Mesopelagic N2 Fixation Related to Organic Matter Composition in the Solomon and Bismarck Seas (Southwest Pacific)

Dinitrogen (N₂) fixation was investigated together with organic matter composition in the mesopelagic zone of the Bismarck (Transect 1) and Solomon (Transect 2) Seas (Southwest Pacific). Transparent exopolymer particles (TEP) and the presence of compounds sharing molecular formulae with saturated fatty acids and sugars, as well as dissolved organic matter (DOM) compounds containing nitrogen (N) and phosphorus (P) were higher on Transect 1 than on Transect 2, while oxygen concentrations showed an opposite pattern. N₂ fixation rates (up to ~1 nmol N L^-1 d^-1) were higher in Transect 1 than in Transect 2, and correlated positively with TEP, suggesting a dependence of diazotroph activity on organic matter. The scores of the multivariate ordination of DOM molecular formulae and their relative abundance correlated negatively with bacterial abundances and positively with N₂ fixation rates, suggesting an active bacterial exploitation of DOM and its use to sustain diazotrophic activity. Sequences of the nifH gene clustered with Alpha-, Beta-, Gamma- and Deltaproteobacteria, and included representatives from Clusters I, III and IV. A third of the clone library included sequences close to the potentially anaerobic Cluster III, suggesting that N₂ fixation was partially supported by presumably particle-attached diazotrophs. Quantitative polymerase chain reaction (qPCR) primer-probe sets were designed for three phylotypes and showed low abundances, with a phylotype within Cluster III at up to 10³ nifH gene copies L^-1. These results provide new insights into the ecology of non-cyanobacterial diazotrophs and suggest that organic matter sustains their activity in the mesopelagic ocean.     

Systemic induction of proteinase-inhibitor-II gene expression in potato plants by wounding

The systemic induction of expression of the gene for proteinase inhibitor II after wounding different parts of potato (Solanum tuberosum L.) plants was analysed at the RNA level. Wounding of either leaves or tubers led to an induction of expression of this gene in non-wounded upper and lower leaves as well as in the upper stem segment, whereas no expression was observed in nonwounded roots or in the lower stem segment. The signal mediating the systemic induction in nonwounded tissue must therefore be able to move both acropetally and basipetally. The systemic wound response is specific for the expression of the proteinase-inhibitor-II gene as no influence was observed for the expression of genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase and the tuber storage protein patatin which were examined in parallel with the proteinase-inhibitor-II gene.Abbreviation ssRubisco small subunit of ribulose-1,5-bis-phosphate carboxylase     

Unravelling biodiversity,evolution and threats to conservation in the Sahara‐Sahel

Deserts and arid regions are generally perceived as bare and rather homogeneous areas of low diversity. The Sahara is the largest warm desert in the world and together with the arid Sahel displays high topographical and climatic heterogeneity, and has experienced recent and strong climatic oscillations that have greatly shifted biodiversity distribution and community composition. The large size, remoteness and long‐term political instability of the Sahara‐Sahel, have limited knowledge on its biodiversity. However, over the last decade, there have been an increasing number of published scientific studies based on modern geomatic and molecular tools, and broad sampling of taxa of these regions. This review tracks trends in knowledge about biodiversity patterns, processes and threats across the Sahara‐Sahel, and anticipates needs for biodiversity research and conservation. Recent studies are changing completely the perception of regional biodiversity patterns. Instead of relatively low species diversity with distribution covering most of the region, studies now suggest a high rate of endemism and larger number of species, with much narrower and fragmented ranges, frequently limited to micro‐hotspots of biodiversity. Molecular‐based studies are also unravelling cryptic diversity associated with mountains, which together with recent distribution atlases, allows identifying integrative biogeographic patterns in biodiversity distribution. Mapping of multivariate environmental variation (at 1 km × 1 km resolution) of the region illustrates main biogeographical features of the Sahara‐Sahel and supports recently hypothesised dispersal corridors and refugia. Micro‐scale water‐features present mostly in mountains have been associated with local biodiversity hotspots. However, the distribution of available data on vertebrates highlights current knowledge gaps that still apply to a large proportion of the Sahara‐Sahel. Current research is providing insights into key evolutionary and ecological processes, including causes and timing of radiation and divergence for multiple taxa, and associating the onset of the Sahara with diversification processes for low‐mobility vertebrates. Examples of phylogeographic patterns are showing the importance of allopatric speciation in the Sahara‐Sahel, and this review presents a synthetic overview of the most commonly hypothesised diversification mechanisms. Studies are also stressing that biodiversity is threatened by increasing human activities in the region, including overhunting and natural resources prospection, and in the future by predicted global warming. A representation of areas of conflict, landmines, and natural resources extraction illustrates how human activities and regional insecurity are hampering biodiversity research and conservation. Although there are still numerous knowledge gaps for the optimised conservation of biodiversity in the region, a set of research priorities is provided to identify the framework data needed to support regional conservation planning.     

Dynein self-organizes while translocating the centrosome in T-cells

T-cells massively restructure their internal architecture upon reaching an antigen-presenting cell (APC) to form the immunological synapse (IS), a cell–cell interface necessary for efficient elimination of the APC. This reorganization occurs through tight coordination of cytoskeletal processes: actin forms a peripheral ring, and dynein motors translocate the centrosome toward the IS. A recent study proposed that centrosome translocation involves a microtubule (MT) bundle that connects the centrosome perpendicularly to dynein at the synapse center: the “stalk.” The synapse center, however, is actin-depleted, while actin was assumed to anchor dynein. We propose that dynein is attached to mobile membrane anchors, and investigate this model with computer simulations. We find that dynein organizes into a cluster in the synapse when translocating the centrosome, aligning MTs into a stalk. By implementing both a MT-capture-shrinkage and a MT-sliding mechanism, we explicitly demonstrate that this organization occurs in both systems. However, results obtained with MT-sliding dynein are more robust and display a stalk morphology consistent with our experimental data obtained with expansion microscopy. Thus, our simulations suggest that actin organization in T-cells during activation defines a specific geometry in which MT-sliding dynein can self-organize into a cluster and cause stalk formation.     

An Improved Protocol for Intact Chloroplasts and cpDNA Isolation in Conifers

Background

Performing chloroplast DNA (cpDNA) isolation is considered a major challenge among different plant groups, especially conifers. Isolating chloroplasts in conifers by such conventional methods as sucrose gradient and high salt has not been successful. So far, plastid genome sequencing protocols for conifer species have been based mainly on long-range PCR, which is known to be time-consuming and difficult to implement.

Methodology/Principal Findings

We developed a protocol for cpDNA isolation using three different conifer families: Araucaria angustifolia and Araucaria bidwilli (Araucariaceae), Podocarpus lambertii (Podocarpaceae) and Pinus patula (Pinaceae). The present protocol is based on high salt isolation buffer followed by saline Percoll gradient. Combining these two strategies allowed enhanced chloroplast isolation, along with decreased contamination caused by polysaccharides, polyphenols, proteins, and nuclear DNA in cpDNA. Microscopy images confirmed the presence of intact chloroplasts in high abundance. This method was applied to cpDNA isolation and subsequent sequencing by Illumina MiSeq (2×250 bp), using only 50 ng of cpDNA. Reference-guided chloroplast genome mapping showed that high average coverage was achieved for all evaluated species: 24.63 for A. angustifolia, 135.97 for A. bidwilli, 1196.10 for P. lambertii, and 64.68 for P. patula.

Conclusion

Results show that this improved protocol is suitable for enhanced quality and yield of chloroplasts and cpDNA isolation from conifers, providing a useful tool for studies that require isolated chloroplasts and/or whole cpDNA sequences.     

Humidity levels drive reproductive modes and phylogenetic diversity of amphibians in the Brazilian Atlantic Forest

Aim The diversity of reproductive modes among amphibians constitutes a striking example of how differences in the biology of species provide important explanations for species distribution patterns on a broad scale. We hypothesize that sites with a higher humidity level will support more modes of reproduction than drier sites and will consequently exhibit a higher phylogenetic diversity. Furthermore, if there is a gradient in the tolerance of reproductive modes to desiccation, there will be a nested pattern in the composition of reproductive modes among sites. Location Twenty‐seven forest sites in the Brazilian Atlantic Forest. Methods Through a path analysis approach, we evaluated the direct and indirect effects of the humidity level on the number of reproductive modes, as well as the relative importance of both variables on amphibian phylogenetic diversity. A nestedness analysis was used to quantify the extent to which the compositions of both species and reproductive modes in drier sites correspond to subsets of those in sites with higher annual precipitation. Results We found that the reproductive modes present in drier sites are non‐random subsets of those present in sites with higher humidity levels. Because reproductive modes are phylogenetically conserved among amphibians, sites with a greater number of reproductive modes supported greater phylogenetic diversity. Sites with high precipitation throughout the year provided suitable environmental conditions for a larger number of reproductive modes, whereas sites with low precipitation and typical seasonal climates supported only those reproductive modes specialized to resist desiccation. Main conclusions Our results show that humidity‐related variables are key environmental factors related to both the richness of reproductive modes and phylogenetic diversity. Our results support the hypothesis that the higher phylogenetic diversity found in moister sites reflects differences in the tolerance to desiccation among different reproductive modes. Given that reproductive modes are associated with susceptibility to desiccation, their incorporation into explanatory models may trigger a significant advance in the understanding of the mechanisms regulating the species richness and composition of amphibian communities.     

IGF-1 Regulates Cyr61 Induced Breast Cancer Cell Proliferation and Invasion

Background

Studies from our laboratory and others have shown that cysteine-rich 61 (Cyr61) may be involved in tumor proliferation and invasion. In earlier studies, we demonstrated increased insulin-like growth factor-I (IGF-1) is associated with breast tumor formation and poor clinical outcomes. In our current study we have investigated IGF-1 regulation of Cyr61 and whether targeting IGF-1 could inhibit Cyr61 induced tumor growth and proliferation.

Methods

Several ATCC derived normal and breast cancer cell lines were used in this study: MDA-MB231, BT474, MCF-7, and SKBR3. We also tested cells stably transfected in our laboratory with active Akt1 (pAkt; SKBR3/AA and MCF-7/AA) and dominant negative Akt1 (SKBR3/DN and MCF-7/DN). In addition, we used MCF-7 cells transfected with full length Cyr61 (CYA). Monolayer cultures treated with IGF-1 were analyzed for Cyr61 expression by RT-PCR and immunohistochemical staining. Migration assays and MTT based proliferation assays were used to determine invasive characteristics in response to IGF-1/Cyr61 activation.

Results

Cells with activated Akt have increased levels of Cyr61. Conversely, cells with inactive Akt have decreased levels of Cyr61. IGF-1 treatment increased Cyr61 expression significantly and cells with high level of Cyr61 demonstrate increased invasiveness and proliferation. Cyr61 overexpression and activation led to decrease in E-cadherin and decrease in FOXO1. Inhibition of the PI3K and MAPK pathways resulted in significant decrease in invasiveness and proliferation, most notably in the PI3K pathway inhibited cells.

Conclusion

The findings of this study show that IGF-1 upregulates Cyr61 primarily through activation of the Akt-PI3K pathway. IGF-1 induced MAPK plays a partial role. Increase in Cyr61 leads to increase in breast cancer cell growth and invasion. Hence, targeting Cyr61 and associated pathways may offer an opportunity to inhibit IGF-1 mediated Cyr61 induced breast cancer growth and invasion.     

Analysis of the expression of the Trichoderma harzianum ech42 gene in two isogenic clones of Escherichia coli by surface response methodology

The endochitinase gene ech42 from Trichoderma harzianum was cloned and expressed in Escherichia coli BL21(D3)pLysS using a pET-19b expression system. The effect of temperature, cell concentrations and inductor concentration on protein expression were evaluated by response surface methodology. Applying a second-order model to describe the amount of endochitinase produced by two selected clones, demonstrated that different optimal conditions of expression were found among clones, indicating that the best environmental conditions for expression among the several isogenic clones differed.     

Aflatoxins: Production of the Toxins In Vitro in Relation to Temperature

The production or accumulation of aflatoxins in vitro by four isolates on three substrates (acid-delinted cottonseed, shelled Spanish peanut, and rough rice) was studied in relation to temperature in the range of 10 to 40 C. Within the first 10 days after inoculation, the optimal temperature range for aflatoxin production was between 20 and 35 C. Only small amounts of the toxins were produced at 10 and 40 C. Within the optimal temperature range, the time required for toxin production and for significant accumulation decreased as the temperature increased. More aflatoxin G was produced or accumulated in relation to aflatoxin B at low temperatures (within the optimal range), and the G aflatoxins were metabolized more rapidly at the higher temperatures.