Plastid genomes reveal support for deep phylogenetic relationships and extensive rate variation among palms and other commelinid monocots.

Despite progress based on multilocus, phylogenetic studies of the palms (order Arecales, family Arecaceae), uncertainty remains in resolution/support among major clades and for the placement of the palms among the commelinid monocots. Palms and related commelinids represent a classic case of substitution rate heterogeneity that has not been investigated in the genomic era. To address questions of relationships, support and rate variation among palms and commelinid relatives, 39 plastomes representing the palms and related family Dasypogonaceae were generated via genome skimming and integrated within a monocot-wide matrix for phylogenetic and molecular evolutionary analyses. Support was strong for 'deep' relationships among the commelinid orders, among the five palm subfamilies, and among tribes of the subfamily Coryphoideae. Additionally, there was extreme heterogeneity in the plastid substitution rates across the commelinid orders indicated by model based analyses, with c. 22 rate shifts, and significant departure from a global clock. To date, this study represents the most comprehensively sampled matrix of plastomes assembled for monocot angiosperms, providing genome-scale support for phylogenetic relationships of monocot angiosperms, and lays the phylogenetic groundwork for comparative analyses of the drivers and correlates of such drastic differences in substitution rates across a diverse and significant clade.

Investigating the path of plastid genome degradation in an early-transitional clade of heterotrophic orchids, and implications for heterotrophic angiosperms.

Parasitic organisms exemplify morphological and genomic reduction. Some heterotrophic, parasitic plants harbor drastically reduced and degraded plastid genomes resulting from relaxed selective pressure on photosynthetic function. However, few studies have addressed the initial stages of plastome degradation in groups containing both photosynthetic and nonphotosynthetic species. Corallorhiza is a genus of leafless, heterotrophic orchids that contains both green, photosynthetic species and nongreen, putatively nonphotosynthetic species, and represents an ideal system in which to assess the beginning of the transition to a "minimal plastome." Complete plastomes were generated for nine taxa of Corallorhiza using Illumina paired-end sequencing of genomic DNA to assess the degree of degradation among taxa, and for comparison with a general model of degradation among angiosperms. Quantification of total chlorophyll suggests that nongreen Corallorhiza still produce chlorophyll, but at 10-fold lower concentrations than green congeners. Complete plastomes and partial nuclear rDNA cistrons yielded a fully resolved tree for Corallorhiza, with at least two independent losses of photosynthesis, evidenced by gene deletions and pseudogenes in Co. striata and nongreen Co. maculata. All Corallorhiza show some evidence of degradation in genes of the NAD(P)H dehydrogenase complex. Among genes with open reading frames, photosynthesis-related genes displayed evidence of neutral evolution in nongreen Corallorhiza, whereas genes of the ATP synthase complex displayed some evidence of positive selection in these same groups, though for reasons unknown. Corallorhiza spans the early stages of a general model of plastome degradation and has added critical insight for understanding the process of plastome evolution in heterotrophic angiosperms.

Biomarkers associated with periimplant diseases.

PURPOSE: Recently, implantology has shifted its focus from implant placement to periimplant disease early detection, prevention, and treatment. The purpose of this article was to review the current understanding of the biomarkers associated with periimplant diseases. MATERIALS AND METHODS: A search of PubMed was conducted up to August 2013 with keywords "peri-implantitis" and "biomarkers." Selected articles addressed the relationship between biomarkers and periimplant mucositis or peri-implantitis. RESULTS: Biomarkers have been shown to possess potential in detecting periimplant diseases. For example, interleukin (IL)-1ß levels were shown to be a good marker to detect periimplant mucositis lesions before they progress to peri-implantitis. Matrix metalloproteinase (MMP)-8 levels in periimplant sulcus fluid may be useful for monitoring the progression of periimplant disease. Osteoprotegerin (OPG) and receptor activator of NFκB ligand (RANKL) were found to be significantly higher in peri-implantitis sites compared with healthy implant sites. CONCLUSION: Biomarkers such as IL-1ß, MMP-8, OPG, RANKL, and others have shown promising outcomes in differentiating from periimplant disease to health. However, because of varying results, additional evidence is needed to validate the links reported.

Flightless I is a focal adhesion-associated actin-capping protein that regulates cell migration.

The role of adhesion-associated actin-binding proteins in cell migration is not well defined. In mouse fibroblasts we screened for focal adhesion-associated proteins that were isolated with collagen-coated beads and detected by tandem mass spectrometry. We identified flightless I (FliI) as an actin-binding protein in focal adhesion fractions, which was verified by immunoblotting. By confocal microscopy most FliI was distributed throughout the cytosol and in focal adhesions. By sedimentation assays and in vitro binding assays, we found that FliI associates with actin filaments and actin monomers. Assays using purified proteins showed that FliI inhibits actin polymerization and caps but does not sever actin filaments. Cells with FliI knockdown or cells overexpressing FliI migrated more or less rapidly, respectively, than wild-type controls. Compared with controls, cells with FliI knockdown were less adherent than wild-type cells, exhibited reduced numbers of focal adhesions containing activated ß1 integrins and vinculin, and exhibited increased incorporation of actin monomers into nascent filaments at focal adhesions. These data indicate that FliI regulates cell migration through its localization to focal adhesions and its ability to cap actin filaments, which collectively affect focal adhesion maturation.

Tumor necrosis factor-alpha mediates cardiac remodeling and ventricular dysfunction after pressure overload state.

BACKGROUND: Pressure overload is accompanied by cardiac myocyte apoptosis, hypertrophy, and inflammatory/fibrogenic responses that lead to ventricular remodeling and heart failure. Despite incomplete understanding of how this process is regulated, the upregulation of tumor necrosis factor (TNF)-alpha after aortic banding in the myocardium is known. In the present study, we tested our hypothesis that TNF-alpha regulates the cardiac inflammatory response, extracellular matrix homeostasis, and ventricular hypertrophy in response to mechanical overload and contributes to ventricular dysfunction. METHODS AND RESULTS: C57/BL wild-type mice and TNF-knockout (TNF-/-) mice underwent descending aortic banding or sham operation. Compared with sham-operated mice, wild-type mice with aortic banding showed a significant increase in cardiac TNF-alpha levels, which coincided with myocyte apoptosis, inflammatory response, and cardiac hypertrophy in week 2 and a significant elevation in matrix metalloproteinase-9 activity and impaired cardiac function in weeks 2 and 6. Compared with wild-type mice with aortic banding, TNF-/- mice with aortic banding showed attenuated cardiac apoptosis, hypertrophy, inflammatory response, and reparative fibrosis. These mice also showed reduced cardiac matrix metalloproteinase-9 activity and improved cardiac function. CONCLUSIONS: Findings from the present study have suggested that TNF-alpha contributes to adverse left ventricular remodeling during pressure overload through regulation of cardiac repair and remodeling, leading to ventricular dysfunction.

Using molecular beacons for sensitive fluorescence assays of the enzymatic cleavage of nucleic acids.

A novel method for DNA enzymatic cleavage assays using molecular beacons (MBs) as the substrate for nuclease is described. An MB is a hairpin-shaped DNA probe that is labeled with a fluorescent dye at one end and a quencher at the other end. The loop sequence of the MB can be used as the substrate for single-stranded specific nucleases, whereas the stem of the MB can be designed as the substrate for restriction enzymes. The enzymatic cleavage breaks the MB into fragments and leads to the distance separation of the quencher and the fluorophore, resulting in an increase in the fluorescent signal. Up to an 80-fold signal-to-noise ratio was observed when these probes were cleaved by nucleases. Taking advantage of the MB's detection-without-separation property, this method allows for the real-time detection of DNA cleavage, which is useful for the characterization of DNA nuclease activity as well as the study of steady-state cleavage reaction kinetics. With its simplicity, convenience, high sensitivity, and excellent reproducibility, this method has the potential to be used in the study of both natural and artificial nucleic acid-cleaving enzymes.

The Effect of Vertical Implant Position in Relation to Adjacent Teeth on Marginal Bone Loss in Posterior Arches: A Retrospective Study.

PURPOSE: The aim of this retrospective study was to investigate the possible association between peri-implant marginal bone loss and the apicocoronal (vertical) positioning of dental implants placed adjacent to a tooth. MATERIALS AND METHODS: Dental records at the University of Michigan, School of Dentistry, were screened. To be included in the study, the patient had to have at least one implant in the premolar or molar region, unilaterally or bilaterally, in either arch, with an immediately mesial adjacent tooth. The implant had to have been functionally loaded for at least 3 years after crown insertion, and clear, nondistorted periapical films had to be available. Several landmarks were identified: the cementoenamel junction (CEJ) and crestal bone (CB) of the tooth adjacent to the implant, the implant platform (PL), and the first radiographic implant-bone contact (BL). The following parameters were measured: CEJ-PL, CEJ-CB, CB-PL, horizontal distance between the adjacent tooth and PL (HD), and vertical distance between BL and the first implant thread at the mesial (BL-m) and distal (BL-d) implant surfaces. RESULTS: A total of 139 patients with a mean age of 62.1 ± 9.3 years were included. The mean follow-up period was 4.42 ± 2.5 years. When the implant was placed more than 3 mm apical to the CEJ of the adjacent tooth, significantly greater peri-implant bone loss occurred at the mesial (difference of means = 0.57 mm) and distal (difference of means = 0.83 mm) implant surfaces. CONCLUSION: In this study population, implants placed in the posterior area with a vertical distance greater than 3 mm from the CEJ of the adjacent tooth displayed more peri-implant bone loss. Further investigation is required to determine whether this increased peri-implant bone loss predisposes a site to peri-implantitis.

Mouse snail is a target gene for HIF.

The transcriptional inhibitor Snail is a critical regulator for epithelial-mesenchymal transition (EMT). Although low oxygen induces Snail transcription, thereby stimulating EMT, a direct role of hypoxia-inducible factor (HIF) in this process remains to be demonstrated. Here we show that hypoxia induces the expression of Snail via HIF. In silico analysis identified a potential hypoxia-response element (HRE) close to the minimal promoter of the human and mouse genome of the snail gene. Gel shift assays demonstrated that a specific hypoxia-inducible complex is formed with the putative HRE and that the complex contains HIF proteins. ChIP assays confirmed the interaction of HIF proteins with the putative HRE in vivo. Reporter gene analyses showed that the putative HRE responds to hypoxia in its natural position as well as in front of a heterologous promoter and that the HRE is directly activated by HIF-1α or HIF-2α. HIF knockdown with siRNA at 2% oxygen and overexpression of an oxygen-insensitive HIF (HIF-ΔODD) mutant at 21% oxygen showed that HIF regulates Snail activation and subsequent cell migration. Our findings identify snail as a HIF target gene and provide novel insights into the regulation of snail and hypoxia-induced EMT.

Flexible automated approach for quantitative liquid handling of complex biological samples.

A fully automated protein precipitation technique for biological sample preparation has been developed for the quantitation of drugs in various biological matrixes. All liquid handling during sample preparation was automated using a Hamilton MicroLab Star Robotic workstation, which included the preparation of standards and controls from a Watson laboratory information management system generated work list, shaking of 96-well plates, and vacuum application. Processing time is less than 30 s per sample or approximately 45 min per 96-well plate, which is then immediately ready for injection onto an LC-MS/MS system. An overview of the process workflow is discussed, including the software development. Validation data are also provided, including specific liquid class data as well as comparative data of automated vs manual preparation using both quality controls and actual sample data. The efficiencies gained from this automated approach are described.