Comparative analysis of bones, mites, soil chemistry, nematodes and soil micro-Eukaryotes from a suspected homicide to estimate the post-mortem interval

Criminal investigations of suspected murder cases require estimating the post-mortem interval (PMI, or time after death) which is challenging for longer periods. Here we present the case of human remains found in a Swiss forest. We have used a multidisciplinary approach involving the analysis of bones, soil chemical characteristics, mites and nematodes (by microscopy) and micro-Eukaryotes (by Illumina high throughput sequencing). We analysed soil samples collected beneath the remains of the head, upper and lower body and “control” samples taken a few meters away. The PMI estimated on hair 14C-data via bomb peak radiocarbon dating gave a time range of 1 to 2 years before the finding of the remains on site. Cluster analyses for chemical constituents, nematodes, mites and micro- Eukaryotes revealed two clusters 1) head and upper body and 2) lower body and controls. From mite evidence, we conclude that the body was likely to have been brought to the site after death. However, chemical analyses, nematode community analyses and the analyses of micro-Eukaryotes indicate that decomposition took place at least partly on site. This study illustrates the usefulness of combining several lines of evidence for the study of homicide cases to better calibrate PMI inference tools

Analysis of structural determinants of the stability of thermolysin-like proteases by molecular modelling and site-directed mutagenesis

The thermolysin-like protease (TLP) produced by Bacillus stearothermophilus CU21 (TLP-ste) differs at 43 positions from the more thermally stable thermolysin (containing 316 residues in total), Of these differences, 26 were analysed by studying the effect of replacing residues in TLP-ste by the corresponding residues in thermolysin, Several stabilizing mutations were identified but, remarkably, considerable destabilizing mutational effects were also found, A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertional difference between the two enzymes) appeared to make an important contribution to the stability of the enzyme. Mutations with large effects on stability were all localized in the beta-pleated N-terminal domain of TLP-ste, confirming;observations that this domain has a lower intrinsic stability than the largely alpha-helical C-terminal domain, Rigidifying mutations such as Gly58 --> Ala and Ala69 --> Pro were among the most stabilizing ones, Apart from this observation, the analyses did not reveal general rules for stabilizing proteins. Instead, the results highlight the importance of context in evaluating the stability effects of mutations

Eukaryotic transporters for hydroxyderivatives of benzoic acid

Several yeast species catabolize hydroxyderivatives of benzoic acid. However, the nature of carriers responsible for transport of these compounds across the plasma membrane is currently unknown. In this study, we analyzed a family of genes coding for permeases belonging to the major facilitator superfamily (MFS) in the pathogenic yeast Candida parapsilosis. Our results revealed that these transporters are functionally equivalent to bacterial aromatic acid: H+ symporters (AAHS) such as GenK, MhbT and PcaK. We demonstrate that the genes HBT1 and HBT2 encoding putative transporters are highly upregulated in C. parapsilosis cells assimilating hydroxybenzoate substrates and the corresponding proteins reside in the plasma membrane. Phenotypic analyses of knockout mutants and hydroxybenzoate uptake assays provide compelling evidence that the permeases Hbt1 and Hbt2 transport the substrates that are metabolized via the gentisate (3-hydroxybenzoate, gentisate) and 3-oxoadipate pathway (4-hydroxybenzoate, 2,4-dihydroxybenzoate and protocatechuate), respectively. Our data support the hypothesis that the carriers belong to the AAHS family of MFS transporters. Phylogenetic analyses revealed that the orthologs of Hbt permeases are widespread in the subphylum Pezizomycotina, but have a sparse distribution among Saccharomycotina lineages. Moreover, these analyses shed additional light on the evolution of biochemical pathways involved in the catabolic degradation of hydroxyaromatic compounds.We would like to thank Ladislav Kováč and Jordan Kolarov (Comenius University in Bratislava) for long-term support, Peter Polčic and our lab members for discussions. This work was supported by the Slovak grant agencies VEGA (1/0333/15 and 1/0052/16) and APVV (14-0253 and 15-0022) and the Comenius University grant (UK/429/2015). TG was supported in part by a grant from the Spanish Ministry of Economy and Competitiveness grants, 'Centro de Excelencia Severo Ochoa 2013-2017' SEV-2012-0208, and BFU2015-67107 cofounded by European Regional Development Fund (ERDF); from the European Union and ERC Seventh Framework Programme (FP7/2007-2013) under grant agreements FP7-PEOPLE-2013-ITN-606786 and ERC-2012-StG-310325; from the Catalan Research Agency (AGAUR) SGR857, and grant from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No H2020-MSCA-ITN-2014-642095. AG was funded by NKFIH NN 113153, by GINOP 2.3.2-15-2016-00035 and by GINOP 2.3.3-15-2016-00006