Deciliation interferes with cell-cycle progression in Tetrahymena

The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied. It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation. Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later. This was shown by pulse-labelling the cells with [3H]thymidine at various times after deciliation. Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose. At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment. This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division. The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure. The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells. This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation. Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells.     

High resolution detection and analysis of CpG dinucleotides methylation using MBD-Seq technology

Methyl-CpG binding domain protein sequencing (MBD-seq) is widely used to survey DNA methylation patterns. However, the optimal experimental parameters for MBD-seq remain unclear and the data analysis remains challenging. In this study, we generated high depth MBD-seq data in MCF-7 cell and developed a bi-asymmetric-Laplace model (BALM) to perform data analysis. We found that optimal efficiency of MBD-seq experiments was achieved by sequencing ～100 million unique mapped tags from a combination of 500 mM and 1000 mM salt concentration elution in MCF-7 cells. Clonal bisulfite sequencing results showed that the methylation status of each CpG dinucleotides in the tested regions was accurately detected with high resolution using the proposed model. These results demonstrated the combination of MBD-seq and BALM could serve as a useful tool to investigate DNA methylome due to its low cost, high specificity, efficiency and resolution.     

Non-cell autonomous control of apoptosis by ligand-independent Hedgehog signaling in Drosophila

Hedgehog (Hh) signaling is important for development and homeostasis in vertebrates and invertebrates. Ligand-independent, deregulated Hh signaling caused by loss of negative regulators such as Patched causes excessive cell proliferation, leading to overgrowth in Drosophila and tumors in humans, including basal-cell carcinoma and medulloblastoma. We show that in Drosophila deregulated Hh signaling also promotes cell survival by increasing the resistance to apoptosis. Surprisingly, cells with deregulated Hh activity do not protect themselves from apoptosis; instead, they promote cell survival of neighboring wild-type cells. This non-cell autonomous effect is mediated by Hh-induced Notch signaling, which elevates the protein levels of Drosophila inhibitor of apoptosis protein-1 (Diap-1), conferring resistance to apoptosis. In summary, we demonstrate that deregulated Hh signaling not only promotes proliferation but also cell survival of neighboring cells. This non-cell autonomous control of apoptosis highlights an underappreciated function of deregulated Hh signaling, which may help to generate a supportive micro-environment for tumor development.     

Lipopolysaccharide pretreatment of the udder protects against experimental Escherichia coli mastitis

Exposure to pathogen-associated molecular patterns such as LPS can cause an immune refractory state in mammals known as endotoxin tolerance (ET), resulting in a decreased inflammatory response after pathogen contact. This ET concept was used to reduce the severity of an experimentally-induced clinical mastitis. Cows were pretreated with 1?μg LPS per udder quarter and challenged 72?h (group L72EC) or 240?h (group L240EC) later with 500 CFU Escherichia coli. Pretreated animals showed no leukopenia after challenge, no (L72EC), or only slightly (L240EC), elevated body temperature and significantly reduced systemic and local clinical scores compared with cows that were not pretreated. Whereas an increase of milk somatic cell count after the E. coli challenge was abrogated in L72EC animals, it was significantly delayed in the L240EC group. In both pretreated groups the bacterial load in milk was markedly reduced. Based on the expression of inflammation-related genes in lobulo-alveolar mammary tissue, the tolerizing effect of LPS pretreatment is based on the inhibited up-regulation of inflammatory (TNF-α, IL-6, CXCL8, CCL20) and anti-inflammatory genes (IL-10, IRAK-M). These findings indicate that the concept of ET may be usefully applied as mastitis prophylaxis facilitating a rapid response to microbial infection and avoiding dysregulated inflammation.     

Increased Numbers of IL-7 Receptor Molecules on CD4+CD25?CD107a+ T-Cells in Patients with Autoimmune Diseases Affecting the Central Nervous System

Background

High content immune profiling in peripheral blood may reflect immune aberrations associated with inflammation in multiple sclerosis (MS) and other autoimmune diseases affecting the central nervous system.

Methods and Findings

Peripheral blood mononuclear cells from 46 patients with multiple sclerosis (MS), 9 patients diagnosed with relapsing remitting MS (RRMS), 13 with secondary progressive multiple sclerosis (SPMS), 9 with other neurological diseases (OND) and well as 15 healthy donors (HD) were analyzed by 12 color flow cytometry (TCRαβ, TCRγδ, CD4, CD8α, CD8β, CD45RA, CCR7, CD27, CD28, CD107a, CD127, CD14) in a cross-sectional study to identify variables significantly different between controls (HD) and patients (OND, RRMS, SPMS). We analyzed 187 individual immune cell subsets (percentages) and the density of the IL-7 receptor alpha chain (CD127) on 59 individual immune phenotypes using a monoclonal anti-IL-7R antibody (clone R34.34) coupled to a single APC molecule in combination with an APC-bead array. A non-parametric analysis of variance (Kruskal-Wallis test) was conducted in order to test for differences among the groups in each of the variables. To correct for the multiplicity problem, the FDR correction was applied on the p-values. We identified 19 variables for immune cell subsets (percentages) which allowed to segregate healthy individuals and individuals with CNS disorders. We did not observe differences in the relative percentage of IL-7R-positive immune cells in PBMCs. In contrast, we identified significant differences in IL-7 density, measured on a single cell level, in 2/59 variables: increased numbers of CD127 molecules on TCRαβ+CD4+CD25 (intermed) T-cells and on TCRαβ+CD4+CD25−CD107a+ T-cells (mean: 28376 Il-7R binding sites on cells from HD, 48515 in patients with RRMS, 38195 in patients with SPMS and 33692 IL-7 receptor binding sites on cells from patients with OND).

Conclusion

These data show that immunophenotyping represents a powerful tool to differentiate healthy individuals from individuals suffering from neurological diseases and that the number of IL-7 receptor molecules on differentiated TCRαβ+CD4+CD25−CD107a+ T-cells, but not the percentage of IL-7R-positive cells, segregates healthy individuals from patients with neurological disorders.