127 research outputs found
Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.
DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.This study was supported by funds obtained from The Evelyn Trust, Crohn’s in Childhood Research Association (CICRA) and Crohn’s and Colitis in Childhood (3Cs) charity. J.K. was funded by a PhD studentship from CICRA. Funding for E.C. was provided by the Deutsche Forschungsgemeinschaft (Grant CA226/4-3) and Interne Forschungsförderung Essen (IFORES).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/mi.2015.8
Air–sea interaction and formation of the Asian summer monsoon onset vortex over the Bay of Bengal
Lactation Defect in a Widely Used MMTV-Cre Transgenic Line of Mice
MMTV-Cre mouse lines have played important roles in our understanding about the functions of numerous genes in mouse mammary epithelial cells during mammary gland development and tumorigenesis. However, numerous studies have not included MMTV-Cre mice as controls, and many investigators have not indicated which of the different MMTV-Cre founder lines were used in their studies. Here, we describe a lactation defect that severely limits the use of one of the most commonly used MMTV-Cre founder lines.To explore the role of protein tyrosine phosphatase Shp1 in mammary gland development, mice bearing the floxed Shp1 gene were crossed with MMTV-Cre mice and mammary gland development was examined by histological and biochemical techniques, while lactation competency was assessed by monitoring pup growth. Surprisingly, both the Shp1fl/+;MMTV-Cre and MMTV-Cre female mice displayed a severe lactation defect when compared to the Shp1 fl/+ control mice. Histological and biochemical analyses reveal that female mice expressing the MMTV-Cre transgene, either alone or in combination with floxed genes, exhibit defects in lobuloalveolar expansion, presence of large cytoplasmic lipid droplets in luminal alveolar epithelial cells postpartum, and precocious induction of involution. Using a PCR-based genotyping method, the three different founder lines can be distinguished, and we determined that the MMTV-Cre line A, the most widely used MMTV-Cre founder line, exhibits a profound lactation defect that limits its use in studies on mammary gland development.The identification of a lactation defect in the MMTV-Cre line A mice indicates that investigators must use MMTV-Cre alone mice as control in studies that utilize Cre recombinase to excise genes of interest from mammary epithelial cells. Our results also suggest that previous results obtained in studies using the MMTV-Cre line A line should be re-evaluated if the controls did not include mice expressing only Cre recombinase
Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination
Background Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l β-glycerophosphate, 8 mmol/l CaCl2, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/ml ascorbic acid, and 100 nmol/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. Results Calcium deposition was stimulated dose-dependently by β-glycerophosphate, CaCl2, and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01–1 μmol/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2–50 μmol/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 μmol/l) induced considerable apoptosis of VSMCs. Conclusion In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations ≥10 μmol/l, which could contribute to the plaque-stabilizing effect reported for statins
Comparative tissue transcriptomics reveal prompt inter-organ communication in response to local bacterial kidney infection
<p>Abstract</p> <p>Background</p> <p>Mucosal infections elicit inflammatory responses via regulated signaling pathways. Infection outcome depends strongly on early events occurring immediately when bacteria start interacting with cells in the mucosal membrane. Hitherto reported transcription profiles on host-pathogen interactions are strongly biased towards <it>in vitro </it>studies. To detail the local <it>in vivo </it>genetic response to infection, we here profiled host gene expression in a recent experimental model that assures high spatial and temporal control of uropathogenic <it>Escherichia coli </it>(UPEC) infection within the kidney of a live rat.</p> <p>Results</p> <p>Transcriptional profiling of tissue biopsies from UPEC-infected kidney tissue revealed 59 differentially expressed genes 8 h post-infection. Their relevance for the infection process was supported by a Gene Ontology (GO) analysis. Early differential expression at 3 h and 5 h post-infection was of low statistical significance, which correlated to the low degree of infection. Comparative transcriptomics analysis of the 8 h data set and online available studies of early local infection and inflammation defined a core of 80 genes constituting a "General tissue response to early local bacterial infections". Among these, 25% were annotated as interferon-γ (IFN-γ) regulated. Subsequent experimental analyses confirmed a systemic increase of IFN-γ in rats with an ongoing local kidney infection, correlating to splenic, rather than renal <it>Ifng </it>induction and suggested this inter-organ communication to be mediated by interleukin (IL)-23. The use of comparative transcriptomics allowed expansion of the statistical data handling, whereby relevant data could also be extracted from the 5 h data set. Out of the 31 differentially expressed core genes, some represented specific 5 h responses, illustrating the value of comparative transcriptomics when studying the dynamic nature of gene regulation in response to infections.</p> <p>Conclusion</p> <p>Our hypothesis-free approach identified components of infection-associated multi-cellular tissue responses and demonstrated how a comparative analysis allows retrieval of relevant information from lower-quality data sets. The data further define marked representation of IFN-γ responsive genes and a prompt inter-organ communication as a hallmark of an early local tissue response to infection.</p
An initial loading-dose vitamin D versus placebo after hip fracture surgery: baseline characteristics of a randomized controlled trial (REVITAHIP)
Immunological properties of Oxygen-Transport Proteins: Hemoglobin, Hemocyanin and Hemerythrin
The Opportunistic Pathogen Propionibacterium acnes: Insights into Typing, Human Disease, Clonal Diversification and CAMP Factor Evolution
We previously described a Multilocus Sequence Typing (MLST) scheme based on eight genes that facilitates population
genetic and evolutionary analysis of P. acnes. While MLST is a portable method for unambiguous typing of bacteria, it is
expensive and labour intensive. Against this background, we now describe a refined version of this scheme based on two
housekeeping (aroE; guaA) and two putative virulence (tly; camp2) genes (MLST4) that correctly predicted the phylogroup
(IA1, IA2, IB, IC, II, III), clonal complex (CC) and sequence type (ST) (novel or described) status for 91% isolates (n = 372) via
cross-referencing of the four gene allelic profiles to the full eight gene versions available in the MLST database (http://
pubmlst.org/pacnes/). Even in the small number of cases where specific STs were not completely resolved, the MLST4
method still correctly determined phylogroup and CC membership. Examination of nucleotide changes within all the MLST
loci provides evidence that point mutations generate new alleles approximately 1.5 times as frequently as recombination;
although the latter still plays an important role in the bacterium’s evolution. The secreted/cell-associated ‘virulence’ factors
tly and camp2 show no clear evidence of episodic or pervasive positive selection and have diversified at a rate similar to
housekeeping loci. The co-evolution of these genes with the core genome might also indicate a role in commensal/normal
existence constraining their diversity and preventing their loss from the P. acnes population. The possibility that members of
the expanded CAMP factor protein family, including camp2, may have been lost from other propionibacteria, but not P.
acnes, would further argue for a possible role in niche/host adaption leading to their retention within the genome. These
evolutionary insights may prove important for discussions surrounding camp2 as an immunotherapy target for acne, and
the effect such treatments may have on commensal lineages
Frequency and typing of Propionibacterium acnes in prostate tissue obtained from men with and without prostate cancer
Control of Propionibacterium acnes by natural antimicrobial substances: Role of the bacteriocin AS-48 and lysozyme
We report the high susceptibility of several clinical isolates of Propionibacterium acnes from different
sources (skin, bone, wound exudates, abscess or blood contamination) to the head-to-tail cyclized
bacteriocin AS-48. This peptide is a feasible candidate for further pharmacological development against
this bacterium, due to its physicochemical and biological characteristics, even when it is growing in a
biofilm. Thus, the treatment of pre-formed biofilms with AS-48 resulted in a dose- and time-dependent
disruption of the biofilm architecture beside the decrease of bacterial viability. Furthermore, we
demonstrated the potential of lysozyme to bolster the inhibitory activity of AS-48 against P. acnes,
rendering high reductions in the MIC values, even in matrix-growing cultures, according to the results
obtained using a range of microscopy and bioassay techniques. The improvement of the activity of
AS-48 through its co-formulation with lysozyme may be considered an alternative in the control of P.
acnes, especially after proving the absence of cytotoxicity demonstrated by these natural compounds
on relevant human skin cell lines. In summary, this study supports that compositions comprising the
bacteriocin AS-48 plus lysozyme must be considered as promising candidates for topical applications
with medical and pharmaceutical purposes against dermatological diseases such as acne vulgaris.This research was funded by a grant
from the Spanish Ministry of Economy and Competitiveness (SAF2013-48971-C2-1-R that included funds from
European Regional Development, ERDF), and the Research Group General (BIO160, UGR)
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