Helicobacter acinonychis: genetic and rodent infection studies of a Helicobacter pylori-like gastric pathogen of cheetahs and other big cats

Insights into bacterium-host interactions and genome evolution can emerge from comparisons among related species. Here we studied Helicobacter acinonychis (formerly H. acinonyx), a species closely related to the human gastric pathogen Helicobacter pylori. Two groups of strains were identified by randomly amplified polymorphic DNA fingerprinting and gene sequencing: one group from six cheetahs in a U.S. zoo and two lions in a European circus, and the other group from a tiger and a lion-tiger hybrid in the same circus. PCR and DNA sequencing showed that each strain lacked the cag pathogenicity island and contained a degenerate vacuolating cytotoxin (vacA) gene. Analyses of nine other genes (glmM, recA, hp519, glr, cysS, ppa, flaB, flaA, and atpA) revealed a approximately 2% base substitution difference, on average, between the two H. acinonychis groups and a approximately 8% difference between these genes and their homologs in H. pylori reference strains such as 26695. H. acinonychis derivatives that could chronically infect mice were selected and were found to be capable of persistent mixed infection with certain H. pylori strains. Several variants, due variously to recombination or new mutation, were found after 2 months of mixed infection. H. acinonychis ' modest genetic distance from H. pylori, its ability to infect mice, and its ability to coexist and recombine with certain H. pylori strains in vivo should be useful in studies of Helicobacter infection and virulence mechanisms and studies of genome evolution.     

Mutation discovery in bacterial genomes: metronidazole resistance in Helicobacter pylori

We developed a microarray hybridization-based method, 'comparative genome sequencing' (CGS), to find mutations in bacterial genomes and used it to study metronidazole resistance in H. pylori. CGS identified mutations in several genes, most likely affecting metronidazole activation, and produced no false positives in analysis of three megabases. We conclude that CGS identifies mutations in bacterial genomes efficiently, should enrich understanding of systems biology and genome evolution, and help track pathogens during outbreaks.     

The effects of ischemia and experimental conditions on the respiration rate of cardiac fibers

The mitochondrial respiratory parameters were measured in situ, i.e. in saponin-skinned rabbit cardiac fibers and in fibers treated with saponin + collagenase. It was found that the decrease of maximal ADP-stimulated respiration rate of saponin-skinned fibers with pyruvate + malate under the conditions of total ischemia (0.5–1.5 h) is less pronounced as compared to isolated mitochondria. Maximal succinate oxidation rate (+ADP), however, was not different from control (1 h ischemia) but it exceeded the control level when measured in the medium supplemented with cytochrome c. It was also demonstrated that treatment of fibers with collagenase alone or in combination with saponin significantly (almost 2 fold) enhanced the maximal ADP-stimulated respiration rate if compared with saponin-skinned fibers. The data obtained suggest that mitochondrial respiration in saponin-skinned rabit cardiac fibers is not completely revealed, most probably, due to insufficient permeabilization of sarcolemma by saponin and, thus, inadequate accessibility of mitochondria to exogenous substrates, ADP in particular. These parameters can be improved by pre-treatment of fibers with collagenase. (Mol Cell Biochem 174: 87–90, 1997)     

Site-directed chemical modification of archaeal Thermococcus litoralis Sh1B DNA polymerase: Acquired ability to read through template-strand uracils

We present site-directed chemical modification (SDCM), a tool for engineering U-resistant archaeal DNA polymerases of family B. The Thermococcus litoralis Sh1B DNA polymerase (GenBank: GQ891548) was chosen as the object of the study. Similar to D.Tok, Kod1, Pfu, Tgo and other archaeal members of this family, the T. litoralis Sh1B DNA polymerase is a domain structured, proofreading-proficient enzyme that has the polymerization and 3′→5′ DNA exonucleolytic activities and contains N-terminally located highly conserved template-strand U-binding pocket. The tight binding of template uracil in the enzyme pocket during polymerization blocks the replication of DNA containing uracils. This effect can be alleviated by mutations in key amino acids of the U-binding pocket. We altered T. litoralis Sh1B DNA polymerase's ability to read through the template-strand uracils by applying SDCM. Specific modification of individual cysteine residues in U-binding pocket — targets introduced into certain positions by site-directed mutagenesis — enables the enzyme to effectively replicate DNA containing uracils. We demonstrate that the acquired resistance of chemically modified T. litoralis Sh1B DNA polymerase to DNA uracil correlates with its decreased affinity for template-strand uracil.     

Effectiveness of enterobacterial repetitive intergenic consensus PCR and random amplified polymorphic DNA fingerprinting for Helicobacter pylori strain differentiation

We compared the robustness and discriminatory power of the enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphic DNA (RAPD) fingerprinting methods for detecting cases of mixed Helicobacter pylori infection in Peruvian shantytown residents. H. pylori isolates from 63 participants were cultured, and five single colonies and a pool of additional colonies from each participant were analyzed by ERIC-PCR and by RAPD tests with four 10-nucleotide primers (one primer per reaction). There was 94% agreement between the ERIC and RAPD profiles in classifying sets of isolates as uniform versus closely related but not identical versus probably unrelated, indicating a high kappa statistic of 0.8942. Subtle differences in related ERIC or RAPD patterns likely reflect gene transfer between strains, recombination, and/or mutation, whereas markedly different patterns reflect infection by unrelated strains. At least half of infected shantytown residents seemed to carry more than one H. pylori strain, although in 19 of 31 persons, the strains were closely related. Three RAPD tests, each with a different primer, were needed to achieve the sensitivity of one ERIC test. ERIC-PCR constitutes a resource- and time-efficient method for H. pylori strain differentiation.     

Contraselectable streptomycin susceptibility determinant for genetic manipulation and analysis of Helicobacter pylori

Many Helicobacter pylori genetic studies would benefit from an ability to move DNA sequences easily between strains by transformation and homologous recombination, without needing to leave a conventional drug resistance determinant at the targeted locus. Presented here is a two-gene cassette that can be selected both (i) against, due to a Campylobacter jejuni rpsL gene (dominant streptomycin susceptibility in cells also carrying an rpsL-str(r) allele), and (ii) for, due to an erm gene (erythromycin resistance). This rpsL,erm cassette's utility was assessed by using it to replace four gene loci (mdaB, frxA, fur, and nikR) in four streptomycin-resistant [Str(r)] strain backgrounds (derivatives of 26695, SS1, X47, and G27MA). The resultant 16 strains (phenotypically erythromycin resistant [Erm(r)] and Str(s)) were each transformed with wild-type genomic DNAs, and Str(r) derivatives were selected. The desired Erm(s) Str(r) isolates were obtained at frequencies that ranged from 17 to 96% among Str(r) transformants, with the Erm(s) yield apparently depending on the strain background and genome location of the targeted locus. The ease of isolating unmarked transformants described here should be valuable for many H. pylori molecular genetic and evolutionary analyses.     

Some Lithuanian ethnobotanical taxa: a linguistic view on Thorn Apple and related plants

Background

The perception and use of plants correspond with common plant names. The study of plant names may give insight into historical and recent use of plants.

Methods

Plant names in dictionaries and folklore have been evaluated. A etymological analysis of the names is provided. Onomasiological and semasiological aspects have been considered. Therefore, species named with names related to each other have been selected.

Results

Plant names containing the stem dag- or deg- may belong to either of two categories: incenses or thorny plants. Plants named in durn- have been in use as psychopharmaca. The name rymo points not to Rome but to the use of plants as anodyne or psychopharmaca.     

Digital immunohistochemistry platform for the staining variation monitoring based on integration of image and statistical analyses with laboratory information system

Background

Digital immunohistochemistry (IHC) is one of the most promising applications brought by new generation image analysis (IA). While conventional IHC staining quality is monitored by semi-quantitative visual evaluation of tissue controls, IA may require more sensitive measurement. We designed an automated system to digitally monitor IHC multi-tissue controls, based on SQL-level integration of laboratory information system with image and statistical analysis tools.

Methods

Consecutive sections of TMA containing 10 cores of breast cancer tissue were used as tissue controls in routine Ki67 IHC testing. Ventana slide label barcode ID was sent to the LIS to register the serial section sequence. The slides were stained and scanned (Aperio ScanScope XT), IA was performed by the Aperio/Leica Colocalization and Genie Classifier/Nuclear algorithms. SQL-based integration ensured automated statistical analysis of the IA data by the SAS Enterprise Guide project. Factor analysis and plot visualizations were performed to explore slide-to-slide variation of the Ki67 IHC staining results in the control tissue.

Results

Slide-to-slide intra-core IHC staining analysis revealed rather significant variation of the variables reflecting the sample size, while Brown and Blue Intensity were relatively stable. To further investigate this variation, the IA results from the 10 cores were aggregated to minimize tissue-related variance. Factor analysis revealed association between the variables reflecting the sample size detected by IA and Blue Intensity. Since the main feature to be extracted from the tissue controls was staining intensity, we further explored the variation of the intensity variables in the individual cores. MeanBrownBlue Intensity ((Brown+Blue)/2) and DiffBrownBlue Intensity (Brown-Blue) were introduced to better contrast the absolute intensity and the colour balance variation in each core; relevant factor scores were extracted. Finally, tissue-related factors of IHC staining variance were explored in the individual tissue cores.

Conclusions

Our solution enabled to monitor staining of IHC multi-tissue controls by the means of IA, followed by automated statistical analysis, integrated into the laboratory workflow. We found that, even in consecutive serial tissue sections, tissue-related factors affected the IHC IA results; meanwhile, less intense blue counterstain was associated with less amount of tissue, detected by the IA tools.

     

Spatial and temporal variations in the White-tailed Eagle Haliaeetus albicilla breeding diet revealed by prey remains

ABSTRACT

Capsule: Despite its population increase, the White-tailed Eagle Haliaeetus albicilla has not become food-limited, and does not pose a serious threat to birds of conservation concern in the region east of the Baltic Sea.

Aims: We aimed to test variations in the main prey groups and dietary niche breadth of White-tailed Eagles through the years and along an environmental gradient as well as to evaluate the extent of predation on bird species of conservation concern.

Methods: Prey remains were collected during nestling ringing from successful nests located in Lithuania.

Results: The frequency of the prey groups varied along an environmental gradient, with a general pattern of increasing avian prey consumed by pairs breeding in areas with less abundant aquatic habitats. The frequency of alternative prey (i.e. terrestrial birds, mammals) and dietary niche breadth did not increase between 2005 and 2018 and during which time the White-tailed Eagle population nearly doubled. Instead, the occurrence of Northern Pike Esox lucius remains, which was the most frequent single prey species, increased throughout the study period. Bird species of conservation concern constituted only a small fraction of all the prey identified.

Conclusions: The White-tailed Eagle has not become more dependent on alternative prey despite its population increase.