Energy Signaling in the Regulation of Gene Expression during Stress

Maintenance of homeostasis is pivotal to all forms of life. In the case of plants, homeostasis is constantly threatened by the inability to escape environmental fluctuations, and therefore sensitive mechanisms must have evolved to allow rapid perception of environmental cues and concomitant modification of growth and developmental patterns for adaptation and survival. Re-establishment of homeostasis in response to environmental perturbations requires reprogramming of metabolism and gene expression to shunt energy sources from growth-related biosynthetic processes to defense, acclimation, and, ultimately, adaptation. Failure to mount an initial 'emergency' response may result in nutrient deprivation and irreversible senescence and cell death. Early signaling events largely determine the capacity of plants to orchestrate a successful adaptive response. Early events, on the other hand, are likely to be shared by different conditions through the generation of similar signals and before more specific responses are elaborated. Recent studies lend credence to this hypothesis, underpinning the importance of a shared energy signal in the transcriptional response to various types of stress. Energy deficiency is associated with most environmental perturbations due to their direct or indirect deleterious impact on photosynthesis and/or respiration. Several systems are known to have evolved for monitoring the available resources and triggering metabolic, growth, and developmental decisions accordingly. In doing so, energy-sensing systems regulate gene expression at multiple levels to allow flexibility in the diversity and the kinetics of the stress response.

Screening of the Open Source Malaria Box Reveals an Early Lead Compound for the Treatment of Alveolar Echinococcosis.

The metacestode (larval) stage of the tapeworm Echinococcus multilocularis causes alveolar echinococcosis (AE), a very severe and in many cases incurable disease. To date, benzimidazoles such as albendazole and mebendazole are the only approved chemotherapeutical treatment options. Benzimidazoles inhibit metacestode proliferation, but do not act parasiticidal. Thus, benzimidazoles have to be taken a lifelong, can cause adverse side effects such as hepatotoxicity, and are ineffective in some patients. We here describe a newly developed screening cascade for the evaluation of the in vitro efficacy of new compounds that includes assessment of parasiticidal activity. The Malaria Box from Medicines for Malaria Venture (MMV), comprised of 400 commercially available chemicals that show in vitro activity against Plasmodium falciparum, was repurposed. Primary screening was carried out at 10 μM by employing the previously described PGI assay, and resulted in the identification of 24 compounds that caused physical damage in metacestodes. Seven out of these 24 drugs were also active at 1 μM. Dose-response assays revealed that only 2 compounds, namely MMV665807 and MMV665794, exhibited an EC50 value below 5 μM. Assessments using human foreskin fibroblasts and Reuber rat hepatoma cells showed that the salicylanilide MMV665807 was less toxic for these two mammalian cell lines than for metacestodes. The parasiticidal activity of MMV665807 was then confirmed using isolated germinal layer cell cultures as well as metacestode vesicles by employing viability assays, and its effect on metacestodes was morphologically evaluated by electron microscopy. However, both oral and intraperitoneal application of MMV665807 to mice experimentally infected with E. multilocularis metacestodes did not result in any reduction of the parasite load.

Systemic and local immune responses in sheep after Neospora caninum experimental infection at early, mid and late gestation.

Besides its importance in cattle, Neospora caninum may also pose a high risk as abortifacient for small ruminants. We have recently demonstrated that the outcome of experimental infection of pregnant sheep with 10(6) Nc-Spain7 tachyzoites is strongly dependent on the time of gestation. In the current study, we assessed peripheral and local immune response in those animals. Serological analysis revealed earlier and higher IFN-γ and IgG responses in ewes infected at early (G1) and mid (G2) gestation, when abortion occurred. IL-4 was not detected in sera from any sheep. Inflammatory infiltrates in the placenta mainly consisted of CD8+ and, to a lesser extent, CD4+ T cells and macrophages (CD163+). The infiltrate was more intense in sheep infected at mid-gestation. In the foetal mesenchyme, mostly free tachyzoites were found in animals infected at G1, while those infected in G2 displayed predominantly particulate antigen, and parasitophorous vacuoles were detected in sheep infected at G3. A similar pattern of placental cytokine mRNA expression was found in all groups, displaying a strengthened upregulation of IFN-γ and IL-4 and milder increases of TNF-α and IL-10, reminiscent of a mixed Th1 and Th2 response. IL-12 and IL-6 were only slightly upregulated in G2, and TGF-β was downregulated in G1 and G2, suggestive of limited T regulatory (Treg) cell activity. No significant expression of TLR2 or TLR4 could be detected. In summary, this study confirms the pivotal role of systemic and local immune responses at different times of gestation during N. caninum infection in sheep.

Maternal separation followed by isolation-housing differentially affects prepulse inhibition of the acoustic startle response in C57BL/6 mice

Exposure to chronic stress is associated with an increased incidence of neuropsychiatric dysfunction. The current study evaluated two competing hypotheses, the cumulative stress and the match/mismatch hypothesis of neuropsychiatric dysfunction, using two paradigms relating to exposure to “stress”: pre-weaning maternal separation and post-weaning isolation-housing. C57BL/6 offspring were reared under four conditions: typical animal facility rearing (AFR, control), early handling (EH, daily 15 min separation from dam), maternal separation (MS, daily 4 hr separation from dam), and maternal and peer separation (MPS, daily 4 hr separation from dam and from littermates). After weaning, mice were either housed socially (2–3/cage) or in isolation (1/cage) and then tested for prepulse inhibition in adulthood. Isolation-housed MPS subjects displayed greater deficits in prepulse inhibition relative to socially-housed MPS subjects while socially-housed AFR subjects displayed greater deficits in prepulse inhibition relative to isolation-housed AFR subjects. The results indicate that these treatment conditions represent a potentially valuable model for evaluating the match/mismatch hypothesis in regards to neuropsychiatric dysfunction.

Zebrafish Caudal Fin Angiogenesis Assay-Advanced Quantitative Assessment Including 3-Way Correlative Microscopy.

BACKGROUND Researchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics. OBJECTIVE To establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way. APPROACH & RESULTS Basic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including "graph energy" and "distance to farthest node". The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level. CONCLUSIONS The improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations.

Figure 2. Downcore variations of total diatom concentration in core MD02-2529

The modern eastern equatorial Pacific (EEP) is a major natural source for atmospheric carbon dioxide and is thought to be connected to high-latitude ocean dynamics by oceanic teleconnections on glacial-interglacial timescales. A wealth of sedimentary records aiming at reconstructing last Quaternary changes in primary productivity and nutrient utilization have been devoted to understanding those linkages between the EEP and other distant oceanic areas. Most of these records are, however, clustered in the pelagic EEP cold tongue, with comparatively little attention devoted to coastal areas. Here we present downcore measurements of the composition and concentration of the diatom assemblage together with opal (biogenic silica) concentration at site MD02-2529 recovered in the coastal Panama Basin. Piston core MD02-2529, collected in an area affected by a multitude of processes, provides evidence for strong variations in diatom production at the millennial timescale during the last glacial cycle. The maxima in total diatom concentration occurred during the early marine isotopic stage (MIS) 4 as well as during the MIS 4/3 transition and MIS 3. Rapid changes in diatom concentrations during the MIS 3 mimics Bond cycles as independently recorded by the SSS estimation derived from planktonic foraminifera from the same core. Such patterns indicate a clear linkage between diatom production in the coastal EEP and rapid climate changes in the high-latitude North Atlantic. In parallel, the long-term succession of the diatom community from coastal diatoms, predominantly thriving during MIS 5 and 4, towards pelagic diatoms, dominant during MIS 3 and 2, points to a long-term change in the surface hydrology. During Heinrich Events, diatoms strongly reduced their production, probably due to enhanced stratification in the upper water column. After the last glacial maximum, diatom production and valve preservation strongly decreased in response to the advection of nutrient (H2SiO4)-depleted, warmer water masses. Our high-resolution record highlights how regional climatic processes can modulate rapid changes in siliceous primary production as triggered by wind-induced local upwelling, indicating that millennial climatic variability can overtake other prominent hydrological processes such as those related to silicic acid leakage.

Late Eocene to early Oligocene carbonate and barite accumulation rates in the Pacific Basin

The late Eocene through earliest Oligocene (40-32 Ma) spans a major transition from greenhouse to icehouse climate, with net cooling and expansion of Antarctic glaciation shortly after the Eocene/Oligocene (E/O) boundary. We investigated the response of the oceanic biosphere to these changes by reconstructing barite and CaCO3 accumulation rates in sediments from the equatorial and North Pacific Ocean. These data allow us to evaluate temporal and geographical variability in export production and CaCO3 preservation. Barite accumulation rates were on average higher in the warmer late Eocene than in the colder early Oligocene, but cool periods within the Eocene were characterized by peaks in both barite and CaCO3 accumulation in the equatorial region. We infer that climatic changes not only affected deep ocean ventilation and chemistry, but also had profound effects on surface water characteristics influencing export productivity. The ratio of CaCO3 to barite accumulation rates, representing the ratio of particulate inorganic C accumulation to Corg export, increased dramatically at the E/O boundary. This suggests that long-term drawdown of atmospheric CO2 due to organic carbon deposition to the seafloor decreased, potentially offsetting decreasing pCO2 levels and associated cooling. The relatively larger increase in CaCO3 accumulation compared to export production at the E/O suggests that the permanent deepening of the calcite compensation depth (CCD) at that time stems primarily from changes in deep water chemistry and not from increased carbonate production.

Stickleback F2 generation transgenerational plasticity experiment: egg and body size data

Nongenetic inheritance mechanisms such as transgenerational plasticity (TGP) can buffer populations against rapid environmental change such as ocean warming. Yet, little is known about how long these effects persist and whether they are cumulative over generations. Here, we tested for adaptive TGP in response to simulated ocean warming across parental and grandparental generations of marine sticklebacks. Grandparents were acclimated for two months during reproductive conditioning, whereas parents experienced developmental acclimation, allowing us to compare the fitness consequences of short-term vs. prolonged exposure to elevated temperature across multiple generations. We found that reproductive output of F1 adults was primarily determined by maternal developmental temperature, but carry-over effects from grandparental acclimation environments resulted in cumulative negative effects of elevated temperature on hatching success. In very early stages of growth, F2 offspring reached larger sizes in their respective paternal and grandparental environment down the paternal line, suggesting that other factors than just the paternal genome may be transferred between generations. In later growth stages, maternal and maternal granddam environments strongly influenced offspring body size, but in opposing directions, indicating that the mechanism(s) underlying the transfer of environmental information may have differed between acute and developmental acclimation experienced by the two generations. Taken together, our results suggest that the fitness consequences of parental and grandparental TGP are highly context dependent, but will play an important role in mediating some of the impacts of rapid climate change in this system.

Early Eocene benthic carbon isotope data of ODP Site 208-1263

The early Eocene represents a time of major changes in the global carbon cycle and fluctuations in global temperatures on both short- and long-time scales. These perturbations of the ocean-atmosphere system have been linked to orbital forcing and changes in net organic carbon burial, but accurate age models are required to disentangle the various forcing mechanisms and assess causal relationships. Discrepancies between the employed astrochronological and radioisotopic dating techniques prevent the construction of a robust time frame between ~49 and ~54 Ma. Here we present an astronomically tuned age model for this critical time period based on a new high-resolution benthic d13C record of ODP Site 1263, SE Atlantic. First, we assess three possible tuning options to the stable long-eccentricity cycle (405-kyr), starting from Eocene Thermal Maximum 2 (ETM2, ~54 Ma). Next we compare our record to the existing bulk carbonate d13C record from the equatorial Atlantic (Demerara Rise, ODP Site 1258) to evaluate our three initial age models and compare them with alternative age models previously established for this site. Finally, we refine our preferred age model by expanding our tuning to the 100-kyr eccentricity cycle of the La2010d solution. This solution appears to accurately reflect the long- and short-term eccentricity-related patterns in our benthic d13C record of ODP Site 1263 back to at least 52 Ma and possibly to 54 Ma. Our time scale not only aims to provide a new detailed age model for this period, but it may also serve to enhance our understanding of the response of the climate system to orbital forcing during this super greenhouse period as well as trends in its background state.

Age estimations of calcareous nannofossil biohorizons of the middle Paleocene to early Eocene at ODP Site 208-1262 (Table 1, Appendix B)

Over the last several decades debates on the 'tempo and mode' of evolution have centered on the question whether morphological evolution preferentially occurs gradually or punctuated, i.e., with long periods of stasis alternating with short periods of rapid morphological change and generation of new species. Another major debate is focused on the question whether long-term evolution is driven by, or at least strongly influenced by changes in the environment, or by interaction with other life forms. Microfossils offer a unique opportunity to obtain the large datasets as well as the precision in dating of subsequent samples to study both these questions.We present high-resolution analyses of selected calcareous nannofossils from the deep-sea section recovered at ODP Site 1262 (Leg 208) in the South-eastern Atlantic. The studied section encompasses nannofossil Zones NP4-NP12 (equivalent to CP3-CP10) and Chrons C27r-C24n. We document more than 70 biohorizons occurring over an about 10 Myr time interval, (~62.5 Ma to ~52.5 Ma), and discuss their reliability and reproducibility with respect to previous data, thus providing an improved biostratigraphic framework, which we relate to magnetostratigraphic information, and present for two possible options of a new Paleocene stratigraphic framework based on cyclostratigraphy. This new framework enabled us to tentatively reconstruct steps in the evolution of early Paleogene calcareous nannoplankton through documentation of transitional morphotypes between genera and/or species and of the phylogenetic relations between the genera Fasciculithus, Heliolithus, Discoasteroides and Discoaster, as well as between Rhomboaster and Tribrachiatus. The exceptional record provided by the continuous, composite sequence recovered at Walvis Ridge allows us to describe the mode of evolution among calcareous nannoplankton: new genera and/or new species usually originated through branching of lineages via gradual, but relatively rapid, morphological transitions, as documented by the presence of intermediate forms between the end-member ancestral and descendant forms. Significant modifications in the calcareous nannofossil assemblages are often "related" to significant changes in environmental conditions, but the appearance of structural innovations and radiations within a single genus also occurred during "stable" environmental conditions. These lines of evidence suggest that nannoplankton evolution is not always directly triggered by stressed environmental conditions but could be also driven by endogenous biotic control.

Age models for sediment cores off Peru

Planktonic foraminiferal faunas of the southeast Pacific indicate that sea surface temperatures (SST) have varied by as much as 8-10°C in the Peru Current, and by ?5-7°C along the equator, over the past 150,000 years. Changes in SST at times such as the Last Glacial Maximum reflect incursion of high-latitude species Globorotalia inflata and Neogloboquadrina pachyderma into the eastern boundary current and as far north as the equator. A simple heat budget model of the equatorial Pacific shows that observed changes in Peru Current advection can account for about half of the total variability in equatorial SSTs. The remaining changes in equatorial SST, which are likely related to local changes in upwelling or pycnocline depth, precede changes in polar climates as recorded by d18O. This partitioning of processes in eastern equatorial Pacific SST reveals that net ice-age cooling here reflects first a rapid response of equatorial upwelling to insolation, followed by a later response to changes in the eastern boundary current associated with high-latitude climate (which closely resembles variations in atmospheric CO2 as recorded in the Vostok ice core). Although precise mechanisms responsible for the equatorial upwelling component of climate change remain uncertain, one likely candidate that may operate independently of the ice sheets is insolation-driven changes in El Niño/Southern Oscillation (ENSO) frequency. Early responses of equatorial SST detected both here and elsewhere highlight the sensitivity of tropical systems to small changes in seasonal insolation. The scale of tropical changes we have observed are substantially greater than model predictions, suggesting a need for further quantitative assessment of processes associated with long-term climate change.