Background: Imaging large volumes such as entire cells or small model organisms at nanoscale resolution seemed an unrealistic, rather tedious task so far. Now, technical advances have lead to several electron microscopy (EM) large volume imaging techniques. One is array tomography, where ribbons of ultrathin serial sections are deposited on solid substrates like silicon wafers or glass coverslips. Results: To ensure reliable retrieval of multiple ribbons from the boat of a diamond knife we introduce a substrate holder with 7 axes of translation or rotation specifically designed for that purpose. With this device we are able to deposit hundreds of sections in an ordered way in an area of 22 × 22 mm, the size of a coverslip. Imaging such arrays in a standard wide field fluorescence microscope produces reconstructions with 200 nm lateral resolution and 100 nm (the section thickness) resolution in z. By hierarchical imaging cascades in the scanning electron microscope (SEM), using a new software platform, we can address volumes from single cells to complete organs. In our first example, a cell population isolated from zebrafish spleen, we characterize different cell types according to their organelle inventory by segmenting 3D reconstructions of complete cells imaged with nanoscale resolution. In addition, by screening large numbers of cells at decreased resolution we can define the percentage at which different cell types are present in our preparation. With the second example, the root tip of cress, we illustrate how combining information from intermediate resolution data with high resolution data from selected regions of interest can drastically reduce the amount of data that has to be recorded. By imaging only the interesting parts of a sample considerably less data need to be stored, handled and eventually analysed. Conclusions: Our custom-designed substrate holder allows reproducible generation of section libraries, which can then be imaged in a hierarchical way. We demonstrate, that EM volume data at different levels of resolution can yield comprehensive information, including statistics, morphology and organization of cells and tissue. We predict, that hierarchical imaging will be a first step in tackling the big data issue inevitably connected with volume EM.
Background: Drought is the most important environmental stress that limits crop yield in a global warming world. Despite the compelling evidence of an important role of oxidized and reduced sulfur-containing compounds during the response of plants to drought stress (e.g. sulfate for stomata closure or glutathione for scavenging of reactive oxygen species), the assimilatory sulfate reduction pathway is almost not investigated at the molecular or at the whole plant level during drought. Results: In the present study, we elucidated the role of assimilatory sulfate reduction in roots and leaves of the staple crop maize after application of drought stress. The time-resolved dynamics of the adaption processes to the stress was analyzed in a physiological relevant situation –when prolonged drought caused significant oxidation stress but root growth should be maintained. The allocation of sulfate was significantly shifted to the roots upon drought and allowed for significant increase of thiols derived from sulfate assimilation in roots. This enabled roots to produce biomass, while leaf growth was stopped. Accumulation of harmful reactive oxygen species caused oxidation of the glutathione pool and decreased glutathione levels in leaves. Surprisingly, flux analysis using [35S]-sulfate demonstrated a significant down-regulation of sulfate assimilation and cysteine synthesis in leaves due to the substantial decrease of serine acetyltransferase activity. The insufficient cysteine supply caused depletion of glutathione pool in spite of significant transcriptional induction of glutathione synthesis limiting GSH1. Furthermore, drought impinges on transcription of membrane-localized sulfate transport systems in leaves and roots, which provides a potential molecular mechanism for the reallocation of sulfur upon prolonged water withdrawal. Conclusions: The study demonstrated a significant and organ-specific impact of drought upon sulfate assimilation. The sulfur metabolism related alterations at the transcriptional, metabolic and enzyme activity level are consistent with a promotion of root growth to search for water at the expense of leaf growth. The results provide evidence for the importance of antagonistic regulation of sulfur metabolism in leaves and roots to enable successful drought stress response at the whole plant level.
Projekte im Rahmen des Wassernetzwerks Baden-Württemberg Zwei Forschungsverbünde zum Thema Wasser, an denen Wissenschaftler des Heidelberg Center for the Environment (HCE) der Ruperto Carola beteiligt sind, erhalten eine Förderung des Landes Baden-Württemberg. Die beiden inter- und transdisziplinären Konsortien gehören zu drei Forschernetzwerken im Rahmen des Programms Wasserforschung Baden-Württemberg. Forschernetzwerk "Eff-Net“ ("Effect Network in Water Research": im Projekt "Eff-Net" werden Wirkungszusammenhänge für die Risikobewertung von Chemikalien in Gewässerökosystemen untersucht.
Background: Recent malaria control and elimination attempts show remarkable success in several parts of sub-Saharan Africa. Vector control via larval source management represents a new and to date underrepresented approach in low income countries to further reduce malaria transmission. Although the positive impact of such campaigns on malaria incidence has been researched, there is a lack of data on which prerequisites are needed for implementing such programs on a routine basis on large scale. Our objectives are to point out important steps in implementing an anti-malaria larviciding campaign in a resource and infrastructure restraint setting and share the lessons learned from our experience during a three-year intervention study in rural Burkina Faso. Methods: We describe the approaches we followed and the challenges that have been encountered during the EMIRA project, a three-year study on the impact of environmental larviciding on vector ecology and human health. An inventory of all performed work packages and associated problems and peculiarities was assembled. Results: Key to the successful implementation of the larviciding program within a health district was the support and infrastructure from the local research center run by the government. This included availability of trained scientific personnel for local project management, data collection and analysis by medical personnel, entomologists and demographers and teams of fieldworkers for the larviciding intervention. A detailed a priori assessment of the environment and vector breeding site ecology was essential to calculate personnel requirements and the need for larvicide and application apparel. In our case of a three-year project, solid funding for the whole duration was an important issue, which restricted the number of possible donors. We found the acquisition of qualified field personnel in fair numbers not to be always easy and training in application techniques and basic entomologic knowledge required several weeks of theoretical and practical formation. A further crucial point was to establish an effective quality control system that ensured the timely verification of larviciding success and facilitated in time data handling. While the experiences of running a larviciding campaign may vary globally, the experiences gained and the methods used in the Nouna health district may be employed in similar settings. Conclusions: Our observations highlight important components and strategies that should be taken into account when planning and running a similar larviciding program against malaria in a resource limited setting. A strong local partnership, meticulous planning with the possibility of ad-hoc adaption of project components and a reliable source of funding turned out to be crucial factors to successfully accomplish such a project.
Background: Glutaredoxins (GRXs) are small proteins which bind glutathione to either reduce disulfide bonds or to coordinate iron sulfur clusters. Whereas these well-established functions are associated with ubiquitously occurring GRXs that encode variants of a CPYC or a CGFS motif in the active center, land plants also possess CCxC/S-type GRXs (named ROXYs) for which the biochemical functions are yet unknown. ROXYs physically and genetically interact with bZIP transcription factors of the TGA family. In Arabidopsis, ectopically expressed ROXY19 (originally named GRX480 or GRXC9) negatively regulates expression of jasmonic acid/ethylene-induced defense genes through an unknown mechanism that requires at least one of the redundant transcription factors TGA2, TGA5 or TGA6. Results: Ectopically expressed ROXY19 interferes with the activation of TGA-dependent detoxification genes. Similar to the tga2 tga5 tga6 mutant, 35S:ROXY19 plants are more susceptible to the harmful chemical TIBA (2,3,5-triiodobenzoic acid). The repressive function of ROXY19 depends on the integrity of the active site, which can be either CCMC or CPYC but not SSMS. Ectopic expression of the related GRX ROXY18/GRXS13 also led to increased susceptibility to TIBA, indicating potential functional redundancy of members of the ROXY gene family. This redundancy might explain why roxy19 knock-out plants did not show a phenotype with respect to the regulation of the TIBA-induced detoxification program. Complementation of the tga2 tga5 tga6 mutant with either TGA5 or TGA5C186S, in which the single potential target-site of ROXY19 had been eliminated, did not reveal any evidence for a critical redox modification that might be important for controlling the detoxification program. Conclusions: ROXY19 and related proteins of the ROXY gene family can function as negative regulators of TGA-dependent promoters controlling detoxification genes.
Background: The key tools in malaria control are early diagnosis and treatment of cases as well as vector control. Current strategies for malaria vector control in sub-Saharan Africa are largely based on long-lasting insecticide-treated nets (LLINs) and to a much smaller extent on indoor residual spraying (IRS). An additional tool in the fight against malaria vectors, larval source management (LSM), has not been used in sub-Saharan Africa on a wider scale since the abandonment of environmental spraying of DDT. Increasing concerns about limitations of LLINs and IRS and encouraging results from large larvicide-based LSM trials make a strong case for using biological larviciding as a complementary tool to existing control measures. Arguments that are often quoted against such a combined approach are the alleged high implementation costs of LSM. This study makes the first step to test this argument. The implementation costs of larval source management based on Bacillus thuringiensis israelensis (Bti) (strain AM65-52) spraying under different implementation scenarios were analysed in a rural health district in Burkina Faso. Methods: The analysis draws on detailed cost data gathered during a large-scale LSM intervention between 2013 and 2015. All 127 villages in the study setup were assigned to two treatment arms and one control group. Treatment either implied exhaustive spraying of all available water collections or targeted spraying of the 50 % most productive larval sources via remote-sensing derived and entomologically validated risk maps. Based on the cost reports from both intervention arms, the per capita programme costs were calculated under the assumption of covering the whole district with either intervention scenario. Cost calculations have been generalized by providing an adaptable cost formula. In addition, this study assesses the sensitivity of per capita programme costs with respect to changes in the underlying cost components. Results: The average annual per capita costs of exhaustive larviciding with Bti during the main malaria transmission period (June–October) in the Nouna health district were calculated to be US$ 1.05. When targeted spraying of the 50% most productive larval sources is used instead, average annual per capita costs decrease by 27% to US$ 0.77. Additionally, a high sensitivity of per capita programme costs against changes in total surface of potential larval sources and the number of spraying repetitions was found. Discussion: The per capita costs for larval source management interventions with Bti are roughly a third of the annual per capita expenditures for anti-malarial drugs and those for LLINs in Burkina Faso which are US$ 3.80 and 3.00, respectively. The average LSM costs compare to those of IRS and LLINs for sub-Saharan Africa. The authors argue that in such a setting LSM based on Bti spraying is within the range of affordable anti-malarial strategies and, consequently, should deserve more attention in practice. Future research includes a cost-benefit calculation, based on entomological and epidemiological data collected during the research project.
Dass es Tierversuche gibt, und aus Sicht vieler Forscher noch geben muss, ist ein heikles Thema in der Wissenschaft. Ein Projekt von Biologen der Uni Heidelberg, das zur Reduzierung von Tierversuchen beitragen soll, wird von Land Baden-Württemberg jetzt mit rund 107.000 Euro gefördert. Bisher wurde, zum Nachweis von Schadstoffen im Wasser, oft der Zebrabärbling, ein in der Forschung etablierter Fisch eingesetzt. Das neue Verfahren soll mehrere 100.000 erwachsene Versuchstiere jährlich durch Fischembryonen ersetzen.