A comparative study of freezing single cells and spheroids: Towards a new model system for optimizing freezing protocols for cryobanking of human tumours

Cryopreservation of human tumour cells and tissue is a valuable tool for retrospective analysis and for the transport and handling of biopsy material. Tumour tissue consists of different cell types, which have different optimal freezing conditions, and extracellular matrix. A well-defined and authentic model system is required for developing new freezing protocols and media. This work describes the use of L929 and PC-3 spheroids as new model systems for freezing human tumours. Cell suspension and spheroids were frozen in different vessels (1 ml cryovials and a special, cryo-compatible 30 × 25 μl multi well plate) at slow rate (1 °C/min). Freezing media were combinations of culture or tumour transport medium (Liforlab^®) with the cryoprotective agents, Me₂SO, trehalose and modified starch. We also present a new method of evaluating the viability of three dimensional multicellular systems to compare thawed spheroids objectively. Best viability (70%) of L929 spheroids occurred with a combination of Liforlab^® and starch hydrolysis product. The best cryopreservation results for spheroids were found with extracellular cryoprotectants, while optimum viability of single cells was achieved with Me₂SO.     

Mitochondrial Haplogroup U5b3: A Distant Echo of the Epipaleolithic in Italy and the Legacy of the Early Sardinians

There are extensive data indicating that some glacial refuge zones of southern Europe (Franco-Cantabria, Balkans, and Ukraine) were major genetic sources for the human recolonization of the continent at the beginning of the Holocene. Intriguingly, there is no genetic evidence that the refuge area located in the Italian Peninsula contributed to this process. Here we show, through phylogeographic analyses of mitochondrial DNA (mtDNA) variation performed at the highest level of molecular resolution (52 entire mitochondrial genomes), that the most likely homeland for U5b3—a haplogroup present at a very low frequency across Europe—was the Italian Peninsula. In contrast to mtDNA haplogroups that expanded from other refugia, the Holocene expansion of haplogroup U5b3 toward the North was restricted by the Alps and occurred only along the Mediterranean coasts, mainly toward nearby Provence (southern France). From there, ∼7,000–9,000 years ago, a subclade of this haplogroup moved to Sardinia, possibly as a result of the obsidian trade that linked the two regions, leaving a distinctive signature in the modern people of the island. This scenario strikingly matches the age, distribution, and postulated geographic source of a Sardinian Y chromosome haplogroup (I2a2-M26), a paradigmatic case in the European context of a founder event marking both female and male lineages.     

Duetting—A mechanism to strengthen pair bonds in a dispersed pair‐living primate (Lepilemur edwardsi)?

Duetting is defined as an interactively organized pair display in which one pair partner coordinates its vocalizations in time with those of the other. It is widespread among tropical birds and cohesive pair‐living primates, in which it is suggested to strengthen pair bonds. We know very little about the presence and function of duetting in dispersed pair‐living mammals. We studied duetting behavior in a solitary foraging, but pair‐sleeping, primate, the Milne Edwards' sportive lemur, in a dry deciduous forest of north‐western Madagascar. We radio‐tracked six pairs throughout 1 year and recorded their sleeping sites and associations, home‐range use, and vocal and behavioral interactions. Three different periods were covered (mating, pregnancy, and offspring care). Sleeping partners form long‐term pair bonds, indicated by an almost exclusive pair‐specific usage of sleeping sites and home‐ranges across periods. We explored three functional hypothesis of duetting: mate reunion, pair reunion, and joint‐territorial defense. Pairs regularly engaged in duet calling. Duetting increased significantly during the offspring care period. Duetting occurred significantly more often at feeding sites than at sleeping sites. Pair partners synchronized behavioral activities after duetting. The activity most often synchronized was locomotion. Pair partners played an equal role in duetting with no difference between sexes in starting or terminating duetting. Altogether, our results provide support for the hypothesis that in dispersed pair‐living primates, duetting evolved as a mechanism to coordinate activities between pair partners dispersed in space, to strengthen pair bonds, and, perhaps, to limit infanticide and nutritional stress in lactating females. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of the kinetics and mechanisms of inhibition of drugs interacting with the S. cerevisiae multidrug resistance pumps Pdr5p and Snq2p

We have developed a novel screening method that measures the kinetics and potencies of inhibitors of the yeast multidrug resistance pumps Pdr5p and Snq2p. The assay uses the potentiometric fluorescent probe diS-C₃(3) (as a benchmark substrate of both pumps) to distinguish drugs with minimal effects on plasma membrane potential as a marker of side-effects on membrane function and integrity. Using FK506, its structural analog rapamycin and enniatin B, we showed that our assay can also be used to determine the minimum drug concentration causing an immediate inhibitory effect and to compare the inhibitory potencies of the drug on the two pumps. We found that the protonophore CCCP effectively inhibits the transport of diS-C₃(3) by both pumps and confirmed the activation of membrane H⁺-ATPase by CCCP.     

Isolation of sixteen autosomal loci and a sex-linked polymorphic microsatellite locus from the Milne-Edwards' sportive lemur (Lepilemur edwardsi)

We isolated 21 microsatellites from the Milne-Edwards' sportive lemur, Lepilemur edwardsi. Eighteen microsatellite sequences possessed sufficient flanking DNA for primer design. Seventeen loci amplified and were found to be polymorphic displaying two to 17 alleles in 32 unrelated individuals from a population from the National Park of Ankarafantsika in northwest Madagascar. One locus (Led-12) was found to be sex linked located on the X chromosome and can be used to sex-type 40% of female L. edwardsi lemurs. These 17 loci were characterized to investigate family structure and the phylogeography of L. edwardsi.     

Fatness-Associated FTO Gene Variant Increases Mortality Independent of Fatness – in Cohorts of Danish Men

Background

The A-allele of the single nucleotide polymorphism (SNP), rs9939609, in the FTO gene is associated with increased fatness. We hypothesized that the SNP is associated with morbidity and mortality through the effect on fatness.

Methodology/Principal Findings

In a population of 362,200 Danish young men, examined for military service between 1943 and 1977, all obese (BMI≥31.0 kg/m²) and a random 1% sample of the others were identified. In 1992–94, at an average age of 46 years, 752 of the obese and 876 of the others were re-examined, including measurements of weight, fat mass, height, and waist circumference, and DNA sampling. Hospitalization and death occurring during the following median 13.5 years were ascertained by linkage to national registers. Cox regression analyses were performed using a dominant effect model (TT vs. TA or AA). In total 205 men died. Mortality was 42% lower (p = 0.001) with the TT genotype than in A-allele carriers. This phenomenon was observed in both the obese and the randomly sampled cohort when analysed separately. Adjustment for fatness covariates attenuated the association only slightly. Exploratory analyses of cause-specific mortality and morbidity prior to death suggested a general protective effect of the TT genotype, whereas there were only weak associations with disease incidence, except for diseases of the nervous system.

Conclusion

Independent of fatness, the A-allele of the FTO SNP appears to increase mortality of a magnitude similar to smoking, but without a particular underlying disease pattern barring an increase in the risk of diseases of the nervous system.     

Impairments of Biological Motion Perception in Congenital Prosopagnosia

Prosopagnosia is a deficit in recognizing people from their faces. Acquired prosopagnosia results after brain damage, developmental or congenital prosopagnosia (CP) is not caused by brain lesion, but has presumably been present from early childhood onwards. Since other sensory, perceptual, and cognitive abilities are largely spared, CP is considered to be a stimulus-specific deficit, limited to face processing. Given that recent behavioral and imaging studies indicate a close relationship of face and biological-motion perception in healthy adults, we hypothesized that biological motion processing should be impaired in CP. Five individuals with CP and ten matched healthy controls were tested with diverse biological-motion stimuli and tasks. Four of the CP individuals showed severe deficits in biological-motion processing, while one performed within the lower range of the controls. A discriminant analysis classified all participants correctly with a very high probability for each participant. These findings demonstrate that in CP, impaired perception of faces can be accompanied by impaired biological-motion perception. We discuss implications for dedicated and shared mechanisms involved in the perception of faces and biological motion.     

Genetic Diversity of the Cestode Echinococcus multilocularis in Red Foxes at a Continental Scale in Europe

Background

Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level.

Methodology/Principal Findings

The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas.

Conclusions/Significance

The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a “mainland–island” system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.     

Pentaschistis (Poaceae) diversity in the Cape mediterranean region: habitat heterogeneity and climate stability

Aim To evaluate the role of habitat heterogeneity on species richness and turnover in the mega species‐rich Cape Floristic Region (Cape), the mediterranean region of southern Africa. Location The Cape and Drakensberg regions of southern Africa. Methods Bioclimatic data were used to estimate habitat amount and habitat heterogeneity in the Cape and Drakensberg regions; these data were then used to explain the patterns of species diversity in the Pentaschistis clade (Poaceae) in these two regions. Habitat variables were used to create ‘bioclimatic units’ to characterize 1′× 1′ cells in southern Africa and to describe the niches of species. Using these bioclimatic units, the niche and range sizes of species in the two regions were compared. A phylogram was used to test for age and lineage effects. Results Pentaschistis species diversity and turnover are higher in the Cape than the Drakensberg. There is no significant difference in the habitat amount and heterogeneity between the two regions. Species occupy the same range of estimated niche sizes, yet there are significantly more range‐restricted Pentaschistis species in the Cape. Main conclusions The roles of age‐ and lineage‐related effects are rejected; biodiversity differences lie in the regions. Current macrohabitat does not explain the differences in biodiversity between the two regions. The larger number of range‐restricted species in the Cape cannot be explained by dispersal mechanism or the occupation of range‐restricted habitats. Species of Pentaschistis and other Cape clades share characteristics associated with species from historically climatically stable areas, and palaeoclimatic and palaeontological evidence indicates the Cape climate has been more stable than the Drakensberg climate throughout the Pleistocene. We conclude that the corresponding lack of extinction might have allowed an accumulation of species in the Cape. Similar climatic and biological evidence for the south‐west Australian Floristic and Mediterranean regions indicate that the same mechanism might explain the high species richness of these mediterranean regions.