Marginal Accuracy of Monolithic and Veneered Zirconia Crowns Fabricated by Conventional and Digital Workflows.

PURPOSE: To compare the marginal accuracy of zirconia crowns fabricated by different workflows (conventional and digital) and designs (monolithic and veneered). MATERIALS AND METHODS: A prepared maxillary first molar was used for the study. Four workflow combinations were evaluated: (1) intraoral scanning and monolithic zirconia (IOS-M), (2) intraoral scanning and veneered zirconia (IOS-V), (3) conventional impression and monolithic zirconia (IMP-M), and (4) conventional impression and veneered zirconia (IMP-V). All of the specimens had similar designs. The veneered groups had a buccal cutback for esthetic veneer application. A total of 10 crowns were produced in each workflow. The vertical and horizontal marginal accuracies were measured with a traveling microscope. Depending on the normality of the data, one-way analysis of variance test or Kruskal-Wallis test were applied to evaluate the differences among the groups (α = 0.05). RESULTS: The most superior vertical marginal accuracy was observed for IOS-V (mean = 22.5 µm; SD = 6.7 µm), followed by IMP-V (mean = 23.9 µm; SD = 7.8 µm), IOS-M (mean = 28.7 µm; SD = 10.3 µm), and IMP-M (mean = 39.8 µm; SD = 22.0 µm), respectively (p < 0.001). The IOS-M had the greatest mean horizontal discrepancies (mean = 23.9 µm; SD = 4.3 µm) followed by IMP-M (mean = 21.3 µm; SD = 5.7 µm), IMP-V (mean = 19.2 µm; SD = 5.3 µm) and IOS-V (mean = 17.6 µm; SD = 5.7 µm) (p < 0.001). CONCLUSIONS: Monolithic zirconia crowns fabricated digitally had superior marginal accuracy than monolithic zirconia crowns fabricated conventionally. Esthetic buccal veneering of predominantly monolithic zirconia copings improved the vertical and horizontal marginal accuracies.

Quantitative Features of the Choriocapillaris in Healthy Individuals Using Swept-Source Optical Coherence Tomography Angiography.

BACKGROUND AND OBJECTIVE: To quantify vessel density (VD) and grey value (GV) as a measure of flow in the choriocapillaris (CC) in healthy subjects with optical coherence tomography angiography (OCTA). PATIENTS AND METHODS: In this prospective, noncomparative case series, 3 mm × 3 mm OCTA images of 36 eyes of 22 healthy individuals were obtained using a swept-source instrument. VD and GV levels were calculated on CC en face slabs in the central 1-mm (subfoveal field) and surrounding 2.5-mm parafoveal ring. VD was calculated as a ratio of vessel area over nonvessel area following image binarization. GV was computed as the mean, un-normalized greyscale intensity value for all pixels in the region of interest. For each eye, the procedure was repeated 1 minute to 2 minutes later and intersession repeatability was analyzed. The choroidal thickness (CT) was automatically measured in the subfoveal and parafoveal regions and compared to VD and GV values. RESULTS: The VD ratio and GV was lower in the subfoveal field than in the parafoveal four sectors. The intersession intraclass correlation coefficients were high for both VD and GV measurements. There was no correlation observed between CT and VD or GV. CONCLUSIONS: Quantitative metrics can be obtained from CC OCTA en face images. These values show moderate to good intersession repeatability. These normative data may be of value as a reference of comparison in future studies of eyes with disease. [Ophthalmic Surg Lasers Imaging Retina. 2017;48:623-631.].

Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes.

Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-ß-activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2-mediated ubiquitination and TAB1-TAB2. TAB1-TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption.

Antimicrobial susceptibility of well-characterised multiresistant CTX-M-producing Escherichia coli: failure of automated systems to detect resistance to piperacillin/tazobactam.

This study was designed to determine the in vitro activities of several antimicrobial agents against well-characterised CTX-M-producing Escherichia coli strains isolated from clinical specimens. Minimum inhibitory concentrations (MICs) were determined for 202 extended-spectrum beta-lactamase (ESBL)-producing E. coli using microbroth dilution and Vitek methods according to Clinical and Laboratory Standards Institute criteria. Molecular characterisation was performed using isoelectric focusing and polymerase chain reaction (PCR) with sequencing, whilst strain relatedness was determined by pulsed-field gel electrophoresis (PFGE) using XbaI. Of the 202 ESBL-producing E. coli, 2 produced VEB-1, 12 produced TEM-52, 32 produced SHV types (including SHV-2 and -12) and 156 produced CTX-M types (including CTX-M-2, -3, -14, -15, -24, -27 and -30). The MIC(50) and MIC(90) (MIC for 50% and 90% of the organisms, respectively) for the antimicrobial agents tested were, respectively: piperacillin/tazobactam (TZP), 32mg/L and >256mg/L; ciprofloxacin, 16mg/L and 32mg/L; gentamicin, 8mg/L and 128mg/L; amikacin, 2mg/L and 16mg/L; ertapenem, 0.03mg/L and 0.12mg/L; imipenem, 0.03mg/L and 0.12mg/L; meropenem, 0.03mg/L and 0.03mg/L; and tigecycline 0.12mg/L and 0.5mg/L. Vitek Legacy and Vitek 2 failed to detect TZP resistance in 91 (90%) and 75 (74%) of 101 TZP-resistant ESBL-producing strains, respectively, especially CTX-M-15-producing isolates that co-produced OXA-1. The carbapenems, amikacin and tigecycline had good in vitro activities against multiresistant CTX-M-producing E. coli. We recommend that laboratories using Vitek should employ alternative susceptibility testing methods for TZP before reporting the activity of this agent against ESBL-producing E. coli.

Impaired male fertility and atrophy of seminiferous tubules caused by haploinsufficiency for Foxa3.

Foxa1, 2 and 3 (formerly HNF-3alpha, -beta and -gamma) constitute a sub-family of winged helix transcription factors with multiple roles in mammalian organ development. While all three Foxa mRNAs are present in endoderm derivatives including liver and pancreas, only Foxa3 is expressed in the testis. Here we demonstrate by genetic lineage tracing that Foxa3 is expressed in postmeiotic germ and interstitial Leydig cells. The germinal epithelium of Foxa3-deficient testes is characterized by a loss of germ cells secondary to an increase in germ cell apoptosis that ultimately leads to a Sertoli cell-only syndrome. Remarkably, not only the Foxa3(-/-) mice but also Foxa3(+/-) mice exhibited loss of germ cells. This cellular phenotype caused significantly reduced fertility and testis weight of both Foxa3(-/-) and Foxa3(+/-) mice. Using microarray analysis, we found a dramatic downregulation of the zinc finger protein 93 and the testicular tumor-associated paraneoplastic Ma antigen (PNMA) and increased expression of a number of genes including zinc finger protein 94 and several kallikrein 1-related peptidases which could account for at least part of the observed phenotype. In summary, we have identified Foxa3 as a transcriptional regulator with a dominant phenotype in germ cell maintenance and suggest FOXA3 as a potential candidate gene for subfertility in man.

Transcriptional networks in the liver: hepatocyte nuclear factor 6 function is largely independent of Foxa2.

A complex network of hepatocyte nuclear transcription factors, including HNF6 and Foxa2, regulates the expression of liver-specific genes. The current model, based on in vitro studies, suggests that HNF6 and Foxa2 interact physically. This interaction is thought to synergistically stimulate Foxa2-dependent transcription through the recruitment of p300/CBP by HNF6 and to inhibit HNF6-mediated transcription due to the interference of Foxa2 with DNA binding by HNF6. To test this model in vivo, we utilized hepatocyte-specific gene ablation to study the binding of HNF6 to its targets in the absence of Foxa2. Chromatin immunoprecipitation using anti-HNF6 antibodies was performed on chromatin isolated from Foxa2(loxP/loxP) Alfp.Cre and control mouse livers, and HNF6 binding to its target, Glut2, was determined by quantitative PCR. In contrast to the current model, we found no significant difference in HNF6 occupancy at the Glut2 promoter between Foxa2-deficient and control livers. In order to evaluate the Foxa2/HNF6 interaction model on a global scale, we performed a location analysis using a microarray with 7,000 mouse promoter fragments. Again, we found no evidence that HNF6 binding to its targets in chromatin is reduced in the presence of Foxa2. We also examined the mRNA levels of HNF6 targets in the liver using a cDNA array and found that their expression was similar in Foxa2-deficient and control mice. Overall, our studies demonstrate that HNF6 binds to and regulates its target promoters in vivo in the presence and absence of Foxa2.

Glucocorticoid receptor-dependent gene regulatory networks.

While the molecular mechanisms of glucocorticoid regulation of transcription have been studied in detail, the global networks regulated by the glucocorticoid receptor (GR) remain unknown. To address this question, we performed an orthogonal analysis to identify direct targets of the GR. First, we analyzed the expression profile of mouse livers in the presence or absence of exogenous glucocorticoid, resulting in over 1,300 differentially expressed genes. We then executed genome-wide location analysis on chromatin from the same livers, identifying more than 300 promoters that are bound by the GR. Intersecting the two lists yielded 53 genes whose expression is functionally dependent upon the ligand-bound GR. Further network and sequence analysis of the functional targets enabled us to suggest interactions between the GR and other transcription factors at specific target genes. Together, our results further our understanding of the GR and its targets, and provide the basis for more targeted glucocorticoid therapies.

Detection of Pseudomonas aeruginosa producing metallo-beta-lactamases in a large centralized laboratory.

Metallo-beta-lactamases (MBLs) have been increasingly recognized from clinical isolates worldwide, but the laboratory detection of these strains is not well defined. We report a study that developed an EDTA disk screen test and a molecular diagnostic assay for the detection of MBL-producing Pseudomonas aeruginosa. Using NCCLS disk methodology, inhibition zone diameters were determined in tests with imipenem (IPM) and meropenem (MEM) disks alone and in combination with 930 microg of EDTA. This test was compared with the MBL Etest. The duplex PCR assay showed 100% sensitivity and specificity for detecting MBL-producing control strains. Of the 241 clinical strains of IPM-nonsusceptible P. aeruginosa from the Calgary Health Region isolated from 2002 to 2004, 110/241 (46%) were MBL positive using phenotypic methods while 107/241 (45%) were PCR positive for MBL genes: 103/241 (43%) for bla(VIM) and 4/241 (2%) for bla(IMP). The EDTA disk screen test using MEM showed 100% sensitivity and 97% specificity for detecting MBLs in control and clinical strains. The EDTA disk screen test is simple to perform and to interpret and can easily be introduced into the workflow of a clinical laboratory. We recommend that all IPM-nonsusceptible P. aeruginosa isolates be routinely screened for MBL production using the EDTA disk screen test and that PCR confirmation be performed at a regional laboratory.

Orthogonal analysis of C/EBPbeta targets in vivo during liver proliferation.

CCAAT enhancer-binding protein beta (C/EBPbeta), a basic-leucine zipper transcription factor, is an important effector of signals in physiologic growth and cancer. The identification of direct C/EBPbeta targets in vivo has been limited by functional compensation by other C/EBP family proteins and the low stringency of the consensus sequence. Here we use the combined power of expression profiling and high-throughput chromatin immunoprecipitation to identify direct and biologically relevant targets of C/EBPbeta. We identified 25 potential C/EBPbeta targets, of which 88% of those tested were confirmed as in vivo C/EBPbeta-binding sites. Six of these genes also displayed differential expression in C/EBPbeta-/- livers. Computational analysis revealed that bona fide C/EBPbeta target genes can be distinguished by the presence of binding motifs for specific additional transcription factors in the vicinity of the C/EBPbeta site. This approach is generally applicable to the discovery of direct, biologically relevant targets of mammalian transcription factors.

Using prior knowledge to improve genetic network reconstruction from microarray data.

The use of Bayesian Network methods to recover transcriptional regulatory networks from static microarray data is an active area of bioinformatics research. However, early work in this area lacked realistic analysis of the effects of data set size on learning performance and ignored the potentially immense benefits of using prior biological knowledge. More recent work which has utilized such information has tended to focus on qualitative descriptions of the results. In this paper, we construct a detailed, realistic model for glucose homeostasis and use this model to generate static, synthetic gene expression data. We then use a Bayesian Network method to reconstruct this genetic network from the synthetic microarray data utilizing various amounts and types of prior knowledge. By quantitatively analyzing the effects of data set size and the incorporation of different types of prior biological knowledge on our ability to reconstruct the original network, we show that characteristic portions of genetic networks can be reconstructed from microarray data. Incorporating prior knowledge into the learning scheme greatly reduces the data required, allowing these reverse engineering techniques to be used to learn regulatory interactions from microarray data sets of realistic size.

Transcriptional program of the endocrine pancreas in mice and humans.

The Endocrine Pancreas Consortium was formed in late 1999 to derive and sequence cDNA libraries enriched for rare transcripts expressed in the mammalian endocrine pancreas. Over the past 3 years, the Consortium has generated 20 cDNA libraries from mouse and human pancreatic tissues and deposited >150,000 sequences into the public expressed sequence tag databases. A special effort was made to enrich for cDNAs from the endocrine pancreas by constructing libraries from isolated islets. In addition, we constructed a library in which fetal pancreas from Neurogenin 3 null mice, which consists of only exocrine and duct cells, was subtracted from fetal wild-type pancreas to enrich for the transcripts from the endocrine compartment. Sequence analysis showed that these clones cluster into 9,464 assembly groups (approximating unique transcripts) for the mouse and 13,910 for the human sequences. Of these, >4,300 were unique to Consortium libraries. We have assembled a core clone set containing one cDNA for each assembly group for the mouse and have constructed the corresponding microarray, termed "PancChip 4.0," which contains >9,000 nonredundant elements. We show that this PancChip is highly enriched for genes expressed in the endocrine pancreas. The mouse and human clone sets and corresponding arrays will be important resources for diabetes research.