ALBERT

Description: Experts providing evidence in legal cases are universally recommended to be transparent, particularly in their reasoning, so that legal practitioners can critically check whether the conclusions are adequately supported by the results. However, when exploring the practical meaning of this recommendation it becomes clear that people have different things in mind. The UK appeal court case R v T painfully exposes the different views. In this article we argue that there can be a trade-off between clarity and transparency, and that in some cases it is impossible for the legal practitioner to be able to follow the expert’s reasoning in full detail because of the level of complexity. All that can be expected in these cases is that the legal practitioner is able to understand the reasoning up to a certain level. We propose that experts should only report the main arguments, but must make this clear and provide further details on request. Reporting guidelines should address the reasoning in more detail. Legal practitioners and scientists should not be telling each other what to do in the setting of a legal case, but in other settings more discussion will be beneficial to both. We see the likelihood ratio framework and Bayesian networks as tools to promote transparency and logic. Finally, we argue that transparency requires making clear whether a conclusion is a consensus and reporting diverging opinions on request.

Description: Identifying specific human body fluids and establishing their presence in traces can be crucial to help reconstructing alleged incidents in criminal cases. It is up to the forensic practitioner to test for the presence of body fluids, interpret the test results and draw scientifically supported conclusions that can be used in a court of law. This study presents a Bayesian network for the interpretation of test results for human saliva based on the presence of human salivary α-amylase. The Bayesian network can be used by forensic practitioners as an exploratory tool to form their expert opinion on the presence or absence of saliva in a trace.

Description: Nucleotide positions in the hypervariable V4 and V9 regions of the small subunit (SSU)-rDNA locus are normally difficult to align and are usually removed before standard phylogenetic analyses. Yet, with next-generation sequencing data, amplicons of these regions are all that are available to answer ecological and evolutionary questions that rely on phylogenetic inferences. With ciliates, we asked how inclusion of the V4 or V9 regions, regardless of alignment quality, affects tree topologies using distinct phylogenetic methods (including PairDist that is introduced here). Results show that the best approach is to place V4 amplicons into an alignment of full-length Sanger SSU-rDNA sequences and to infer the phylogenetic tree with RAxML. A sliding window algorithm as implemented in RAxML shows, though, that not all nucleotide positions in the V4 region are better than V9 at inferring the ciliate tree. With this approach and an ancestral-state reconstruction, we use V4 amplicons from European nearshore sampling sites to infer that rather than being primarily terrestrial and freshwater, colpodean ciliates may have repeatedly transitioned from terrestrial/freshwater to marine environments.

Description: Targeted genome engineering has become an important research area for diverse disciplines, with site-specific recombinases (SSRs) being among the most popular genome engineering tools. Their ability to trigger excision, integration, inversion and translocation has made SSRs an invaluable tool to manipulate DNA in vitro and in vivo . However, sophisticated strategies that combine different SSR systems are ever increasing. Hence, the demand for additional precise and efficient recombinases is dictated by the increasing complexity of the genetic studies. Here, we describe a novel site-specific recombination system designated Vika/ vox . Vika originates from a degenerate bacteriophage of Vibrio coralliilyticus and shares low sequence similarity to other tyrosine recombinases, but functionally carries out a similar type of reaction. We demonstrate that Vika is highly specific in catalyzing vox recombination without recombining target sites from other SSR systems. We also compare the recombination activity of Vika/ vox with other SSR systems, providing a guideline for deciding on the most suitable enzyme for a particular application and demonstrate that Vika expression does not cause cytotoxicity in mammalian cells. Our results show that Vika/ vox is a novel powerful and safe instrument in the ‘genetic toolbox’ that can be used alone or in combination with other SSRs in heterologous hosts.

Description: In long-baseline interferometry, bandwidth smearing of an extended source occurs at finite bandwidth when its different components produce interference packets that only partially overlap. In this case, traditional model fitting or image reconstruction using standard formulas and tools lead to biased results. In this paper, we propose and implement a method to overcome this effect by calculating analytically a corrective term for the conventional interferometric observables: the visibility amplitude and closure phase. For that purpose, we model the interferogram taking into account the finite bandwidth and the instrumental differential phase. We obtain generic expressions for the visibility and closure phase in the case of temporally modulated interferograms, either processed using Fourier analysis or with the ABCD method. The expressions can be used to fit arbitrary models to the data. We then apply our results to the search and charactrization of stellar companions with the Precision Integrated-Optics Near-infrared Imaging ExpeRiment at the Very Large Telescope Interferometer, assessing the bias on observables and model-fitted parameters of a binary star. Finally, we consider the role of the atmosphere, first with an analytic model to identify the main contributions to bias and, secondly, by confirming the model with a numerical simulation of the atmospheric turbulence. In addition to the analytic expressions, the main results of our study are as follows: (i) the chromatic dispersion in the beam transport in the instrument has a strong impact on the closure phase and introduces additional biases even at separations where smearing is not expected to play an important role; (ii) the atmospheric turbulence introduces additional biases when smearing is present, but the impact is important only at very low spectral resolution; (iii) the bias on the observables strongly depends on the recombination scheme and data processing; (iv) the goodness of model fits is improved by modelling a Gaussian bandpass as long as the smearing is moderate.

Description: It is shown that, relative to Bishop-style constructive mathematics, the boundedness principle BD - N is equivalent both to a general result about the convergence of double sequences and to a particular one about Cauchyness in a semi-metric space.

Description: X-linked adrenoleukodystrophy (X-ALD) is a fatal neurodegenerative disease caused by mutations in the ABCD1 gene, encoding a member of the peroxisomal ABC transporter family. The ABCD1 protein transports CoA-activated very long-chain fatty acids (VLCFAs) into peroxisomes for degradation via β-oxidation. In the severest form, X-ALD patients suffer from inflammatory demyelination of the brain. As the extent of the metabolic defect in the main immune cells is unknown, we explored their phenotypes concerning mRNA expression pattern of the three peroxisomal ABC transporters, VLCFA accumulation and peroxisomal β-oxidation. In controls, ABCD1 expression was high in monocytes, intermediate in B cells and low in T cells; ABCD2 expression was extremely low in monocytes, intermediate in B cells and highest in T cells; ABCD3 mRNA was equally distributed. In X-ALD patients, the expression patterns remained unaltered; accordingly, monocytes, which lack compensatory VLCFA transport by ABCD2, displayed the severest biochemical phenotype with a 6-fold accumulation of C26:0 and a striking 70% reduction in peroxisomal β-oxidation activity. In contrast, VLCFA metabolism was close to control values in B cells and T cells, supporting the hypothesis that sufficient ABCD2 is present to compensate for ABCD1 deficiency. Thus, the vulnerability of the main immune cell types is highly variable in X-ALD. Based on these results, we propose that in X-ALD the halt of inflammation after allogeneic hematopoietic stem cell transplantation relies particularly on the replacement of the monocyte lineage. Additionally, these findings support the concept that ABCD2 is a target for pharmacological induction as an alternative therapeutic strategy.

Description: Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.

Description: Isolated defects of ether lipid (EL) biosynthesis in humans cause rhizomelic chondrodysplasia punctata type 2 and type 3, serious peroxisomal disorders. Using a previously described mouse model [Rodemer, C., Thai, T.P., Brugger, B., Kaercher, T., Werner, H., Nave, K.A., Wieland, F., Gorgas, K., and Just, W.W. (2003) Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet. , 12 , 1881–1895], we investigated the effect of EL deficiency in isolated murine nerve terminals (synaptosomes) on the pre-synaptic release of the neurotransmitters (NTs) glutamate and acetylcholine. Both Ca 2+ -dependent exocytosis and Ca 2+ -independent efflux of the transmitters were affected. EL-deficient synaptosomes respire at a reduced rate and exhibit a lowered adenosin-5'-triphosphate/adenosine diphosphate (ATP/ADP) ratio. Consequently, ATP-driven processes, such as synaptic vesicle cycling and maintenance of Na + , K + and Ca 2+ homeostasis, might be disturbed. Analyzing reactive oxygen species in EL-deficient neural and non-neural tissues revealed that plasmalogens (PLs), the most abundant EL species in mammalian central nervous system, considerably contribute to the generation of the lipid peroxidation product malondialdehyde. Although EL-deficient tissue contains less lipid peroxidation products, fibroblasts lacking ELs are more susceptible to induced oxidative stress. In summary, these results suggest that due to the reduced energy state of EL-deficient tissue, the Ca 2+ -independent efflux of NTs increases while the Ca 2+ -dependent release declines. Furthermore, lack of PLs is mainly compensated for by an increase in the concentration of phosphatidylethanolamine and results in a significantly lowered level of lipid peroxidation products in the brain cortex and cerebellum.

Description: Type IIA topoisomerases control DNA supercoiling and separate newly replicated chromosomes using a complex DNA strand cleavage and passage mechanism. Structural and biochemical studies have shown that these enzymes sharply bend DNA by as much as 150°; an invariant isoleucine, which has been seen structurally to intercalate between two base pairs outside of the DNA cleavage site, has been suggested to promote deformation. To test this assumption, we examined the role of isoleucine on DNA binding, bending and catalytic activity for a bacterial type IIA topoisomerase, Escherichia coli topoisomerase IV (topo IV), using a combination of site-directed mutagenesis and biochemical assays. Our data show that alteration of the isoleucine (Ile 172 ) did not affect the basal ATPase activity of topo IV or its affinity for DNA. However, the amino acid was important for DNA bending, DNA cleavage and supercoil relaxation. Moreover, an ability to bend DNA correlated with efficacy with which nucleic acid substrates stimulate ATP hydrolysis. These data show that DNA binding and bending by topo IV can be uncoupled, and indicate that the stabilization of a highly curved DNA geometry is critical to the type IIA topoisomerase catalytic cycle.