Consensus Statement for the Management and Communication of High Risk Laboratory Results

Ineffective test follow-up is a major source of harm for patients around the world. Unreliable communication from medical laboratories (henceforth termed ‘laboratories’) to clinicians of results that represent critical or significant risk to patients (collectively termed ‘high risk results’) is a contributing factor to this problem. Throughout Australasia, management practices for such results vary considerably. The recommendations presented in this document are based on best practice derived from the published literature and follow consultation with a wide range of stakeholders. These recommendations were created to harmonise Australasian practices by guiding laboratories in the design and implementation of safe and effective communication procedures for managing high risk results which require timely notification.     

Classification and gradient analysis of the beech forest vegetation of the southern Rodopi (northeast Greece)

Pure and mixed beech forest vegetation of the southern Rodopi range (northeast Greece) was studied using 614 relevés and multivariate analyses (TWINSPAN and DCA). Classification of the relevés resulted in 12 vegetation units, 8 of which were ranked as associations or communities and the rest as subcommunities and variants. DCA diagrams of relevés and taxa indicated that floristic differentiation was attributed mainly to factors such as altitude (affecting temperature and humidity), soil nutrient content and substrate type (affecting physical and chemical soil properties). Differential taxa of vegetation units were chosen based on their phi coefficient values, which were calculated from three different percentage synoptic tables that corresponded to three ranks (ecological groups, associations and communities, and subcommunities and variants) of floristic differentiation. The calculation of phi coefficient on the basis of relative constancy of taxa helps to overcome the problem of the dependence of fidelity values on the number of relevés per vegetation unit and to facilitate the better investigation of the floristic differentiation even of rare vegetation units represented by a small number of relevés. Furthermore, the calculation of fidelity values for different hierarchical levels enables a more detailed and thorough investigation of the floristic differentiation of the vegetation units.     

Pleurotus opuntiae revisited – An insight to the phylogeny of dimitic Pleurotus species with emphasis on the P. djamor complex

The name Pleurotus opuntiae is indiscriminately used for describing mushrooms with white to off-white to white-grey pilei with short or absent stipe and dimitic hyphal system, which grow on plants of the genera Opuntia, Yucca, Agave, Phytolacca etc. However, the outcome of the present study evidences that this name should be reserved for specimens deriving from the Mediterranean area only; an epitype originating from Italy on Opuntia ficus-indica is designated. Pertinent material was sequenced by using the internal transcribed spacer region (ITS) and found to be phylogenetically related to P. djamor from Kenya and Nigeria, while members of the P. djamor complex from other continents were clearly more distant. Results were further corroborated by examining the large subunit of nuclear ribosomal DNA (LSU) and the second subunit of RNA polymerase II (RPB2). The P. djamor complex shows high intraspecific polymorphism evidenced by sequence divergence and genetic distance values, presents a cosmopolitan distribution and also comprises material initially identified as P. flabellatus, P. opuntiae, P. ostreatoroseus, P. parsonsiae and P. salmoneostramineus. An ITS tree including representative specimens from all major Pleurotus species is provided for the first time and ambiguous taxa are discussed in the context of new findings.     

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor

Cellular senescence is acknowledged as a key contributor to organismal ageing and late-life disease. Though popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and by its paracrine effects. To address these issues, we repurposed a small molecule inhibitor, inflachromene (ICM), to induce senescence to human primary cells. Within 6 days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered a high similarity of the two states. Notably though, ICM suppresses the pro-inflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM rapidly and synchronously induces a senescent-like phenotype thereby allowing the study of its core regulatory program without confounding heterogeneity.     

Cometabolism of chlorinated solvents and binary chlorinated solvent mixtures using M. trichosporium OB3b PP358.

The mutant methanotroph, Methylosinus trichosporium OB3b PP358, which constitutively expresses soluble methane monooxygenase (sMMO), was used to study the degradation kinetics of individual chlorinated solvents and binary solvent mixtures. Although sMMO's broad specificity permits a wide range of chlorinated solvents to be degraded, it creates the potential for competitive inhibition of degradation rates in mixtures because multiple chemicals are simultaneously available to the enzyme. To effectively design both ex-situ and in-situ groundwater bioremediation systems using strain PP358, kinetic parameters for chlorinated solvent degradation and accurate kinetic expressions to account for inhibition in mixtures are required. Toward this end, the degradation parameters for six prevalent chlorinated solvents and the verification of enzyme competition model for binary mixtures were the focus of this investigation. M. trichosporium OB3b PP358 degraded trichloroethylene (TCE), chloroform, cis-1,2-dichloroethylene (c-DCE), trans-1,2-dichloroethylene (t-DCE), and 1, 1-dichloroethylene (1,1-DCE) rapidly, with maximum substrate transformation rates of >20.8, 3.1, 9.5 24.8, and >7.5 mg/mg-day, respectively. 1,1,1-trichloroethane (TCA) was not significantly degraded. Half-saturation coefficients ranged from 1 to greater than 10 mg/L. Competition experiments were carried out to observe the effect of a second solvent on degradation rates and to verify the applicability of the Monod model adjusted for competitive inhibition. Binary mixtures of 0.3->0.5 mg/L TCE with up to 5 mg/L c-DCE and up to 7 mg/L 1,1,1-TCA were studied with 20 mM of formate and no growth substrate. No competition was observed at any of these concentrations. Additional competition experiments, using binary mixtures of t-DCE with TCE and t-DCE with c-DCE, were conducted at higher concentrations (i.e., 7-18 mg/L) and enzyme competition was observed. Predictions from a competitive inhibition model compared well with experimental data for these mixtures.     

Gene Fusion Analysis in the Battle against the African Endemic Sleeping Sickness

The protozoan Trypanosoma brucei causes African Trypanosomiasis or sleeping sickness in humans, which can be lethal if untreated. Most available pharmacological treatments for the disease have severe side-effects. The purpose of this analysis was to detect novel protein-protein interactions (PPIs), vital for the parasite, which could lead to the development of drugs against this disease to block the specific interactions. In this work, the Domain Fusion Analysis (Rosetta Stone method) was used to identify novel PPIs, by comparing T. brucei to 19 organisms covering all major lineages of the tree of life. Overall, 49 possible protein-protein interactions were detected, and classified based on (a) statistical significance (BLAST e-value, domain length etc.), (b) their involvement in crucial metabolic pathways, and (c) their evolutionary history, particularly focusing on whether a protein pair is split in T. brucei and fused in the human host. We also evaluated fusion events including hypothetical proteins, and suggest a possible molecular function or involvement in a certain biological process. This work has produced valuable results which could be further studied through structural biology or other experimental approaches so as to validate the protein-protein interactions proposed here. The evolutionary analysis of the proteins involved showed that, gene fusion or gene fission events can happen in all organisms, while some protein domains are more prone to fusion and fission events and present complex evolutionary patterns.     

Evolution of the F0F1 ATP Synthase Complex in Light of the Patchy Distribution of Different Bioenergetic Pathways across Prokaryotes

Bacteria and archaea are characterized by an amazing metabolic diversity, which allows them to persist in diverse and often extreme habitats. Apart from oxygenic photosynthesis and oxidative phosphorylation, well-studied processes from chloroplasts and mitochondria of plants and animals, prokaryotes utilize various chemo- or lithotrophic modes, such as anoxygenic photosynthesis, iron oxidation and reduction, sulfate reduction, and methanogenesis. Most bioenergetic pathways have a similar general structure, with an electron transport chain composed of protein complexes acting as electron donors and acceptors, as well as a central cytochrome complex, mobile electron carriers, and an ATP synthase. While each pathway has been studied in considerable detail in isolation, not much is known about their relative evolutionary relationships. Wanting to address how this metabolic diversity evolved, we mapped the distribution of nine bioenergetic modes on a phylogenetic tree based on 16S rRNA sequences from 272 species representing the full diversity of prokaryotic lineages. This highlights the patchy distribution of many pathways across different lineages, and suggests either up to 26 independent origins or 17 horizontal gene transfer events. Next, we used comparative genomics and phylogenetic analysis of all subunits of the F₀F₁ ATP synthase, common to most bacterial lineages regardless of their bioenergetic mode. Our results indicate an ancient origin of this protein complex, and no clustering based on bioenergetic mode, which suggests that no special modifications are needed for the ATP synthase to work with different electron transport chains. Moreover, examination of the ATP synthase genetic locus indicates various gene rearrangements in the different bacterial lineages, ancient duplications of atpI and of the beta subunit of the F₀ subcomplex, as well as more recent stochastic lineage-specific and species-specific duplications of all subunits. We discuss the implications of the overall pattern of conservation and flexibility of the F₀F₁ ATP synthase genetic locus.     

In vitro scanning saturation mutagenesis of all the specificity determining residues in an antibody binding site.

For the first time, each specificity determining residue (SDR) in the binding site of an antibody has been replaced with every other possible single amino acid substitution, and the resulting mutants analyzed for binding affinity and specificity. The studies were conducted on a variant of the 26-10 antidigoxin single chain Fv (scFv) using in vitro scanning saturation mutagenesis, a new process that allows the high throughput production and characterization of antibody mutants [Burks,E.A., Chen,G., Georgiou,G. and Iverson,B.L. (1997) Proc. Natl Acad. Sci. USA, 94, 412-417]. Single amino acid mutants of 26-10 scFv were identified that modulated specificity in dramatic fashion. The overall plasticity of the antibody binding site with respect to amino acid replacement was also evaluated, revealing that 86% of all mutants retained measurable binding activity. Finally, by analyzing the physical properties of amino acid substitutions with respect to their effect on hapten binding, conclusions were drawn regarding the functional role played by the wild-type residue at each SDR position. The reported results highlight the value of in vitro scanning saturation mutagenesis for engineering antibody binding specificity, for evaluating the plasticity of proteins, and for comprehensive structure-function studies and analysis.