Functional Implications of DNA Methylation in Adipose Biology.

The twin epidemics of obesity and type 2 diabetes (T2D) are a serious health, social, and economic issue. The dysregulation of adipose tissue biology is central to the development of these two metabolic disorders, as adipose tissue plays a pivotal role in regulating whole-body metabolism and energy homeostasis (1). Accumulating evidence indicates that multiple aspects of adipose biology are regulated, in part, by epigenetic mechanisms. The precise and comprehensive understanding of the epigenetic control of adipose tissue biology is crucial to identifying novel therapeutic interventions that target epigenetic issues. Here, we review the recent findings on DNA methylation events and machinery in regulating the developmental processes and metabolic function of adipocytes. We highlight the following points: 1) DNA methylation is a key epigenetic regulator of adipose development and gene regulation, 2) emerging evidence suggests that DNA methylation is involved in the transgenerational passage of obesity and other metabolic disorders, 3) DNA methylation is involved in regulating the altered transcriptional landscape of dysfunctional adipose tissue, 4) genome-wide studies reveal specific DNA methylation events that associate with obesity and T2D, and 5) the enzymatic effectors of DNA methylation have physiological functions in adipose development and metabolic function

Signaling pathways in osteogenesis and osteoclastogenesis: Lessons from cranial sutures and applications to regenerative medicine.

One of the simplest models for examining the interplay between bone formation and resorption is the junction between the cranial bones. Although only roughly a quarter of patients diagnosed with craniosynostosis have been linked to known genetic disturbances, the molecular mechanisms elucidated from these studies have provided basic knowledge of bone homeostasis. This work has translated to methods and advances in bone tissue engineering. In this review, we examine the current knowledge of cranial suture biology derived from human craniosynostosis syndromes and discuss its application to regenerative medicine

The Direct Effect of Low-Magnitude High-Frequency Mechanical Vibration on Osteoclast Formation from RAW267.4 Monocytes

Low-magnitude high-frequency (LMHF) mechanical vibration has been demonstrated to enhance bone formation possibly through inhibition of osteoclastogenesis of bone. Earlier research has demonstrated osteoclast formation from RAW264.7 monocytes was inhibited by a chewing cycle mimicking vibration through inhibition of dendritic cell-specific transmembrane protein (DC-STAMP). We hypothesize that application of LMHF mechanical vibration directly inhibits osteoclast formation from RAW264.7 monocytes in a frequency dependent manner. RAW264.7 monocytes (ATCC) were cultured in alpha minimal essential medium (MEM) with 10% fetal bovine serum (FBS ) and 1% Pen/Strep at 37°C and 5% CO2. The cells were seeded at a density of 2000 cells/well in 96-well cell culture plates. After allowing growth overnight, the cells were treated with 20ng/ml receptor activator nuclear factor kappa-B ligand (RANKL) and refreshed every 2 days to induce osteoclast formation. In the meantime, the cells were subjected to a low-magnitude (0.3g acceleration) mechanical vibration at various frequencies (0, 30, 60 and 90 Hz) respectively. For each frequency group, the vibration was applied for 1 hour per day for 5 consecutive days. By the end of the 5th day, the cells were rinsed with 1X PBS and fixed in 4% formaldehyde for 5 minutes. Tartrate-resistant Acidic Phosphatase (TRAP, a marker enzyme of osteoclast) staining was performed. The TRAP+ multi nuclei (\u3e = 3) cells were counted and calculated. For statistical analysis, one-way ANOVA was used to test differences among the different frequency groups with Tukey post hoc comparison was used to compare between the groups, with p value being set at 0.05. Three days after RANKL stimulation, osteoclasts started to form from RAW264.7 monocytes, with a peak observed on the 5th day. After 5 days, the cells underwent apoptosis and death. Compared to the control group (0 Hz), the 30 Hz but not 60 Hz and 90 Hz frequencies of vibration group showed significant reduction of osteoclast formation by approximately 21% (p \u3c 0.05, n = 6). No significant difference was found among the three frequency groups. Low-magnitude high-frequency mechanical vibration directly inhibits osteoclast formation from RAW264.7 monocytes, and is frequency dependent

CRISPR-TSKO : a technique for efficient mutagenesis in specific cell types, tissues, or organs in Arabidopsis

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function

The regulation of differentiation in mesenchymal stem cells

Peer reviewedPublisher PD

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis.

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5' end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development

Shuffling of mobile genetic elements (MGEs) in successful healthcare-associated MRSA (HA-MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) CC22 SCCmecIV is a successful hospital-associated (HA-) MRSA, widespread throughout the world, and now the dominant clone in UK hospitals. We have recently shown that MRSA CC22 is a particularly fit clone, and it rose to dominance in a UK hospital at the same time as it began acquiring an increased range of antibiotic resistances. These resistances were not accumulated by individual CC22 isolates, but appear to shuffle frequently between isolates of the MRSA CC22 population. Resistances are often encoded on mobile genetic elements (MGEs) that include plasmids, transposons, bacteriophage and S. aureus pathogenicity islands (SaPIs). Using multi-strain whole genome microarrays, we show that there is enormous diversity of MGE carried within a MRSA CC22 SCCmecIV population, even among isolates from the same hospital and time period. MGE profiles were so variable that they could be used to track the spread of variant isolates within the hospital. We exploited this to show that the majority of patients colonised with MRSA at hospital admission that subsequently became infected were infected with their own colonising isolate. Our studies reveal MGE spread, stability, selection and clonal adaptation to the healthcare setting may be key to the success of HA-MRSA clones, presumably by allowing rapid adaptation to antibiotic exposure and new hosts