A high-throughput glow-type aequorin assay for measuring receptor-mediated changes in intracellular calcium levels

A glow-type aequorin luminescence assay for measuring receptor-mediated stimulation of intracellular calcium levels is described and characterized. The human 5-hydroxytryptamine(2A) receptor stably coexpressed in human embryonic kidney cells with apoaequorin was used to characterize the system and showed that following the flash reaction, a stable luminescence signal could be measured using a microplate scintillation counter for between 3 and 7 h after the addition of receptor agonist. Furthermore, this luminescence was dependent on the concentration of agonist used and gave potency values that were stable over this time period. Testing a range of 5-hydroxytryptamine(2A) receptor agonists gave the expected rank order of potency for this receptor. The glow luminescence could also be inhibited by 5-hydroxytryptamine(2A) receptor antagonists, generating affinity values that directly correlated with those determined for inhibition of the flash reaction carried out under the same buffer conditions. The assay therefore gave pharmacologically relevant data and allows a significant improvement of throughput over the traditional flash-type measurements made using an injecting luminometer.     

River macrophyte indices: not the Holy Grail!

1. Recent studies have demonstrated that there is generally no unambiguous relationship between plant species composition and specific environmental conditions in rivers. Nevertheless, indices of environmental pressures based on macrophytes are flourishing, because of the requirements of the Water Framework Directive (WFD). 2. We first reviewed nine such indices against 13 criteria for bioindicators. Then, using data from France and England, we tested whether the IBMR (Macrophyte Biological Index for Rivers) and LEAFPACS (predictions and classification system for macrophytes) methods could reliably indicate nutrient and hydromorphological pressures. Finally, we used an improved bootstrapping method to estimate accuracy. 3. Currently, most indices lack ecological meaning for a variety of reasons, including partial sampling (backwaters are excluded); reliance on list of taxa (there are identification difficulties) rather than structure and functions; correlation rather than causation; application within a limited biogeographical area; reliance on ‘expert’ judgement; high precision but poor accuracy; poorly defined reference conditions; lack of independent tests; and an inability to discriminate reliably between the target pressures of interest from confounding background variables. 4. IBMR was a far better indicator of pH (or HCO₃‐pCO₂) than it was of soluble reactive phosphorus, SRP (or SRP‐NH₄). While there was a highly significant correlation between IBMR and SRP after removing the effect of pH, the relationship was weak (r² = 0.08, n = 215, P < 0.001). 5. LEAFPACS is a multi‐metric method summing up five individual indices, each compliant with the WFD. Its individual metrics were not better correlated with nutrient and hydromorphological pressures (with r² < 0.1, n = 62, P < 0.05) than was the IBMR. The meaning of the overall metric is questionable. 6. There are problems in determining the precision of the indices, owing to uncertainties in recording, but they are less than the uncertainties in determining accuracy (because species optima and tolerances are sometimes poorly known). 7. Reliable information is needed to improve the state of our rivers. Macrophyte indices are able to detect statistically significant pressures from a large population of sites but cannot be applied at specific sites, as required by the WFD, owing to large uncertainties and low explanatory power. Typically, more than 90% of the variability in macrophyte indices is attributed to factors other than human pressure. The WFD would be better served by a simpler, holistic approach based on our current mechanistic understanding of river processes. These findings are likely to apply also to other taxonomic groups (macroinvertebrates, diatoms, fish) used in the assessment of purported ecological quality and to palaeolimnological measures of reference status.     

SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth

Cell growth (accumulation of mass) needs to be coordinated with metabolic processes that are required for the synthesis of macromolecules. The PI3-kinase/Akt signaling pathway induces cell growth via activation of complex 1 of the target of rapamycin (TORC1). Here we show that Akt-dependent lipogenesis requires mTORC1 activity. Furthermore, nuclear accumulation of the mature form of the sterol responsive element binding protein (SREBP1) and expression of SREBP target genes was blocked by the mTORC1 inhibitor rapamycin. We also show that silencing of SREBP blocks Akt-dependent lipogenesis and attenuates the increase in cell size in response to Akt activation in vitro. Silencing of dSREBP in flies caused a reduction in cell and organ size and blocked the induction of cell growth by dPI3K. Our results suggest that the PI3K/Akt/TOR pathway regulates protein and lipid biosynthesis in an orchestrated manner and that both processes are required for cell growth.     

Ecological engineering: A rationale for standardized curriculum and professional certification in the United States

The demand for engineering solutions to ecosystem–level problems has increased as the impact of human activities has expanded to global proportions. While the science of restoration ecology has been developed to address many critical ecosystem management issues, the high degree of complexity and uncertainty associated with these issues demands a more quantitative approach. Ecological engineering uses science-based quantification of ecological processes to develop and apply engineering-based design criteria for sustainable systems. We suggest that in the United States ecological engineering curricula should be offered at the graduate level and should require rigorous Accreditation Board of Engineering and Technology-accredited (or equivalent) undergraduate preparation in engineering fundamentals. In addition to strengthening students’ mastery of engineering theory and application, the graduate curriculum should provide core courses in ecosystem theory including quantitative ecology, systems ecology, restoration ecology, ecological engineering, ecological modeling, and ecological engineering economics. Advanced courses in limnology, environmental plant physiology, ecological economics, and specific ecosystem design should be provided to address students’ specific professional objectives. Finally, professional engineering certification must be developed to insure the credibility of this new engineering specialization.     

Genetic heterogeneity of steroid sulfatase deficiency revealed with cDNA for human steroid sulfatase

Three cDNA clones with inserts of 1.2-1.6 kb that reacted both with antibodies and oligonucleotides specific for steroid sulfatase were isolated from a human placental library in lambda gt11. The 5'-end of one of the inserts, STS-3, was sequenced and colinearity with the amino acid sequence of 3 peptides of steroid sulfatase encompassing 64 amino acids was demonstrated. STS-3 hybridized with 2.5, 4.6 and 6.3 kb species in poly(A)+RNA and with 2.5, 4 and 9 kb fragments of EcoRI digested human DNA. The frequency of the EcoRI fragments in DNA from females was approximately twice that in DNA from males. DNA from two patients with steroid sulfatase deficiency and X-linked ichthyosis did not hybridize with STS-3. DNA from a third patient showed a normal hybridization pattern. It is concluded that steroid sulfatase deficiency is a genetically heterogenous disorder.     

Intra-specific heterogeneity of the rDNA internal transcribed spacer in the Simulium damnosum (Diptera: Simuliidae) complex

The internal transcribed spacer (ITS) of the rRNA gene cluster has been used as a model for the study of the action of concerted evolution and molecular drive on repeated sequence families. In contrast to this general finding, preliminary DNA sequence analysis of cloned representatives of the ITS from the West African black fly species complex Simulium damnosum s.1. demonstrated extensive intra-individual and intra-specific polymorphisms. Variability in the ITS was primarily confined to the ITS1 domain. The degree and type of intra-individual and intra-specific variability within the ITS was further characterized using gel electrophoresis, DNA hybridization, and heteroduplex analysis of the PCR products generated from the ITS1 domain. ITS1 copies from individual S. damnosum s.1. differed in length and sequence composition. These results, when taken together, demonstrate that a large degree of intra-individual and intra-specific heterogeneity exists in the ITS of S. damnosum s.1. The intra-individual heterogeneity was greater in the savanna-dwelling than forest-dwelling sibling species of S. damnosum s.1. This heterogeneity may be due in part to inter-breeding among sympatric sibling species, coupled with disturbance of S. damnosum s.1. populations resulting from intensive vector control efforts.      

Strategies for imaging mitophagy in high-resolution and high-throughput

The selective autophagic removal of mitochondria called mitophagy is an essential physiological signaling for clearing damaged mitochondria and thus maintains the functional integrity of mitochondria and cells. Defective mitophagy is implicated in several diseases, placing mitophagy as a target for drug development. The identification of key regulators of mitophagy as well as chemical modulators of mitophagy requires sensitive and reliable quantitative approaches. Since mitophagy is a rapidly progressing event and sub-microscopic in nature, live cell image-based detection tools with high spatial and temporal resolution is preferred over end-stage assays. We describe two approaches for measuring mitophagy in mammalian cells using stable cells expressing EGFP-LC3 – Mito-DsRed to mark early phase of mitophagy and Mitochondria-EGFP – LAMP1-RFP stable cells for late events of mitophagy. Both the assays showed good spatial and temporal resolution in wide-field, confocal and super-resolution microscopy with high-throughput adaptable capability. A limited compound screening allowed us to identify a few new mitophagy inducers. Compared to the current mitophagy tools, mito-Keima or mito-QC, the assay described here determines the direct delivery of mitochondrial components to the lysosome in real time mode with accurate quantification if monoclonal cells expressing a homogenous level of both probes are established. Since the assay described here employs real-time imaging approach in a high-throughput mode, the platform can be used both for siRNA screening or compound screening to identify key regulators of mitophagy at decisive stages.