Shrews (Sorex spp.) somatometry and reproduction in Slovakia

The morphometrics and reproduction of shrews (Sorex araneus, S. minutus, S. alpinus) was studied in Slovakia. Significant differences in weight, body length and tail length were recorded in adult and subadult shrews. Adult individuals and males had higher mean somatometric values (apart from tail length of subadults). The hind foot length was the least variable characteristic, which can be considered as the most stable taxonomic somatic characteristic of shrews. Weight and body length varied considerably. Values of somatic characteristics in S. araneus and S. minutus increased with increasing altitude, apart from hind foot length in S. araneus, which decreased with increasing altitude. The values of somatic characteristics declined with increasing continentality, but tail length gradually increased in the direction west — east at the highland level. Sorex minutus was characterized by the greatest reproductive activity (length of reproductive cycle April–October, average number of embryos 6.97). The lowest reproductive activity was observed in S. alpinus (average number of embryos 5.72). The mating season of S. araneus began at the end of March and ended in August with an average number of embryos of 6.12.     

Application of Giemsa banding to orchid karyotype analysis

A method for obtaining orchid chromosome squash preparations from ovular tissues and a Giemsa C-band technique are described. Jointly applied, they result in well-defined chromosome banding patterns. Preliminary tests with two species of the genusCephalanthera show that Giemsa banding is also well suited for orchids. Besides aiding in chromosome identification and karyotype analysis, it should prove valuable in studies of chromosomal variation and karyotype evolution of this large family.     

Enriched Bone Marrow Derived Disseminated Neuroblastoma Cells Can Be a Reliable Source for Gene Expression Studies—A Validation Study

Background

Metastases in the bone marrow (BM) in form of disseminated tumor cells (DTCs) are frequent events at diagnosis and also at relapse in high-risk neuroblastoma patients. The frequently highly diluted occurrence of DTCs requires adequate enrichment strategies to enable their detailed characterization. However, to avoid methodical artifacts we tested whether pre-analytical processing steps—including transport duration, temperature and, importantly, tumor cell enrichment techniques—are confounding factors for gene expression analysis in DTCs.

Methods

LAN-1 neuroblastoma cells were spiked into tumor free BM and/or peripheral blood and: i) kept at room temperature or at 4°C for 24, 48 and 72 hours; ii) frozen down at -80°C and thawed; iii) enriched via magnetic beads. The effect on the gene expression signature of LAN-1 cells was analyzed by qPCR arrays and gene expression microarrays.

Results

Neither storage at –80°C in DMSO and subsequent thawing nor enrichment of spiked-in neuroblastoma cells changed the expression of the analyzed genes significantly. Whereas storage at 4°C altered the expression of analyzed genes (14.3%) only at the 72h-timepoint in comparison to the 0h-timepoint, storage at room temperature had a much more profound effect on gene expression by affecting 20% at 24h, 26% at 48h and 43% at 72h of the analyzed genes.

Conclusion

Using neuroblastoma as a model, we show that tumor cell enrichment by magnetic bead separation has virtually no effect on gene expression in DTCs. However, transport time and temperature can influence the expression profile remarkably. Thus, the expression profile of routinely collected BM samples can be analyzed without concern as long as the transport conditions are monitored.     

Physiologically Based Pharmacokinetic Modeling Is Essential in 90Y-Labeled Anti-CD66 Radioimmunotherapy

IntroductionRadioimmunotherapy (RIT) with ⁹⁰Y-labeled anti-CD66 antibody is used to selectively irradiate the red marrow (RM) before blood stem cell transplantation of acute leukemia patients. To calculate the activity to administer, time-integrated activity coefficients are required. These are estimated prior to therapy using gamma camera and serum measurements after injection of ¹¹¹In labeled anti-CD66 antibody. Equal pre-therapeutic and therapeutic biodistributions are usually assumed to calculate the coefficients. However, additional measurements during therapy had shown that this assumption had to be abandoned. A physiologically based pharmacokinetic (PBPK) model was developed to allow the prediction of therapeutic time-integrated activity coefficients in eight patients.AimsThe aims of the study were to demonstrate using a larger patient group 1) the need to perform patient-specific dosimetry in ⁹⁰Y-labeled anti-CD66 RIT, 2) that pre-therapeutic and therapeutic biodistributions differ, and most importantly 3) that this difference in biodistributions can be accurately predicted using a refined model.ResultsVariability of the RM time-integrated activity coefficients ((37.3±7.5) h) indicates the need for patient-specific dosimetry. The relative differences between pre-therapeutic and therapeutic serum time-activity curves were (-25±16)%. The prediction accuracy of these differences using the refined PBPK models was (-3±20)%.ConclusionIndividual treatment is needed due to biological differences between patients in RIT with ⁹⁰Y-labeled anti-CD66 antibody. Differences in pre-therapeutic and therapeutic biokinetics are predominantly caused by different degrees of saturation due to different amounts of administered antibody. These differences could be predicted using the PBPK models.     

Method of releasing and number of animals are determinants for the success of European ground squirrel (Spermophilus citellus) reintroductions

Reintroductions are considered an important part of the action plans and recovery strategies of endangered ground squirrel species, but so far little is known about their proper methodology. We collected primary data on 12 European ground squirrel reintroduction projects carried out at 14 localities in the Czech Republic, Slovakia and Poland since 1989. We focused on seven methodological aspects of each reintroduction: selection of release site, method of releasing, date of releasing, origin of released animals, total number of released animals, mean number of released animals per season and reintroduction site management. The method of releasing was found to be the key factor in determining the settlement of animals at the target locality. Only soft releasing methods, i.e. the use of enclosures and/or artificial burrows, ensure that animals remain at the target locality. The other factors significantly determining reintroduction success are the number of released animals per season (at least 23 animals required) and the total number of released animals (a minimum of 60 individuals). Long-term management of the site and regular monitoring of the newly established population are necessary. Our recommendations, based on experience with the successes and failures of previous reintroductions, could largely improve the efficiency of future reintroductions of highly endangered species.     

MicroRNA pathways in flies and worms: growth,death, fat,stress, and timing

Drosophila geneticists have uncovered roles for microRNAs in the coordination of cell proliferation and cell death during development, and in stress resistance and fat metabolism. In C. elegans, a homolog of the well-known fly developmental regulator hunchback acts downstream of the microRNAs lin-4 and let-7 in a pathway controlling developmental timing.     

Detection and relocation of rare events. A comparative study using the laser scanning cytometer and the Metafer/RCDetect microscope scanning system

We compared instrumental analysis of enriched cord blood nucleated red blood cells (CB-NRBC) out of in vitro contamination preparations of dilutions of minute volumes of male cord blood into peripheral blood from nonpregnant women. This was done using the laser scanning cytometer (LSC) and the Metafer/RCDetect microscope scanning system, both allowing for relocation of positive cells defined on the basis of fluorescence parameters. Both instruments were efficient in performing scanning and relocation; a difference in the recovery of CB-NRBC was not significant and can be explained by the method of preparation used.     

Characterisation of pericentrometric and sticky intercalary heterochromatin in Ornithogalum longibracteatum (Hyacinthaceae)

The hexaploid liliaceous plant Ornithogalum longibracteatum (2n=6x=54) has a heterochromatin-rich bimodal karyotype with large (L) and small (S) chromosomes. The composition and subgenomic distribution of heterochromatin was studied using molecular and cytological methods. The major component of centromeric heterochromatin in all chromosomes is Satl, an abundant satellite DNA with a basic repeat unit of 155 bp and an average A+T content (54%). The major component of the large blocks of intercalary heterochromatin in L chromosomes is Sat2, an abundant satellite DNA with a basic repeat unit of 115 bp and a high A+T content (76%). Additionally, traces of Sat2 can be detected at the centromeric regions of S chromosomes, while minor amounts of Satl are discernible in intercalary heterochromatin of L chromosomes. The chromosomal localisation pattern of Sat2 is consistent with the fluorescent staining pattern obtained with the A+T-specific DNA ligand 4'-6-diamidino-2-phenylindole (DAPI). A+T-rich intercalary heterochromatin is sticky and tends to associate ectopically during mitosis. Sister chromatid exchange clustering was found at the junctions between euchromatin and heterochromatin and at the centromeres. The pattern of mitosis-specific phosphorylation of histone H3 was not uniform along the length of the chromosomes. In all L and S chromosomes, from early prophase to ana-/telophase, there is hyperphosphorylation of histone H3 in the pericentromeric chromatin and a slightly elevated phosphorylated histone H3 level at the intercalary heterochromatin of L chromosomes. Consequently, the overall phosphorylated histone H3 metaphase labelling resembles the distribution of Satl in the karyotype of O. longibracteatum.