Characterization of a cathepsin L-associated protein in Artemia and its relationship to the FAS-I family of cell adhesion proteins.

We reported previously that the major cysteine protease in embryos and larvae of the brine shrimp, Artemia franciscana, is a heterodimeric protein consisting of a catalytic subunit (28.5 kDa) with a high degree of homology with cathepsin L, and a noncatalytic subunit (31.5 kDa) of unknown function. In the study reported here the noncatalytic subunit, or cathepsin L-associated protein (CLAP), was separated from cathepsin L by chromatography on Mono S and found to contain multiple isoforms with pIs ranging from 5.9 to 6.1. Heterodimeric and monomeric cathepsin L showed similar activity between pH 5 and 6.5, while the heterodimer was about twice as active as monomeric cathepsin L below pH 5. The heterodimer was more stable than the monomer between pH 6 and 7.4 and at 30-50 degrees C. Artemia CLAP and cathepsin L are present in nearly equimolar amounts at all stages in the life cycle and most abundant in encysted eggs and embyros. Moreover, CLAP, either free or as a complex with cathepsin L, was resistant to hydrolysis by cathepsin L. Two clones coding for CLAP were isolated from an Artemia embryo cDNA library and sequenced. Both clones have nearly identical open reading frames, but show differences at the 5'- and 3'-termini. Each cDNA clone has an extensive 3'-untranslated region containing 70-72% A+T. The deduced amino acid sequence of CLAP cDNA revealed two domains which were very similar to domains in fasciclin I and other cell adhesion proteins. The nucleotide sequences of clones 1 and 2 have been entered into the NCBI database (AY307377 and AY462276). This study supports the view that the noncatalytic subunit of the heterodimeric cysteine protease in Artemia stabilizes cathepsin L at various pH and temperatures normally inconsistent with cathepsin L from other organisms, and that CLAP serves as a docking mechanism for cathepsin L at nonlysosomal sites in Artemia embryos.     

AU binding proteins recruit the exosome to degrade ARE-containing mRNAs.

Inherently unstable mammalian mRNAs contain AU-rich elements (AREs) within their 3' untranslated regions. Although found 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is not clear. In yeast, 3'-to-5' mRNA degradation is mediated by the exosome, a multisubunit particle. We have purified and characterized the human exosome by mass spectrometry and found its composition to be similar to its yeast counterpart. Using a cell-free RNA decay system, we demonstrate that the mammalian exosome is required for rapid degradation of ARE-containing RNAs but not for poly(A) shortening. The mammalian exosome does not recognize ARE-containing RNAs on its own. ARE recognition requires certain ARE binding proteins that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.     

On the number of Monte Carlo runs in comparative probabilistic LCA

Introduction

The Monte Carlo technique is widely used and recommended for including uncertainties LCA. Typically, 1000 or 10,000 runs are done, but a clear argument for that number is not available, and with the growing size of LCA databases, an excessively high number of runs may be a time-consuming thing. We therefore investigate if a large number of runs are useful, or if it might be unnecessary or even harmful.

Probability theory

We review the standard theory or probability distributions for describing stochastic variables, including the combination of different stochastic variables into a calculation. We also review the standard theory of inferential statistics for estimating a probability distribution, given a sample of values. For estimating the distribution of a function of probability distributions, two major techniques are available, analytical, applying probability theory and numerical, using Monte Carlo simulation. Because the analytical technique is often unavailable, the obvious way-out is Monte Carlo. However, we demonstrate and illustrate that it leads to overly precise conclusions on the values of estimated parameters, and to incorrect hypothesis tests.

Numerical illustration

We demonstrate the effect for two simple cases: one system in a stand-alone analysis and a comparative analysis of two alternative systems. Both cases illustrate that statistical hypotheses that should not be rejected in fact are rejected in a highly convincing way, thus pointing out a fundamental flaw.

Discussion and conclusions

Apart form the obvious recommendation to use larger samples for estimating input distributions, we suggest to restrict the number of Monte Carlo runs to a number not greater than the sample sizes used for the input parameters. As a final note, when the input parameters are not estimated using samples, but through a procedure, such as the popular pedigree approach, the Monte Carlo approach should not be used at all.

     

Numerical Approaches to Life Cycle Interpretation - The case of the Ecoinvent’96 database (10 pp)

Goal, Scope and Background To strengthen the evaluative power of LCA, life cycle interpretation should be further developed. A previous contribution (Heijungs & Kleijn 2001) elaborated five examples of concrete methods within the subset of numerical approaches towards interpretation. These methods were: contribution analysis, perturbation analysis, uncertainty analysis, comparative analysis, and discernibility analysis. Developments in software have enabled the possibility to apply the five example methods to explore the much-used Ecoinvent”96 database.Discussion of Methods The numerical approaches implemented in this study include contribution analysis, perturbation analysis, uncertainty analysis, comparative analysis, discernibility analysis and the newly developed key issue analysis. The data used comes from a very large process database: Ecoinvent’96, containing 1163 processes, 1181 economic flows and 571 environmental flows. Conclusions Results are twofold: they serve as a benchmark to the usefulness and feasibility of these numerical approaches, and they shed light on the question of stability and structure in an often-used large system of interconnected processes. Most of the approaches perform quite well: computation time on a moderate PC is between a few seconds a few minutes. Only Monte Carlo analyses may require much longer, but even then it appears that most questions can be answered within a few hours. Moreover, analytical expressions for error propagation are much faster than Monte Carlo analyses, while giving almost identical results. Despite the fact that many processes are connected to each other, leading to the possibility of a very unstable system and very sensitive coefficients, the overall results show that most results are not extremely uncertain. There are, however, some exceptions to this positive message.     

A greenhouse gas indicator for bioenergy: some theoretical issues with practical implications

Background, aim, and scope  The expectations with respect to biomass as a resource for sustainable energy are sky-high. Many industrialized countries have adopted ambitious policy targets and have introduced financial measures to stimulate the production or use of bioenergy. Meanwhile, the side-effects and associated risks have been pointed out as well. To be able to make a well-informed decision, the Dutch government has expressed the intention to include sustainability criteria into relevant policy instruments. Main features  Among other criteria, it has been proposed to calculate a so-called life-cycle-based greenhouse gas (GHG) indicator, which expresses the reduction of GHG emissions of a bio-based fuel chain in comparison with a fossil-based fuel chain. Life-cycle-based biofuel studies persistently have problems with the handling of biogenic carbon balances and with the treatment of coproducts and recycling. In life-cycle assessments (LCAs) of agricultural products, a distinction between “negative” and “positive” emissions may be relevant. In particular, carbon dioxide, as a naturally occurring compound or an anthropogenic emission, takes part in the so-called geochemical carbon cycle. The most appropriate way to treat carbon cycles is to view them as genuine cycles and, thus, at the systems level, subtract the fixation of CO₂ during tree growth from the CO₂ emitted during waste treatment of discarded wood and to quantify the CH₄ emitted. In solving the multifunctionality problem, two steps may be distinguished. The first concerns the modeling of the product system studied in the inventory analysis. In this step, system boundaries are set, processes are described, and process flows are quantified. Multifunctionality problems can be identified and the model of the product system is drafted. The second step concerns solving the remaining multifunctionality problems. For this step, various ways of solving the multifunctionality problem have been proposed and applied, on the basis of mass, energy, economic value, avoided burdens, etc. As the GHG indicator may constitute the basis for granting subsidies to stimulate the use of bioenergy, for example, and as the method for the GHG indicator provides no guidelines on the handling of biogenic CO₂ and guidelines for solving multifunctionality problems such as with coproducts and recycling that leave room for various choices, this study analyzed whether the current GHG indicator provides results that are a robust basis for granting such subsidies. Results  For the robustness check, a hypothetical case study on wood residue-based electricity was set up in order to illustrate what the effects of different solutions and choices for the two steps mentioned may be. The case dealt with the production of wood pellets (residues of the wood industry) that are cofired in a coal-fired power plant. The functional unit is 1 kWh of electricity. Three possibilities for the places of the multifunctional process, two possibilities for whether or not to include biogenic CO₂, and four possibilities for the allocation method were distinguished and calculated. Varying the options for these three choices in this way appears to have a huge effect on the GHG indicator, while no clear pattern seems to emerge. Discussion  The results found for this hypothetical case indicate that there are several methodological choices that have not sufficiently been fixed by the presently available standards and guidelines for LCA and GHG assessment of bioenergy systems. In particular, we have focused on issues related to biogenic CO₂ and allocation, two issues that play a prominent role in the assessment of bioenergy systems. Moreover, we have demonstrated with a small hypothetical case study that these are not only issues that might theoretically show up, but that they play a decisive role in practice. Conclusions  The present (Dutch) GHG indicator lacks robustness, which will raise problems for providing a sound basis for granting subsidies. This situation can, however, be improved by reducing the freedom of choices for the handling of biogenic CO₂ and allocation to an absolute minimum. Recommendations and perspectives  Even then, however, differences could appear due to different definitions, data sources, and method interpretations. It thus appears that two kinds of guidance are needed: (1) the LCA methodology itself should be expanded with guidelines for those issues that follow from science, logic, or consensus; (2) in the policy regulation that demands LCA to be the basis of the decision, additional guidelines should be specified that perhaps do not (yet) have the status of being scientifically proven or generally agreed upon, but that serve as a set of temporary extra guidelines.

Sensitivity of the Quantiferon-Gold In-Tube Assay in Sputum Smear Positive TB Cases in Indonesia

Background

As part of a formal evaluation of the Quantiferon-Gold in-tube assay (QFT-IT) for latent TB infection we compared its sensitivity to the tuberculin skin test (TST) in confirmed adult TB cases in Indonesia. Smear-positive TB disease was used as a proxy gold standard for latent TB infection.

Methods and Findings

We compared the sensitivity of QFT-IT and TST in 98 sputum smear and chest x-ray positive TB cases and investigated risk factors for negative and discordant results in both tests. Both tests showed high sensitivity; (QFT-IT; 88.7%: TST; 94.9%), not significantly different from each other (p value 0.11). Very high sensitivity was seen when tests were combined (98.9%). There were no variables significantly associated with discordant results or with a negative TST. For QFT-IT which particular staff member collected blood was significantly associated with test positivity (p value 0.01). Study limitations include small sample size and lack of culture confirmation or HIV test results.

Conclusions

The QFT-IT has similar sensitivity in Indonesian TB cases as in other locations. However, QFT-IT, like the TST cannot distinguish active TB disease from LTBI. In countries such as Indonesia, with high background rates of LTBI, test specificity for TB disease will likely be low. While our study was not designed to evaluate the QFT-IT in the diagnosis of active TB disease in TB suspects, the data suggest that a combination of TST and QFT-IT may prove useful for ruling out TB disease. Further research is required to explore the clinical role of QFT-IT in combination with other TB diagnostic tests.     

Maternal antioxidant concentrations after uncomplicated pregnancies

Background: To analyse the post-partum concentrations of intra- and extra-cellular blood antioxidants in women with uncomplicated pregnancies.

Methods: Whole blood and plasma thiols, plasma vitamin E and C, serum cholesterol and triglyceride, ferric reducing ability of plasma (FRAP) concentrations were compared between women delivered by caesarean section (n=17) or spontaneous delivery (n=10). A repeated mixed model was used for statistical analysis.

Results: The majority of whole blood thiols increased significantly in both groups the first days post-partum. However, within the caesarean group free cysteine, oxidised cysteine, homocysteine and glutathione and plasma cysteine and homocysteine levels dropped significantly after 24 h, while FRAP levels peaked significantly in this group. Plasma vitamin E levels decreased significantly in both groups within 24 to 48 h after delivery. Independent of the way of delivery whole blood and plasma thiols were significantly increased and vitamin E levels were significantly decreased 3 months post-partum while plasma vitamin C levels and FRAP were unchanged compared to ante-partum levels.

Discussion: Decreased plasma vitamin E levels shortly post-partum are associated with decreased lipid peroxidation. The 24 h post-partum drop of some plasma and whole blood thiols in the caesarean group may be due to prolonged fasting.