A low-cost, portable generic biotoxicity assay for environmental monitoring applications

Fish chromatophores have been shown to be promising biosensors for the detection of hostile agents in the environment. However, state-of-art methods for such applications are still based on extensive use of data/signal processing, in conjunction with need for a skilled human observer to carry out the detection. As a result, conventional methods are complex, costly and cumbersome rendering them useless for field applications requiring low-cost portable solutions capable of fast detection. A new technique is proposed based on the popular scheme of observing the aggregation response in chromatophores for detection of toxicity, and a solution using optical detection and electronic processing is outlined. This scheme has the advantage of being low in cost while providing simple, fast and reliable detection.     

Waste Treatment and Assessment of Long-Term Emissions (8pp)

Goal, Scope and Background The disposal phase of a product’s life cycle in LCA is often neglected or based on coarse indicators like ‘kilogram waste’. The goal of report No. 13 of the ecoinvent project (Doka 2003) is to create detailed Life Cycle Inventories of waste disposal processes. The purpose of this paper is to give an overview of the models behind the waste disposal inventories in ecoinvent, to present exemplary results and to discuss the assessment of long-term emissions. This paper does not present a particular LCA study. Inventories are compiled for many different materials and various disposal technologies. Considered disposal technologies are municipal incineration and different landfill types, including sanitary landfills, hazardous waste incineration, waste deposits in deep salt mines, surface spreading of sludges, municipal wastewater treatment, and building dismantling. The inventoried technologies are largely based on Swiss plants. Inventories can be used for assessment of the disposal of common, generic waste materials like paper, plastics, packaging etc. Inventories are also used within the ecoinvent database itself to inventory the disposal of specific wastes generated during the production phase. Inventories relate as far as possible to the specific chemical composition of the waste material (waste-specific burdens). Certain expenditures are not related to the waste composition and are inventoried with average values (process-specific burdens). Methods The disposal models are based on previous work, partly used in earlier versions of ecoinvent/ETH LCI data. Important improvements were the extension of the number of considered chemical elements to 41 throughout all disposal models and new landfill models based on field data. New inventories are compiled for waste deposits in deep salt mines and building material disposal. Along with the ecoinvent data and the reports, also Excel-based software tools were created, which allow ecoinvent members to calculate waste disposal inventories from arbitrary waste compositions. The modelling of long-term emissions from landfills is a crucial part in any waste disposal process. In ecoinvent long-term emissions are defined as emissions occurring 100 years after present. They are reported in separate emission categories. The landfill inventories include long-term emissions with a time horizon of 60’000 years after present. Results and Discussion As in earlier studies, the landfills prove to be generally relevant disposal processes, as also incineration and wastewater treatment processes produce landfilled wastes. Heavy metals tend to concentrate in landfills and are washed out to a varying degree over time. Long-term emissions usually represent an important burden from landfills. Comparisons between burdens from production of materials and the burdens from their disposal show that disposal has a certain relevance. Conclusion The disposal phase should by default be included in LCA studies. The use of a material not only necessitates its production, but also requires its disposal. The created inventories and user tools facilitate heeding the disposal phase with a similar level of detail as production processes. The risk of LCA-based decisions shifting burdens from the production or use phase to the disposal phase because of data gaps can therefore be diminished. Recommendation and Perspective Future improvements should include the modelling of metal ore refining waste (tailings) which is currently neglected in ecoinvent, but is likely to be relevant for metals production. The disposal technologies considered here are those of developed Western countries. Disposal in other parts of the World can differ distinctly, for logistic, climatic and economic reasons. The cross-examination of landfill models to LCIA soil fate models could be advantageous. Currently only chemical elements, like copper, zinc, nitrogen etc. are heeded by the disposal models. A possible extension could be the modelling of the behaviour of chemical compounds, like dioxins or other hydrocarbons.     

An alpha/beta-fold C--C bond hydrolase is involved in a central step of nicotine catabolism by Arthrobacter nicotinovorans

The enzyme catalyzing the hydrolytic cleavage of 2,6-dihydroxypseudooxynicotine to 2,6-dihydroxypyridine and gamma-N-methylaminobutyrate was found to be encoded on pAO1 of Arthrobacter nicotinovorans. The new enzyme answers an old question about nicotine catabolism and may be the first C--C bond hydrolase that is involved in the biodegradation of a heterocyclic compound.     

Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse

Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.     

Habitat use of nightjar (Caprimulgus europaeus) in an Austrian pine forest

The Steinfeld in Lower Austria supports a population of European Nightjar (Caprimulgus europaeus) which was extensively studied during 1997 and 1998. The study area encompassed a pine forest of 20 km². The population densities of 1.05 and 1.25 territories/km², respectively, lies within the range found in central European populations. Annual monitoring until 2001 has shown the population to be stable. To gain an insight into habitat use of the species, various habitat-related parameters were measured inside and outside the territories, namely structure of trees, density of trees, structure of undergrowth vegetation and structure of clearings. Discriminant analysis was applied to assess the factors responsible for habitat choice of the Nightjar population. The findings showed that the Nightjars territories were frequently centered on a large clearing with an area of at least 0.7 ha. Clearings less than 50 m wide were not colonized. The requirement for a minimum width of a clearing in addition to a minimum area probably relates to better hunting conditions. Nightjars prefer trees where the lower edge of the crown is on average 4.38 m higher than at control points so that males can churr from dead branches immediately below the canopy. Such trees were found on the edge of clearings in the forest, and the edge of a clearing thus had a pronounced effect on the quality of a territory. In contrast to reports in the literature, neither the proportion of bare patches of ground nor the average height of undergrowth vegetation was found to be decisive for territory selection.     

Simian Virus 40 Activates ATR-Δp53 Signaling To Override Cell Cycle and DNA Replication Control

During infection, simian virus 40 (SV40) attempts to take hold of the cell, while the host responds with various defense systems, including the ataxia-telangiectasia mutated/ATM-Rad3 related (ATM/ATR)-mediated DNA damage response pathways. Here we show that upon viral infection, ATR directly activates the p53 isoform Δp53, leading to upregulation of the Cdk inhibitor p21 and downregulation of cyclin A-Cdk2/1 (AK) activity, which force the host to stay in the replicative S phase. Moreover, downregulation of AK activity is a prerequisite for the generation of hypophosphorylated, origin-competent DNA polymerase α-primase (hypo-Polα), which is, unlike AK-phosphorylated Polα (P-Polα), recruited by SV40 large T antigen (T-Ag) to initiate viral DNA replication. Prevention of the downregulation of AK activity by inactivation of ATR-Δp53-p21 signaling significantly reduced the T-Ag-interacting hypo-Polα population and, accordingly, SV40 replication efficiency. Moreover, the ATR-Δp53 pathway facilitates the proteasomal degradation of the 180-kDa catalytic subunit of the non-T-Ag-interacting P-Polα, giving rise to T-Ag-interacting hypo-Polα. Thus, the purpose of activating the ATR-Δp53-p21-mediated intra-S checkpoint is to maintain the host in S phase, an optimal environment for SV40 replication, and to modulate the host DNA replicase, which is indispensable for viral amplification.Infection of quiescent CV-1 cells with the primate polyomavirus simian virus 40 (SV40) induces cell cycle progression and stimulates host cell DNA replication, which is mandatory for viral amplification. SV40 uses only a single viral protein, T antigen (T-Ag), for its own replication; all other components have to be provided by the host. Initially, a specifically phosphorylated subclass of T-Ag binds to a palindromic sequence in the SV40 origin (43), and in the presence of ATP, T-Ag forms a double-hexamer nucleoprotein complex leading to structural distortion and unwinding of origin DNA sequences (5). In concert with the cellular single-strand DNA binding protein RPA and topoisomerase I, the DNA helicase activity of T-Ag promotes more-extensive origin unwinding, forming a preinitiation complex (pre-RC), resulting in an initiation complex (53). Once the initiation complex forms, the primase activity of the heterotetrameric DNA polymerase α-primase (Polα) complex, consisting of the p180 catalytic subunit, the p70 regulatory subunit, and the p48/58 primase subunits, synthesizes a short RNA primer on each template strand, which is extended by the DNA polymerase activity of Polα (6, 17). Immediately after the first nascent RNA/DNA primer is synthesized, the complete replication machinery is assembled, and elongation at both forks by the processive DNA polymerase δ ensues (62). Thus, during the initiation of SV40 replication, T-Ag performs many of the functions attributed to the eukaryotic pre-RC complex proteins, including Orc, Cdc6, Cdt1, and kinase-independent cyclin E, which facilitates loading of the putative replication helicase Mcm2-7 onto the eukaryotic origin (4, 18). Biochemical evidence shows that initiation of SV40 and eukaryotic DNA replication occurs by the physical interaction of Polα with the appropriate pre-RC in the immediate vicinity of the origin. In SV40, Polα is loaded onto the origin by direct physical contact between the helicase T-Ag and its p180 N-terminal domain C (14, 15, 16). In eukaryotes, Cdc45, Mcm10, and And-1 cooperate to recruit Polα to the origin-initiation complex, thereby tethering the replicase to the origin-loaded Mcm2-7 helicase (34, 61).Although SV40 and chromosomal DNA replication share the same essential replication factors that are recruited to the appropriate pre-RC, there are noticeable differences between the SV40 and eukaryotic replication systems. The viral system allows unregulated multiple firing of the origin, whereas in the eukaryotic system, origin-dependent initiation of replication is regulated and restricted to firing only once per cell cycle. Reinitiation at origins within a cell cycle is prevented by the inactivation of pre-RC components in S and G₂. The cyclin-dependent kinases (Cdks) play a central role in establishing a block to rereplication through phosphorylation of each of the components. At present, several proteins of the mammalian pre-RC, such as Orc1, Cdt1, Cdc6, and the Mcm complex are phosphorylated by cyclin A (cycA)-Cdk2/1 (AK) and, as a result, are degraded or inactivated (1, 26, 30, 33, 40). Nevertheless, not all of the pre-RC components mentioned above are utilized by SV40, and accordingly, not all are involved in viral initiation control. However, in both replication systems, DNA synthesis is initiated by Polα and its initiation activity is regulated by Cdks (55). Moreover, AK-phosphorylated Polα is not recruited to mammalian origins in vivo (13) and is unable to initiate SV40 replication in vitro (47, 57, 58). Considering that cellular mechanisms blocking the rereplication of DNA act by AK phosphorylation of the replication factors mentioned above, especially Polα, it is feasible to suggest that downregulation or even inhibition of this kinase activity promotes dysregulation of replication control in SV40-infected cells.One pathway that leads to downregulation of AK activity in response to cellular stress is the intra-S checkpoint, which employs the novel p53 isoform Δp53 (45). In UV-damaged S-phase cells, ATR (ataxia-telangiectasia mutated [ATM]-Rad3 related)-activated Δp53 upregulates the Cdk inhibitor p21, resulting in a transient reduction in AK activity and decelerated S-phase progression (45). Here we demonstrate that SV40 lytic infection triggers the ATR signaling cascade, leading to the activation of Δp53 and accordingly a p21-mediated drop in AK activity to prevent AK-catalyzed inactivation of the hypophosphorylated, T-Ag-interacting Polα (hypo-Polα) subclass, which is essential for the initiation of viral DNA replication.     

Decreased Soluble Adenylyl Cyclase Activity in Cystic Fibrosis Is Related to Defective Apical Bicarbonate Exchange and Affects Ciliary Beat Frequency Regulation

Human airway cilia contain soluble adenylyl cyclase (sAC) that produces cAMP upon HCO₃⁻/CO₂ stimulation to increase ciliary beat frequency (CBF). Because apical HCO₃⁻ exchange depends on cystic fibrosis transmembrane conductance regulator (CFTR), malfunctioning CFTR might impair sAC-mediated CBF regulation in cells from patients with cystic fibrosis (CF). By Western blot, sAC isoforms are equally expressed in normal and CF airway epithelial cells, but CBF decreased more in CF than normal cells upon increased apical HCO₃⁻/CO₂ exposure in part because of greater intracellular acidification from unbalanced CO₂ influx (estimated by 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence). Importantly, ciliated cell-specific cAMP production (estimated by FRET fluorescence ratio changes of tagged cAMP-dependent protein kinase (PKA) subunits expressed under a ciliated cell-specific promoter) in response to increased apical HCO₃⁻/CO₂ perfusion was higher in normal compared with CF cells. Inhibition of bicarbonate influx via CFTR (CFTR_inh172) and inhibition of sAC (KH7) and PKA activation (H89) led to larger CBF declines in normal cells, now comparable with changes seen in CF cells. These inhibitors also reduced FRET changes in normal cells to the level of CF cells with the expected exception of H89, which does not prevent dissociation of the fluorescently tagged PKA subunits. Basolateral permeabilization and subsequent perfusion with HCO₃⁻/CO₂ rescued CBF and FRET changes in CF cells to the level of normal cells. These results suggest that CBF regulation by sAC-produced cAMP could be impaired in CF, thereby possibly contributing to mucociliary dysfunction in this disease, at least during disease exacerbations when airway acidification is common.     

Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death

Histolysis refers to a widespread disintegration of tissues that is morphologically distinct from apoptosis and often associated with the stimulation of autophagy. Here, we establish that a component of the apoptosome, and pivotal regulator of apoptosis, is also required for histolytic cell death. Using in vivo and ex vivo assays, we demonstrate a global apoptogenic requirement for dark, the fly ortholog of Apaf1, and show that a required focus of dark(-) organismal lethality maps to the central nervous system. We further demonstrate that the Dark protein itself is a caspase substrate and find that alterations of this cleavage site produced the first hypermorphic point mutation within the Apaf1/Ced-4 gene family. In a model of ;autophagic cell death', dark was essential for histolysis but dispensable for characteristic features of the autophagic program, indicating that the induction of autophagy occurs upstream or parallel to histolytic cell death. These results demonstrate that stimulation of autophagy per se is not a ;killing event' and, at the same time, establish that common effector pathways, regulated by the apoptosome, can underlie morphologically distinct forms of programmed cell death.     

Interactions of granisetron with an agonist-free 5-HT3A receptor model

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.