Molecular and biochemical characterisation of a novel mutation in POLG associated with Alpers syndrome

Background DNA polymerase γ (POLG) is the only known mitochondrial DNA (mtDNA) polymerase. It mediates mtDNA replication and base excision repair. Mutations in the POLG gene lead to reduction of functional mtDNA (mtDNA depletion and/or deletions) and are therefore predicted to result in defective oxidative phosphorylation (OXPHOS). Many mutations map to the polymerase and exonuclease domains of the enzyme and produce a broad clinical spectrum. The most frequent mutation p.A467T is localised in the linker region between these domains. In compound heterozygote patients the p.A467T mutation has been described to be associated amongst others with fatal childhood encephalopathy. These patients have a poorer survival rate compared to homozygotes. Methods mtDNA content in various tissues (fibroblasts, muscle and liver) was quantified using quantitative PCR (qPCR). OXPHOS activities in the same tissues were assessed using spectrophotometric methods and catalytic stain of BN-PAGE. Results We characterise a novel splice site mutation in POLG found in trans with the p.A467T mutation in a 3.5 years old boy with valproic acid induced acute liver failure (Alpers-Huttenlocher syndrome). These mutations result in a tissue specific depletion of the mtDNA which correlates with the OXPHOS-activities. Conclusions mtDNA depletion can be expressed in a high tissue-specific manner and confirms the need to analyse primary tissue. Furthermore, POLG analysis optimises clinical management in the early stages of disease and reinforces the need for its evaluation before starting valproic acid treatment.

Lipid metabolism in Trypanosoma brucei

Trypanosoma brucei membranes consist of all major eukaryotic glycerophospholipid and sphingolipid classes. These are de novo synthesized from precursors obtained either from the host or from catabolised endocytosed lipids. In recent years, substantial progress has been made in the molecular and biochemical characterisation of several of these lipid biosynthetic pathways, using gene knockout or RNA interference strategies or by enzymatic characterization of individual reactions. Together with the completed genome, these studies have highlighted several possible differences between mammalian and trypanosome lipid biosynthesis that could be exploited for the development of drugs against the diseases caused by these parasites.

Entwicklung einer PCR zum Nachweis der EAV-Infektion

The goal of this work was the development of suitable (real-time) RT-PCR techniques for fast and sensitive diagnosis of EAV and for molecular-epidemiological characterisation of viral strains, as an alternative to virus isolation. To this purpose two conventional RT-PCR methods and one real-time RT-PCR were adapted to detect the broadest possible spectrum of viral strains. Several dilutions with Bucyrus strain showed a 100-fold higher sensitivity of real-time RT-PCR and heminested RT-PCR compared to simple RT-PCR. Making use of 11 cell culture supernatants of different EAV isolates and 7 semen samples of positive stallions, the suitability of the techniques could be shown. Phylogenetic analysis of sequences of the newly analysed samples compared with known sequences indicated that more EAV-lineages exist than presently described.

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite-host interactions during host cell division.

Characterisation of six novel A-subunit mutations leading to congenital factor XIII deficiency and molecular analysis of the first diagnosed patient with this rare bleeding disorder

In 1960, the first case report on factor XIII deficiency was published describing a seven-year-old Swiss boy with a so far unknown bleeding disorder. Today, more than 60 mutations in the factor XIIIA- and B-subunit genes are known leading to congenital factor XIII deficiency. In the present study, we describe six novel mutations in the factor XIII A-subunit gene. Additionally, we present the molecular characterisation of the first described patient with congenital factor XIII deficiency. The six novel mutations include a small deletion, Glu202 delG, leading to a premature stop codon and truncation of the protein, and a splice site mutation at the exon 10/intron 10 boundary, +1G/A, giving rise to an incorrect spliced mRNA lacking exons 10 and 11. The remaining four mutations are characterised by the single amino acid changes Met159Arg, Gly215Arg, Trp375Cys, and His716Arg, and were expressed in COS-1 cells. Antigen levels and activity of the mutants were significantly reduced compared to the wild-type. The patient described in 1960 also shows a single amino acid change, Arg77Cys. Structural analysis of all mutant enzymes suggests several mechanisms leading to destabilisation of the protein.

Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum

Here we present the identification and cloning of the NcBSR4 gene, the putative Neospora caninum orthologue to the Toxoplasma gondii TgBSR4 gene. To isolate NcBSR4, genome walking PCR was performed on N. caninum genomic DNA using the expressed sequence tag NcEST3c28h02.y1 sequence, which shares a 44% identity with the TgBSR4 gene, as a framework. Nucleotide sequencing of amplified DNA fragments revealed a single uninterrupted 1227 bp open reading frame that encodes a protein of 408 amino acids with 66% similarity to the TgBSR4 antigen. A putative 39-residue signal peptide was found at the NH2-terminus, followed by a hydrophilic region. At the COOH-terminus, a potential site for a glycosylphosphatidylinositol anchor was identified at amino acid 379. A polyclonal serum against recombinant NcBSR4 protein was raised in rabbits, and immunolabelling demonstrated stage-specific expression of the NcBSR4 antigen in N. caninum bradyzoites produced in vitro and in vivo. Furthermore, RT-PCR analysis showed a slight increase of NcBSR4 transcripts in bradyzoites generated during in vitro tachyzoite-to-bradyzoite stage-conversion, suggesting that this gene is specifically expressed at the bradyzoite stage and that its transcription relies on the switch to this stage.

Molecular characterisation and expression of Atlantic cod (Gadus morhua) myoglobin from two populations held at two different acclimation temperatures.

Much previous research has demonstrated the plasticity of myoglobin concentrations in both cardiac and skeletal myocytes in response to hypoxia and training. No study has yet looked at the effect of thermal acclimation on myoglobin in fish. Atlantic cod (Gadus morhua) from two different populations, i.e. the North Sea and the North East Arctic, were acclimated to 10 and 4 degrees C. Both the myoglobin mRNA and myoglobin protein in cod hearts increased significantly by up to 3.7 and 2.3 fold respectively as a result of acclimation to 4 degrees C. These increments were largest in the Arctic population, which in earlier studies have been shown to possess cold compensated metabolic demands at low temperatures. These metabolic demands associated with higher mitochondrial capacities may have driven the increase in cardiac myoglobin concentrations, in order to support diffusive oxygen supply. At the same time the increase in myoglobin levels may serve further functions during cold acclimation, for example, protection of the cell against reactive oxygen species, and scavenging nitric oxide, thereby contributing to the regulation of mitochondrial volume density.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Identification and characterisation of ncRNAs in dormant bacteria

Recent years have led to increasing interest and appreciation of the possible importance of single cell heterogeneity in various biological processes. One of the examples of phenotypic heterogeneity in bacterial populations is antibiotic tolerant persister cells. Such an antibiotic tolerance phenotype is of considerable clinical relevance since dormant bacteria can re-establish infections rapidly after the antibiotic treatment has been terminated. Up to now mechanisms for establishing the persistence phenomenon in bacteria have remained largely enigmatic. Persisters are cells considered to be in a dormant state with down regulated gene expression. Only recently small regulatory RNAs (sRNAs) have been appreciated as important regulators of gene expression in response to environmental stimuli and several theoretical studies have suggested a possible involvement of sRNAs in the mechanisms of regulated heterogeneity in bacteria. We have experimentally addressed this potential link between sRNAs and persistence/dormancy in E. coli as an example of heterogeneity. Beside classical sRNAs we are focusing also on sRNAs directly associating with and possibly regulating the ribosome, the central enzyme of gene expression. The persister and dormant cell specific sRNA profile is studied by the comparative analysis of sRNA profile changes of the whole bacterial population after antibiotic killing. From RNA-Seq data ~ 25 000 potentially stable RNA fragments were identified and initial analysis predicted ~300 of them to be dormant/persister cell specific. After further evaluation the most prominent dormant/persister cell specific sRNAs are functionally characterized and their potential role in the persistence/dormancy will be evaluated by applying genetic, molecular and biochemical tools. The potential results of this project will provide a better understanding on the molecular mechanism of bacterial persistence/dormancy and on the role of ribosome-bound sRNA molecules in fine-tuning gene expression.

Molecular characterisation of a predominant antigenic region of Giardia lamblia variant surface protein H7.

During infection, the intestinal protozoan parasite Giardia lamblia undergoes continuous antigenic variation which is determined by diversification of the parasite's major surface antigen, named VSP (variant surface protein). One member from this protein family, VSP H7, is expressed by G. lamblia clone GS/M-83-H7. In the present study, we characterised a highly antigenic portion of VSP H7 which is positioned inside a 130 amino acid C-terminal region of the protein. This region overlaps with a cysteine-rich motif that is rather conserved within the VSP family. Detailed molecular dissection of the antigenic portion monitored a 12 amino acid peptidyl structure which constitutes a non-conformational epitope of VSP H7. In the murine host, this epitope is recognised relatively early (before day 10 p.i.) during infection and stimulates a strong intestinal immunoglobulin A response. At late infective stages (after day 10 p.i.) this immune reaction is progressively complemented by reactions against 'late' antigenic epitopes which are also located inside the 130 amino acid antigenic portion but in closer proximity to the C-terminal end of VSP H7 than the 12 amino acid epitope. Both the high antigenicity and the conserved character suggest that the 12 amino acid epitope is a key factor within the immunological interplay between G. lamblia and the experimental murine host.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

Varying spin state composition by the choice of capping ligand in a family of molecular chains: detailed analysis of magnetic properties of chromium(III) horseshoes

We report a detailed physical analysis on a family of isolated, antiferro-magnetically (AF) coupled, chromium(III) finite chains, of general formula (Cr(RCO(2))(2)F)(n) where the chain length n = 6 or 7. Additionally, the chains are capped with a selection of possible terminating ligands, including hfac (= 1,1,1,5,5,5-hexafluoropentane-2,4-dionate(1-)), acac (= pentane-2,4-dionate(1-)) or (F)(3). Measurements by inelastic neutron scattering (INS), magnetometery and electron paramagnetic resonance (EPR) spectroscopy have been used to study how the electronic properties are affected by n and capping ligand type. These comparisons allowed the subtle electronic effects the choice of capping ligand makes for odd member spin 3/2 ground state and even membered spin 0 ground state chains to be investigated. For this investigation full characterisation of physical properties have been performed with spin Hamiltonian parameterisation, including the determination of Heisenberg exchange coupling constants and single ion axial and rhombic anisotropy. We reveal how the quantum spin energy levels of odd or even membered chains can be modified by the type of capping ligand terminating the chain. Choice of capping ligands enables Cr-Cr exchange coupling to be adjusted by 0, 4 or 24%, relative to Cr-Cr exchange coupling within the body of the chain, by the substitution of hfac, acac or (F)(3) capping ligands to the ends of the chain, respectively. The manipulation of quantum spin levels via ligands which play no role in super-exchange, is of general interest to the practise of spin Hamilton modelling, where such second order effects are generally not considered of relevance to magnetic properties.

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis - A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomerieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated. 102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).