Stage-specific fluorescence intensity of GFP and mCherry during sporulation In Bacillus Subtilis

<p>Abstract</p> <p>Background</p> <p>Fluorescent proteins are powerful molecular biology tools that have been used to study the subcellular dynamics of proteins within live cells for well over a decade. Two fluorescent proteins commonly used to enable dual protein labelling are GFP (green) and mCherry (red). Sporulation in the Gram positive bacterium <it>Bacillus subtilis </it>has been studied for many years as a paradigm for understanding the molecular basis for differential gene expression. As sporulation initiates, cells undergo an asymmetric division leading to differential gene expression in the small prespore and large mother cell compartments. Use of two fluorescent protein reporters permits time resolved examination of differential gene expression either in the same compartments or between compartments. Due to the spectral properties of GFP and mCherry, they are considered an ideal combination for co-localisation and co-expression experiments. They can also be used in combination with fluorescent DNA stains such as DAPI to correlate protein localisation patterns with the developmental stage of sporulation which can be linked to well characterised changes in DNA staining patterns.</p> <p>Findings</p> <p>While observing the recruitment of the transcription machinery into the forespore of sporulating <it>Bacillus subtilis</it>, we noticed the occurrence of stage-specific fluorescence intensity differences between GFP and mCherry. During vegetative growth and the initial stages of sporulation, fluorescence from both GFP and mCherry fusions behaved similarly. During stage II-III of sporulation we found that mCherry fluorescence was considerably diminished, whilst GFP signals remained clearly visible. This fluorescence pattern reversed during the final stage of sporulation with strong mCherry and low GFP fluorescence. These trends were observed in reciprocal tagging experiments indicating a direct effect of sporulation on fluorescent protein fluorophores.</p> <p>Conclusions</p> <p>Great care should be taken when interpreting the results of protein localisation and quantitative gene expression patterns using fluorescent proteins in experiments involving intracellular physiological change. We believe changes in the subcellular environment of the sporulating cell leads to conditions that differently alter the spectral properties of GFP and mCherry making an accurate interpretation of expression profiles technically challenging.</p

Muscular dystrophy in the mdx mouse is a severe myopathy compounded by hypotrophy, hypertrophy and hyperplasia

Background Preclinical testing of potential therapies for Duchenne muscular dystrophy (DMD) is conducted predominantly of the mdx mouse. But lack of a detailed quantitative description of the pathology of this animal limits our ability to evaluate the effectiveness of putative therapies or their relevance to DMD. Methods Accordingly, we have measured the main cellular components of muscle growth and regeneration over the period of postnatal growth and early pathology in mdx and wild-type (WT) mice; phalloidin binding is used as a measure of fibre size, myonuclear counts and BrdU labelling as records of myogenic activity. Results We confirm a two-phase postnatal growth pattern in WT muscle: first, increase in myonuclear number over weeks 1 to 3, then expansion of myonuclear domain. Mdx muscle growth lags behind that of WT prior to overt signs of pathology. Fibres are smaller, with fewer myonuclei and smaller myonuclear domains. Moreover, satellite cells are more readily detached from mdx than WT muscle fibres. At 3 weeks, mdx muscles enter a phase of florid myonecrosis, accompanied by concurrent regeneration of an intensity that results in complete replacement of pre-existing muscle over the succeeding 3 to 4 weeks. Both WT and mdx muscles attain maximum size by 12 to 14 weeks, mdx muscle fibres being up to 50% larger than those of WT as they become increasingly branched. Mdx muscle fibres also become hypernucleated, containing twice as many myonuclei per sarcoplasmic volume, as those of WT, the excess corresponding to the number of centrally placed myonuclei. Conclusions The best-known consequence of lack of dystrophin that is common to DMD and the mdx mouse is the conspicuous necrosis and regeneration of muscle fibres. We present protocols for measuring this in terms both of loss of muscle nuclei previously labelled with BrdU and of the intensity of myonuclear labelling with BrdU administered during the regeneration period. Both measurements can be used to assess the efficacy of putative antinecrotic agents. We also show that lack of dystrophin is associated with a number of previously unsuspected abnormalities of muscle fibre structure and function that do not appear to be directly associated with myonecrosis

The Spatial Association of Gene Expression Evolves from Synchrony to Asynchrony and Stochasticity with Age

For multicellular organisms, different tissues coordinate to integrate physiological functions, although this systematically and gradually declines in the aging process. Therefore, an association exists between tissue coordination and aging, and investigating the evolution of tissue coordination with age is of interest. In the past decade, both common and heterogeneous aging processes among tissues were extensively investigated. The results on spatial association of gene changes that determine lifespan appear complex and paradoxical. To reconcile observed commonality and heterogeneity of gene changes among tissues and to address evolution feature of tissue coordination with age, we introduced a new analytical strategy to systematically analyze genome-wide spatio-temporal gene expression profiles. We first applied the approach to natural aging process in three species (Rat, Mouse and Drosophila) and then to anti-aging process in Mouse. The results demonstrated that temporal gene expression alteration in different tissues experiences a progressive association evolution from spatial synchrony to asynchrony and stochasticity with age. This implies that tissue coordination gradually declines with age. Male mice showed earlier spatial asynchrony in gene expression than females, suggesting that male animals are more prone to aging than females. The confirmed anti-aging interventions (resveratrol and caloric restriction) enhanced tissue coordination, indicating their underlying anti-aging mechanism on multiple tissue levels. Further, functional analysis suggested asynchronous DNA/protein damage accumulation as well as asynchronous repair, modification and degradation of DNA/protein in tissues possibly contributes to asynchronous and stochastic changes of tissue microenvironment. This increased risk for a variety of age-related diseases such as neurodegeneration and cancer that eventually accelerate organismal aging and death. Our study suggests a novel molecular event occurring in aging process of multicellular species that may represent an intrinsic molecular mechanism of aging

Extended Longevity of Reproductives Appears to be Common in Fukomys Mole-Rats (Rodentia, Bathyergidae)

African mole-rats (Bathyergidae, Rodentia) contain several social, cooperatively breeding species with low extrinsic mortality and unusually high longevity. All social bathyergids live in multigenerational families where reproduction is skewed towards a few breeding individuals. Most of their offspring remain as reproductively inactive “helpers” in their natal families, often for several years. This “reproductive subdivision” of mole-rat societies might be of interest for ageing research, as in at least one social bathyergid (Ansell's mole-rats Fukomys anselli), breeders have been shown to age significantly slower than non-breeders. These animals thus provide excellent conditions for studying the epigenetics of senescence by comparing divergent longevities within the same genotypes without the inescapable short-comings of inter-species comparisons. It has been claimed that many if not all social mole-rat species may have evolved similar ageing patterns, too. However, this remains unclear on account of the scarcity of reliable datasets on the subject. We therefore analyzed a 20-year breeding record of Giant mole-rats Fukomys mechowii, another social bathyergid species. We found that breeders indeed lived significantly longer than helpers (ca. 1.5–2.2fold depending on the sex), irrespective of social rank or other potentially confounding factors. Considering the phylogenetic positions of F. mechowii and F. anselli and unpublished data on a third Fukomys-species (F. damarensis) showing essentially the same pattern, it seems probable that the reversal of the classic trade-off between somatic maintenance and sexual reproduction is characteristic of the whole genus and hence of the vast majority of social mole-rats

Effects of Pre-Natal Vitamin D Supplementation with Partial Correction of Vitamin D Deficiency on Early Life Healthcare Utilisation: A Randomised Controlled Trial

Funded by Asthma UK grant number 09/ 36

Distinct Transcriptome Expression of the Temporal Cortex of the Primate Microcebus murinus during Brain Aging versus Alzheimer's Disease-Like Pathology

Aging is the primary risk factor of neurodegenerative disorders such as Alzheimer's disease (AD). However, the molecular events occurring during brain aging are extremely complex and still largely unknown. For a better understanding of these age-associated modifications, animal models as close as possible to humans are needed. We thus analyzed the transcriptome of the temporal cortex of the primate Microcebus murinus using human oligonucleotide microarrays (Affymetrix). Gene expression profiles were assessed in the temporal cortex of 6 young adults, 10 healthy old animals and 2 old, “AD-like” animals that presented ß-amyloid plaques and cortical atrophy, which are pathognomonic signs of AD in humans. Gene expression data of the 14,911 genes that were detected in at least 3 samples were analyzed. By SAM (significance analysis of microarrays), we identified 47 genes that discriminated young from healthy old and “AD-like” animals. These findings were confirmed by principal component analysis (PCA). ANOVA of the expression data from the three groups identified 695 genes (including the 47 genes previously identified by SAM and PCA) with significant changes of expression in old and “AD-like” in comparison to young animals. About one third of these genes showed similar changes of expression in healthy aging and in “AD-like” animals, whereas more than two thirds showed opposite changes in these two groups in comparison to young animals. Hierarchical clustering analysis of the 695 markers indicated that each group had distinct expression profiles which characterized each group, especially the “AD-like” group. Functional categorization showed that most of the genes that were up-regulated in healthy old animals and down-regulated in “AD-like” animals belonged to metabolic pathways, particularly protein synthesis. These data suggest the existence of compensatory mechanisms during physiological brain aging that disappear in “AD-like” animals. These results open the way to new exploration of physiological and “AD-like” aging in primates

Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

Background The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals. Results Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish. Conclusions The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration

Association between a variation in the phosphodiesterase 4D gene and bone mineral density

BACKGROUND: Fragility fractures caused by osteoporosis are a major cause of morbidity and mortality in aging populations. Bone mineral density (BMD) is a useful surrogate marker for risk of fracture and is a highly heritable trait. The genetic variants underlying this genetic contribution are largely unknown. METHODS: We performed a large-scale association study investigating more than 25,000 single nucleotide polymorphisms (SNPs) located within 16,000 genes. Allele frequencies were estimated in contrasting DNA pools from white females selected for low (<0.87 g/cm(2), n = 319) and high (> 1.11 g/cm(2), n = 321) BMD at the lumbar spine. Significant findings were verified in two additional sample collections. RESULTS: Based on allele frequency differences between DNA pools and subsequent individual genotyping, one of the candidate loci indicated was the phosphodiesterase 4D (PDE4D) gene region on chromosome 5q12. We subsequently tested the marker SNP, rs1498608, in a second sample of 138 white females with low (<0.91 g/cm(2)) and 138 females with high (>1.04 g/cm(2)) lumbar spine BMD. Odds ratios were 1.5 (P = 0.035) in the original sample and 2.1 (P = 0.018) in the replication sample. Association fine mapping with 80 SNPs located within 50 kilobases of the marker SNP identified a 20 kilobase region of association containing exon 6 of PDE4D. In a second, family-based replication sample with a preponderance of females with low BMD, rs1498608 showed an opposite relationship with BMD at different sites (p = 0.00044-0.09). We also replicated the previously reported association of the Ser37Ala polymorphism in BMP2, known to interact biologically with PDE4D, with BMD. CONCLUSION: This study indicates that variants in the gene encoding PDE4D account for some of the genetic contribution to bone mineral density variation in humans. The contrasting results from different samples indicate that the effect may be context-dependent. PDE4 inhibitors have been shown to increase bone mass in normal and osteopenic mice, but up until now there have been no reports implicating any member of the PDE4 gene family in human osteoporosis

Prostaglandin E2 Signals Through PTGER2 to Regulate Sclerostin Expression

The Wnt signaling pathway is a robust regulator of skeletal homeostasis. Gain-of-function mutations promote high bone mass, whereas loss of Lrp5 or Lrp6 co-receptors decrease bone mass. Similarly, mutations in antagonists of Wnt signaling influence skeletal integrity, in an inverse relation to Lrp receptor mutations. Loss of the Wnt antagonist Sclerostin (Sost) produces the generalized skeletal hyperostotic condition of sclerosteosis, which is characterized by increased bone mass and density due to hyperactive osteoblast function. Here we demonstrate that prostaglandin E2 (PGE2), a paracrine factor with pleiotropic effects on osteoblasts and osteoclasts, decreases Sclerostin expression in osteoblastic UMR106.01 cells. Decreased Sost expression correlates with increased expression of Wnt/TCF target genes Axin2 and Tcf3. We also show that the suppressive effect of PGE2 is mediated through a cyclic AMP/PKA pathway. Furthermore, selective agonists for the PGE2 receptor EP2 mimic the effect of PGE2 upon Sost, and siRNA reduction in Ptger2 prevents PGE2-induced Sost repression. These results indicate a functional relationship between prostaglandins and the Wnt/β-catenin signaling pathway in bone