry is an automated, laser- or impedance-based, high throughput method that allows very rapid analysis of multiple chemical and physical characteristics of single cells within a cell population. It is an extremely powerful technology that has been used for over four decades with filamentous fungi. Although single cells within a cell population are normally analysed rapidly on a cell-by-cell basis using the technique, flow cytometry can also be used to analyse cell (e.g. spore) aggregates or entire microcolonies. Living or fixed cells can be stained with a wide range of fluorescent reporters to label different cell components or measure different physiological processes. Flow cytometry is also suited for measurements of cell size, interaction, aggregation or shape using non-labelled cells by means of analysing their light scattering characteristics. Fluorescence-activated cell sorting (FACS) is a specialized form of flow cytometry that provides a method for sorting a heterogeneous mixture of cells into two or more containers based upon the fluorescence and/or light scattering properties of each cell. The major advantage of analysing cells by flow cytometry over microscopy is the speed of analysis: thousands of cells can be analysed per second or sorted in minutes. Drawbacks of flow cytometry are that specific cells cannot be followed in time and normally spatial information relating to individual cells is lacking. A big advantage over microscopy is when using FACS, cells with desired characteristics can be sorted for downstream experimentation (e.g. for growth, infection, enzyme production, gene expression assays or ‘omics’ approaches). In this review, we explain the basic concepts of flow cytometry and FACS, define its advantages and disadvantages in comparison with microscopy, and describe the wide range of applications in which these powerful technologies have been used with filamentous fungi.     

In Vivo Evaluation of Blood Based and Reference Tissue Based PET Quantifications of [11C]DASB in the Canine Brain

This first-in-dog study evaluates the use of the PET-radioligand [¹¹C]DASB to image the density and availability of the serotonin transporter (SERT) in the canine brain. Imaging the serotonergic system could improve diagnosis and therapy of multiple canine behavioural disorders. Furthermore, as many similarities are reported between several human neuropsychiatric conditions and naturally occurring canine behavioural disorders, making this tracer available for use in dogs also provide researchers an interesting non-primate animal model to investigate human disorders. Five adult beagles underwent a 90 minutes dynamic PET scan and arterial whole blood was sampled throughout the scan. For each ROI, the distribution volume (V_T), obtained via the one- and two- tissue compartment model (1-TC, 2-TC) and the Logan Plot, was calculated and the goodness-of-fit was evaluated by the Akaike Information Criterion (AIC). For the preferred compartmental model BP_ND values were estimated and compared with those derived by four reference tissue models: 4-parameter RTM, SRTM2, MRTM2 and the Logan reference tissue model. The 2-TC model indicated in 61% of the ROIs a better fit compared to the 1-TC model. The Logan plot produced almost identical V_T values and can be used as an alternative. Compared with the 2-TC model, all investigated reference tissue models showed high correlations but small underestimations of the BP_ND-parameter. The highest correlation was achieved with the Logan reference tissue model (Y = 0.9266 x + 0.0257; R² = 0.9722). Therefore, this model can be put forward as a non-invasive standard model for future PET-experiments with [¹¹C]DASB in dogs.     

Xylem cavitation vulnerability influences tree species’ habitat preferences in miombo woodlands

Although precipitation plays a central role in structuring Africa’s miombo woodlands, remarkably little is known about plant-water relations in this seasonally dry tropical forest. Therefore, in this study, we investigated xylem vulnerability to cavitation for nine principal tree species of miombo woodlands, which differ in habitat preference and leaf phenology. We measured cavitation vulnerability (Ψ₅₀), stem-area specific hydraulic conductivity (K _S), leaf specific conductivity (K _L), seasonal variation in predawn water potential (Ψ_PD) and xylem anatomical properties [mean vessel diameter, mean hydraulic diameter, mean hydraulic diameter accounting for 95 % flow, and maximum vessel length (V _L)]. Results show that tree species with a narrow habitat range (mesic specialists) were more vulnerable to cavitation than species with a wide habitat range (generalists). Ψ₅₀ for mesic specialists ranged between ?1.5 and ?2.2 MPa and that for generalists between ?2.5 and ?3.6 MPa. While mesic specialists exhibited the lowest seasonal variation in Ψ_PD, generalists displayed significant seasonal variations in Ψ_PD suggesting that the two miombo habitat groups differ in their rooting depth. We observed a strong trade-off between K _S and Ψ₅₀ suggesting that tree hydraulic architecture is one of the decisive factors setting ecological boundaries for principal miombo species. While vessel diameters correlated weakly (P > 0.05) with Ψ₅₀, V _L was positively and significantly correlated with Ψ₅₀. Ψ_PD was significantly correlated with Ψ₅₀ further reinforcing the conclusion that tree hydraulic architecture plays a significant role in species’ habitat preference in miombo woodlands.     

Remote Homology between Munc13 MUN Domain and Vesicle Tethering Complexes

Most core components of the neurotransmitter release machinery have homologues in other types of intracellular membrane traffic, likely underlying a universal mechanism of intracellular membrane fusion. However, no clear similarity between Munc13s and protein families generally involved in membrane traffic has been reported, despite the essential nature of Munc13s for neurotransmitter release. This crucial function was ascribed to a minimal Munc13 region called the MUN domain, which likely participates in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex (SNARE) assembly and is also found in Ca²⁺-dependent activator protein for secretion. We have now used comparative sequence and structural analyses to study the structure and evolutionary origin of the MUN domain. We found weak yet significant sequence similarities between the MUN domain and a set of protein subunits from several related vesicle tethering complexes, such as Sec6 from the exocyst complex and Vps53 from the Golgi-associated retrograde protein complex. Such an evolutionary relationship allows structure prediction of the MUN domain and suggests functional similarities between MUN domain-containing proteins and multisubunit tethering complexes such as exocyst, conserved oligomeric Golgi complex, Golgi-associated retrograde protein complex, and Dsl1p. These findings further unify the mechanism of neurotransmitter release with those of other types of intracellular membrane traffic and, in turn, support a role for tethering complexes in soluble N-ethylmaleimide sensitive factor attachment protein receptor complex assembly.