Detection of methicillin-resistant <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4–6 bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1 pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5 ± 1.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohen's kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.     

Histone deacetylase is required for the activation of Wnt/β-catenin signaling crucial for heart valve formation in zebrafish embryos

During vertebrate heart valve formation, Wnt/β-catenin signaling induces BMP signals in atrioventricular canal (AVC) myocardial cells and underlying AVC endocardial cells then undergo endothelial-mesenchymal transdifferentiation (EMT) by receiving this BMP signals. Histone deacetylases (HDACs) have been implicated in numerous developmental processes by regulating gene expression. However, their specific roles in controlling heart valve development are largely unexplored. To investigate the role of HDACs in vertebrate heart valve formation, we treated zebrafish embryos with trichostatin A (TSA), an inhibitor of class I and II HDACs, from 36 to 48 h post-fertilization (hpf) during which heart looping and valve formation occur. Following TSA treatment, abnormal linear heart tube development was observed. In these embryos, expression of AVC myocardial bmp4 and AVC endocardial notch1b genes was markedly reduced with subsequent failure of EMT in the AVC endocardial cells. However, LiCl-mediated activation of Wnt/β-catenin signaling was able to rescue defective heart tube formation, bmp4 and notch1b expression, and EMT in the AVC region. Taken together, our results demonstrated that HDAC activity plays a pivotal role in vertebrate heart tube formation by activating Wnt/β-catenin signaling which induces bmp4 expression in AVC myocardial cells.     

Transmission of Toxocara canis via ingestion of raw cow liver: a cross-sectional study in healthy adults

The aim of this study is to ascertain the relationship between ingestion of raw cow liver and Toxocara canis infection. A total of 150 apparently healthy adults were divided into 2 groups; 1 group consisted of 86 adults with positive results of Toxocara ELISA, and the other group of 64 adults with negative results. One researcher collected the history of ingestion of raw cow liver within 1 year and recent history of keeping dogs. Among 86 seropositive adults for T. canis, 68 (79.1%) had a recent history of ingestion of raw cow liver. Multivariate statistical analysis showed that a recent ingestion of raw cow liver and keeping dogs were related to an increased risk of toxocariasis (odds ratios, 4.4 and 3.7; and 95% confidence intervals, 1.9-10.2 and 1.2-11.6, respectively). A recent history of ingestion of raw cow liver and keeping dogs was significantly associated with toxocariasis.     

Mesenchymal stem cell-conditioned media recovers lung fibroblasts from cigarette smoke-induced damage

Cigarette smoking causes apoptotic death, senescence, and impairment of repair functions in lung fibroblasts, which maintain the integrity of alveolar structure by producing extracellular matrix (ECM) proteins. Therefore, recovery of lung fibroblasts from cigarette smoke-induced damage may be crucial in regeneration of emphysematous lung resulting from degradation of ECM proteins and subsequent loss of alveolar cells. Recently, we reported that bone marrow-derived mesenchymal stem cell-conditioned media (MSC-CM) led to angiogenesis and regeneration of lung damaged by cigarette smoke. In this study, to further investigate reparative mechanisms for MSC-CM-mediated lung repair, we attempted to determine whether MSC-CM can recover lung fibroblasts from cigarette smoke-induced damage. In lung fibroblasts exposed to cigarette smoke extract (CSE), MSC-CM, not only inhibited apoptotic death, but also induced cell proliferation and reversed CSE-induced changes in the levels of caspase-3, p53, p21, p27, Akt, and p-Akt. MSC-CM also restored expression of ECM proteins and collagen gel contraction while suppressing CSE-induced expression of cyclooxygenase-2 and microsomal PGE(2) synthase-2. The CSE-opposing effects of MSC-CM on cell fate, expression of ECM proteins, and collagen gel contraction were partially inhibited by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. In rats, MSC-CM administration also resulted in elevation of p-Akt and restored proliferation of lung fibroblasts, which was suppressed by exposure to cigarette smoke. Taken together, these data suggest that MSC-CM may recover lung fibroblasts from cigarette smoke-induced damage, possibly through inhibition of apoptosis, induction of proliferation, and restoration of lung fibroblast repair function, which are mediated in part by the PI3K/Akt pathway.     

Viral perturbations of host networks reflect disease etiology

Many human diseases, arising from mutations of disease susceptibility genes (genetic diseases), are also associated with viral infections (virally implicated diseases), either in a directly causal manner or by indirect associations. Here we examine whether viral perturbations of host interactome may underlie such virally implicated disease relationships. Using as models two different human viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), we find that host targets of viral proteins reside in network proximity to products of disease susceptibility genes. Expression changes in virally implicated disease tissues and comorbidity patterns cluster significantly in the network vicinity of viral targets. The topological proximity found between cellular targets of viral proteins and disease genes was exploited to uncover a novel pathway linking HPV to Fanconi anemia.     

8-Hydroxy-2-deoxyguanosine prevents plaque formation and inhibits vascular smooth muscle cell activation through Rac1 inactivation

8-Hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress, has been recently rediscovered to inhibit Rac1 in neutrophils and macrophages, thereby inhibiting Rac1-linked functions of these cells, including reactive oxygen species production through NADPH oxidase activation, phagocytosis, chemotaxis, and cytokine release. In vascular smooth muscle cells (VSMCs), reactive oxygen species also induce abnormal proliferation and migration leading to progression of atherosclerosis. Based upon the involvement of reactive oxygen species in phagocytic cells and VSMCs during the atherosclerotic process, we hypothesized that 8-OHdG could have antiatherosclerotic action and tested this hypothesis in an experimentally induced atherosclerosis in mice. Partially ligated ApoE knockout mice, a more physiologically relevant model of low and oscillatory flow, developed an advanced lesion in 2 weeks, and orally administered 8-OHdG significantly reduced plaque formation along with reduced superoxide formation, monocyte/macrophage infiltration, and extracellular matrix (ECM) accumulation. The effects of 8-OHdG observed in primary VSMCs were consistent with the in vivo effects of 8-OHdG and were inhibitory to angiotensin II or platelet-derived growth factor-induced production of reactive oxygen species, proliferation, migration, and ECM production. Also, angiotensin II-induced Rac1 activity in VSMCs was significantly inhibited by 8-OHdG, and transfection of constitutively active Rac1 reversed the inhibitory effect of 8-OHdG on VSMC activation. Molecular docking study showed that 8-OHdG stabilizes Rac1-GEF complex, indicating the physical contact of 8-OHdG with Rac1. These findings highly suggest that the antiatherosclerotic effect of 8-OHdG is mediated by inhibition of Rac1 activity. In conclusion, our results show a novel action of orally active 8-OHdG in suppressing atherosclerotic plaque formation in vivo and VSMC activation in vitro through inhibition of Rac1, which emphasizes a new therapeutic avenue to benefit atherosclerosis.     

Ectodysplasin regulates activator-inhibitor balance in murine tooth development through Fgf20 signaling

Uncovering the origin and nature of phenotypic variation within species is the first step in understanding variation between species. Mouse models with altered activities of crucial signal pathways have highlighted many important genes and signal networks regulating the morphogenesis of complex structures, such as teeth. The detailed analyses of these models have indicated that the balanced actions of a few pathways regulating cell behavior modulate the shape and number of teeth. Currently, however, most mouse models studied have had gross alteration of morphology, whereas analyses of more subtle modification of morphology are required to link developmental studies to evolutionary change. Here, we have analyzed a signaling network involving ectodysplasin (Eda) and fibroblast growth factor 20 (Fgf20) that subtly affects tooth morphogenesis. We found that Fgf20 is a major downstream effector of Eda and affects Eda-regulated characteristics of tooth morphogenesis, including the number, size and shape of teeth. Fgf20 function is compensated for by other Fgfs, in particular Fgf9 and Fgf4, and is part of an Fgf signaling loop between epithelium and mesenchyme. We showed that removal of Fgf20 in an Eda gain-of-function mouse model results in an Eda loss-of-function phenotype in terms of reduced tooth complexity and third molar appearance. However, the extra anterior molar, a structure lost during rodent evolution 50 million years ago, was stabilized in these mice.     

Roles of MMP/TIMP in regulating matrix swelling and cell migration during chick corneal development

Tissue remodeling is central to embryonic development. Here, we used immunohistochemistry, Western blotting, and RT-PCR analysis to investigate the roles of matrix metalloproteinases (MMPs) and the related "a disintegrin and metalloproteinase" (ADAM) family proteinases in chick corneal development. While MMP-13 was expressed in developing chick corneas from embryonic day (ED) 5 to ED 10, its inhibitor, tissue inhibitors of metalloproteinase-1 (TIMP-1), was expressed from ED 18 to 2 days post-hatching (P2). Early MMP-13 activity may be associated with degradation of type IX collagen from the primary stroma, which loosens the collagen fibrils and facilitates neural crest (NC) cell migration. The membrane-bound and secreted forms of ADAM10 were both detected throughout corneal development, and active ADAM10 formed a cleavage complex with CD44v6, a CD44 splice variant that is a major cell surface adhesion molecule for hyaluronic acid (HA) and has been implicated in cell migration. Both CD44v6 and its ectodomain cleavage products were detected from ED 5 to ED 14, and a broad-spectrum MMP inhibitor blocked ectodomain cleavage in cultured stromal cells. These findings suggest that ADAM10 mediates CD44v6 cleavage in the developing cornea, facilitating NC cell-derived mesenchymal cell migration. Finally, we identified high levels of active membrane-type 3-MMP (MT3-MMP) in developing corneas at ED 7, ED 14, and ED 18. MT3-MMP takes part in MMP-2 activation and possibly also CD44v6 shedding, suggesting that this pathway may be involved in cell migration. These findings collectively show for the first time that multiple MMPs, ADAMs, and TIMPs appear to functionally interact during corneal development.