Silurian (Ludlow) acastid trilobites from Gotland and Scania,Sweden

Acastid trilobites from Ludlow strata on Gotland include three stratigraphically non-overlapping species ofAcastella Reed:Acastella madidipes n. sp. (Hemse Marl and Eke Beds),A. breviceps (Angelin 1851) (Burgsvik Beds), andA. amatrix n. sp. (Hamra Beds, and probably Burgsvik Beds). Type and other material ofA. breviceps (Angelin) from the Klinta Formation, Scania, is revised. Meraspid and early holaspid material ofAcastella is described. Some characters supportAcaste and allied taxa of mostly Wenlock age as a monophyletic subfamily Acastinae.Acastella as widely recognized is a grade with Devonian species more closely related to Asteropyginae than to the Ludlow type species. Baltic Ludlow-P?00ED;dolí species previously placed inScotiella Delo form a distinct group,Ewacaste n. gen., that includes a species from the uppermost Eke Beds on Gotland.     

Antigen-driven interactions with dendritic cells and expansion of Foxp3+ regulatory T cells occur in the absence of inflammatory signals

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.     

A mouse model for monitoring calpain activity under physiological and pathological conditions

Calpains are Ca(2+)-dependent cysteine proteases known to be important for the regulation of cell functions and which aberrant activation causes cell death in a number of degenerative disorders. To provide a tool for monitoring the status of calpain activity in vivo under physiological and pathological conditions, we created a mouse model that expresses ubiquitously a fluorescent reporter consisting of eCFP and eYFP separated by a linker cleavable by the ubiquitous calpains. We named this mouse CAFI for calpain activity monitored by FRET imaging. Our validation studies demonstrated that the level of calpain activity correlates with a decrease in FRET (fluorescence resonance energy transfer) between the two fluorescent proteins. Using this model, we observed a small level of activity after denervation and fasting, a high level of activity during muscle regeneration and ischemia, and local activity in damaged myofibers after exercise. Finally, we crossed the CAFI mouse with the alpha-sarcoglycan-deficient model, demonstrating an increase of calpain activity at the steady state. Altogether, our results present evidence that CAFI mice could be a valuable tool in which to follow calpain activity at physiological levels and in disease states.     

Combination of quantification and observation methods for study of "Side Population" cells in their "in vitro" microenvironment.

BACKGROUND: Qualitative and quantitative analyses of the rare phenotypic variants in in vitro culture systems is necessary for the understanding of cell differentiation in cell culture of primary cells or cell lines. Slide-based cytometry combines image acquisition and data treatment, and associates the power of flow cytometry (FCM) and the resolution of the microscopic studies making it suitable for the analysis of cells with rare phenotype. In this paper we develop a method that applies these principles to a particularly hot problem in cell biology, the study of stem cell like cells in cultures of primary cells, cancer cells, and various cell lines. METHODS: The adherent cells were labeled by the fluorescent dye Hoechst 33342. The images of cell populations were collected by a two-photon microscope and processed by a software developed by us. The software allows the automated segmentation of the nuclei in a very dense cell environment, the measurement of the fluorescence intensity of each nucleus and the recording of their position in the plate. The cells with a given fluorescence intensity can then be located easily on the recorded image of the culture plate for further analysis. RESULTS: The potential of our method is illustrated by the identification and localization of SP cells in the cultures of the C2C12 cell line. Although these cells represent only about 1% of the total population as calculated by flow cytometry, they can be identified in the culture plate with high precision by microscopy. CONCLUSION: Cells with the rare stem-cell like phenotype can be efficiently identified in the undisturbed cultures. Since the fluorescence intensity of rare events and the position of thousands of surrounding cells are recorded at the same time, the method associates the advantage of the FCM analysis and the microscopic observation.     

Loss of calpain 3 proteolytic activity leads to muscular dystrophy and to apoptosis-associated IkappaBalpha/nuclear factor kappaB pathway perturbation in mice

Calpain 3 is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. It was previously shown that defects in the human calpain 3 gene are responsible for limb girdle muscular dystrophy type 2A (LGMD2A), an inherited disease affecting predominantly the proximal limb muscles. To better understand the function of calpain 3 and the pathophysiological mechanisms of LGMD2A and also to develop an adequate model for therapy research, we generated capn3-deficient mice by gene targeting. capn3-deficient mice are fully fertile and viable. Allele transmission in intercross progeny demonstrated a statistically significant departure from Mendel's law. capn3-deficient mice show a mild progressive muscular dystrophy that affects a specific group of muscles. The age of appearance of myopathic features varies with the genetic background, suggesting the involvement of modifier genes. Affected muscles manifest a similar apoptosis-associated perturbation of the IkappaBalpha/nuclear factor kappaB pathway as seen in LGMD2A patients. In addition, Evans blue staining of muscle fibers reveals that the pathological process due to calpain 3 deficiency is associated with membrane alterations.     

Bistable cell fate specification as a result of stochastic fluctuations and collective spatial cell behaviour

Background

In culture, isogenic mammalian cells typically display enduring phenotypic heterogeneity that arises from fluctuations of gene expression and other intracellular processes. This diversity is not just simple noise but has biological relevance by generating plasticity. Noise driven plasticity was suggested to be a stem cell-specific feature.

Results

Here we show that the phenotypes of proliferating tissue progenitor cells such as primary mononuclear muscle cells can also spontaneously fluctuate between different states characterized by the either high or low expression of the muscle-specific cell surface molecule CD56 and by the corresponding high or low capacity to form myotubes. Although this capacity is a cell-intrinsic property, the cells switch their phenotype under the constraints imposed by the highly heterogeneous microenvironment created by their own collective movement. The resulting heterogeneous cell population is characterized by a dynamic equilibrium between “high CD56” and “low CD56” phenotype cells with distinct spatial distribution. Computer simulations reveal that this complex dynamic is consistent with a context-dependent noise driven bistable model where local microenvironment acts on the cellular state by encouraging the cell to fluctuate between the phenotypes until the low noise state is found.

Conclusions

These observations suggest that phenotypic fluctuations may be a general feature of any non-terminally differentiated cell. The cellular microenvironment created by the cells themselves contributes actively and continuously to the generation of fluctuations depending on their phenotype. As a result, the cell phenotype is determined by the joint action of the cell-intrinsic fluctuations and by collective cell-to-cell interactions.     

Lentiviral Transduction of CD34+ Cells Induces Genome-Wide Epigenetic Modifications

Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34⁺ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34⁺ cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34⁺ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.