Phenotypic and genotypic approach to characterize a Trueperella pecoris strain isolated from necrotic vestibulitis of a camel (Camelus dromedarius).

The present study was designed to characterize phenotypically and genotypically a Trueperella (T.) pecoris strain isolated from necrotic vestibulitis of a 10-year-old camel (Camelus dromedarius). The species identity of T. pecoris 203/7 investigated in the present study could be confirmed by phenotypic properties and by phylogenetic analyses based on partial sequencing of the 16S ribosomal RNA (rRNA) gene, the 16S-23S rDNA intergenic spacer region, the glyceraldehyde 3-phosphate dehydrogenase encoding gene gap, elongation factor Tu encoding gene tuf and the target gene rpoB encoding the ß-subunit of bacterial RNA polymerase. T. pecoris strain 203/7 was grouped within the genus Trueperella in the family Arcanobacteriaceae. The 16S rRNA gene analysis showed a sequence identity of 99·9% to reference strain T. pecoris DSM 111392T . The present isolate was clearly identified as T. pecoris, the most recently described species of the genus Trueperella. Strain T. pecoris 203/7 was isolated in moderate numbers from necrotic vestibulitis of the camel and could be of some importance for the infectious process. However, the investigated strain represents the first isolation of T. pecoris from a camel.

Evaluation of different target genes for the detection of Salmonella sp. by loop-mediated isothermal amplification.

The loop-mediated isothermal amplification (LAMP) technique was used to investigate six salmonella-specific sequences for their suitability to serve as targets for the pathogen identification. Sequences selected for designing LAMP primers were genes invA, bcfD, phoP, siiA, gene62181533 and a region within the ttrRSBCA locus. Primers including single nucleotide polymorphisms were configured as degenerate primers. Specificity of the designed primer sets was determined by means of 46 salmonella and 32 other food- and waterborne bacterial reference species and strains. Primers targeting the ttrRSBCA locus showed 100 % inclusivity of target and exclusivity of other test species and strains. Other primer sets revealed deficiencies, especially regarding Salmonella enterica subsp. II-IV and Salmonella bongori. Additionally, primers targeting the siiA gene failed to detect S. enterica subsp. enterica serotypes Newport and Stanley, whereas bcfD primers did not amplify DNA of S. enterica subsp. enterica serotype Schleissheim. TtrRSBCA primers, providing short detection times and constant melting temperatures of amplification products, achieved best overall performance.

Loop-mediated isothermal amplification: Development, validation and application of simple and rapid assays for quantitative detection of species of Arcobacteraceae family- and species-specific Aliarcobacter faecis and Aliarcobacter lanthieri.

AIM: The family Arcobacteraceae formerly genus Arcobacter has recently been reclassified into six genera. Among nine species of the genus Aliarcobacter, Aliarcobacter faecis and Aliarcobacter lanthieri have been identified as emerging pathogens potentially cause health risks to humans and animals. This study was designed to develop/optimize, validate and apply Arcobacteraceae family- and two species-specific (A. faecis and A. lanthieri) loop-mediated isothermal amplification (LAMP) assays to rapidly detect and quantify total number of cells in various environmental niches. METHODS AND RESULTS: Three sets of LAMP primers were designed from conserved and variable regions of 16S rRNA (family-specific) and gyrB (species-specific) genes. Optimized Arcobacteraceae family-specific LAMP assay correctly amplified and detected 24 species, whereas species-specific LAMP assays detected A. faecis and A. lanthieri reference strains as well as 91 pure and mixed culture isolates recovered from aquatic and faecal sources. The specificity of LAMP amplification of A. faecis and A. lanthieri was further confirmed by restriction fragment length polymorphism analysis. Assay sensitivities were tested using variable DNA concentrations extracted from simulated target species cells in an autoclaved agricultural water sample by achieving a minimum detection limit of 10 cells mL-1 (10 fg). Direct DNA-based quantitative detection, from agricultural surface water, identified A. faecis (17%) and A. lanthieri (1%) at a low frequency compared to family-level (93%) with the concentration ranging from 2·1 × 101 to 2·2 × 105 cells 100 mL-1 . CONCLUSIONS: Overall, these three DNA-based rapid and cost-effective novel LAMP assays are sensitive and can be completed in less than 40 min. They have potential for on-site quantitative detection of species of family Arcobacteraceae, A. faecis and A. lanthieri in food, environmental and clinical matrices. SIGNIFICANCE AND IMPACT OF THE STUDY: The newly developed LAMP assays are specific, sensitive, accurate with higher reproducibility that have potential to facilitate in a less equipped lab setting and can help in early quantitative detection and rate of prevalence in environmental niches. The assays can be adopted in the diagnostic labs and epidemiological studies.

Sensitization of melanoma cells for TRAIL-induced apoptosis by BMS-345541 correlates with altered phosphorylation and activation of Bax.

Resistance to TRAIL (TNF-related apoptosis-inducing ligand)- induced apoptosis limits its therapeutic use. Different strategies of TRAIL sensitization and a dependency on Bax have been reported, but common principles of TRAIL resistance and the way of Bax activation remained poorly understood. Applying a melanoma model of TRAIL-sensitive and -resistant cell lines, efficient sensitization for TRAIL-induced apoptosis is demonstrated by the kinase inhibitor BMS-345541 (N-(1,8-dimethylimidazo(1,2-a)quinoxalin-4-yl)-1,2-ethanediamine hydrochloride), which targets IκB (inhibitor of κB proteins) kinase ß (IKKß). This effect was completely abrogated by Bax knockout as well as by Bcl-2 overexpression, in accordance with a Bax dependency. Early loss of the mitochondrial membrane potential, release of cytochrome c and Smac (second mitochondria-derived activator of caspases) clearly indicated the activation of mitochondrial apoptosis pathways. Of note, BMS-345541 alone resulted in an early Bax activation, seen by conformational changes and by Bax translocation. The synergistic effects can be explained by Bid activation through TRAIL, which inhibits Bcl-2, and the activation of Bax through BMS-345541. The critical roles of XIAP (X-chromosome-linked inhibitor of apoptosis protein), Smac and Bid were clearly proven by overexpression and siRNA knockdown, respectively. The way of Bax activation by BMS-345541 was unraveled by establishing new assays for Bax activation. These showed reduction of the inactivating Bax phosphorylation at serine-184, while the activating Bax phosphorylation at threonine-167 was enhanced. Thus, modulation of Bax phosphorylation appeared as tightly related to TRAIL sensitivity/resistance in melanoma cells, and therapeutic strategies may be considered.

Disruption of the VDAC2-Bak interaction by Bcl-x(S) mediates efficient induction of apoptosis in melanoma cells.

The proapoptotic B-cell lymphoma (Bcl)-2 protein Bcl-x(S) encloses the Bcl-2 homology (BH) domains BH3 and BH4 and triggers apoptosis via the multidomain protein Bak, however, the mechanism remained elusive. For investigating Bcl-x(S) efficacy and pathways, an adenoviral vector was constructed with its cDNA under tetracycline-off control. Bcl-x(S) overexpression resulted in efficient apoptosis induction and caspase activation in melanoma cells. Indicative of mitochondrial apoptosis pathways, Bcl-x(S) translocated to the mitochondria, disrupted the mitochondrial membrane potential and induced release of cytochrome c, apoptosis-inducing factor and second mitochondria-derived activator of caspases. In melanoma cells, Bcl-x(S) resulted in significant Bak activation, and Bak knockdown as well as Bcl-x(L) overexpression abrogated Bcl-x(S)-induced apoptosis, whereas Mcl-1 (myeloid cell leukemia-1) knockdown resulted in a sensitization. With regard to the particular role of voltage-dependent anion channel 2 (VDAC2) for inhibition of Bak, we identified here a notable interaction between Bcl-x(S) and VDAC2 in melanoma cells, which was proven in reciprocal coimmunoprecipitation analyses. On the other hand, Bcl-x(S) showed no direct interaction with Bak, and its binding to VDAC2 appeared as also independent of Bak expression. Suggesting a new proapoptotic mechanism, Bcl-x(S) overexpression resulted in disruption of the VDAC2-Bak interaction leading to release of Bak. Further supporting this pathway, overexpression of VDAC2 strongly decreased apoptosis by Bcl-x(S). New proapoptotic pathways are of principle interest for overcoming apoptosis deficiency of melanoma cells.

Electron microscopic and 31-P NMR studies of ischemic injured rat livers during preservation and reflow.

Ischemic injury induced during preservation and reperfusion contributes to post-operative failure in liver transplantation. Hepatic injury and recovery from preservation was studied in an isolated rat liver model reperfused with oxygenated erythrocytes. In order to correlate morphological and functional findings, 31-P nuclear magnetic resonance spectroscopy and electron microscopy were used to investigate metabolic and ultrastructural changes during 6 hours of reperfusion. Following cold preservation, EM's showed a primary sinusoidal cell injury, whereas the hepatocytes were well maintained. During reperfusion, hepatocytes displayed further damage. The simultaneous presence of vacuolarly degenerated mitochondria and mitochondria of increased activity was noted. 31-P NMP spectra demonstrated initially a partial ATP-recovery. The maximum level of 60% of the control ATP-value could not be further increased. EM and 31-P NMR indicate that the progressive injury to the liver is due to microcirculatory malfunction induced by an endothelial cell damage, followed by injured hepatocytes themselves, and the consequent intracellular energy crisis that is produced.

Effect of pentoxiphylline on the recovery of the preserved rat liver: 31P NMR and ultrastructural studies.

Hepatic failure often occurs following transplantation. This is primarily due to cold ischemia during preservation, warm ischemia during implantation, and finally reperfusion damage after transplantation and reflow. The possibility that this ischemia and reperfusion-induced damage can be reduced by preischemic application of a xanthine derivative (pentoxiphylline) was examined using 31P NMR spectroscopy and electron microscopy (EM) studies of bioenergetic and ultrastructural changes in oxygenated erythrocyte-perfused rat livers. EM illustrated that the hepatocytes and the mitochondria appeared to be relatively unaffected by cold preservation of the liver, whereas the endothelial cells lining the sinusoids became disrupted. After reperfusion, NMR spectroscopy showed a partial recovery of ATP levels, and EM indicated progressive mitochondrial injury. This progressive injury to the liver was probably due to endothelial cell damage which resulted in microcirculatory malfunction and free radical formation during reperfusion. Pentoxiphylline pretreated livers showed better preservation of the cell morphology and exhibited better ATP recovery than untreated livers. Pentoxiphylline is known to prevent the loss of precursors of ATP resynthesis by inhibiting AMP dephosphorylation during ischemia and improves the microcirculation via vasodilatory properties following ischemia. Thus, it is concluded that pentoxiphylline may ameliorate ischemia-induced cell damage during transplantation.

[Magnet resonance spectroscopy of tumor-bearing rat livers: magnetite particles as an aid in volume selection]. / Magnetresonanzspektroskopie der tumortragenden Rattenleber: Magnetitpartikel als Hilfe bei der Volumenselektion.

Magnetite, an RES-specific contrast medium, has been used in animal experiments and in a few patients for MRI of the liver. We examined the use of magnetite particles for 31P-MR spectroscopy using a phantom, and perfused tumour-bearing rat livers and liver tumours in living rats. As expected, there is homogeneous uptake of the magnetite in normal liver leading to extinction of the signal when one uses suitable spectroscopic parameters. Since the ferrite particles do not penetrate non-hepatic tissue, such as metastases, a signal remains uniquely from the tumour and this can be used, for instance, for following the effect of cytostatic therapy. The use of magnetite produces a selective effect and interference from normal liver is thereby avoided. Multiple lesions with irregular configuration can be examined simultaneously by this method. It remains to be seen how useful the application of magnetite will be for avoiding motion artifacts during spectroscopy.