Estimating the Number of Essential Genes in a Genome by Random Transposon Mutagenesis

We describe a Bayesian method for estimating the number of essential genes in a genome, on the basis of data on viable mutants for which a single transposon was inserted after a random TA site in a genome,potentially disrupting a gene. The prior distribution for the number of essential genes was taken to be uniform. A Gibbs sampler was used to estimate the posterior distribution. The method is illustrated with simulated data. Further simulations were used to study the performance of the procedure.

Influence of diurnal hyperosmotic loading on the metabolism and matrix gene expression of a whole-organ intervertebral disc model

It is generally agreed that the mechanical environment of intervertebral disc cells plays an important role in maintaining a balanced matrix metabolism. The precise mechanism by which the signals are transduced into the cells is poorly understood. Osmotic changes in the extracellular matrix (ECM) are thought to be involved. Current in-vitro studies on this topic are mostly short-term and show conflicting data on the reaction of disc cells subjected to osmotic changes which is partially due to the heterogenous and often substantially-reduced culture systems. The aim of the study was therefore to investigate the effects of cyclic osmotic loading for 4 weeks on metabolism and matrix gene expression in a full-organ intervertebral disc culture system. Intervertebral disc/endplate units were isolated from New Zealand White Rabbits and cultured either in iso-osmotic media (335 mosmol/kg) or were diurnally exposed for 8 hours to hyper-osmotic conditions (485 mosmol/kg). Cell viability, metabolic activity, matrix composition and matrix gene expression profile (collagen types I/II and aggrecan) were monitored using Live/Dead cell viability assay, tetrazolium reduction test (WST 8), proteoglycan and DNA quantification assays and quantitative PCR. The results show that diurnal osmotic stimulation did not have significant effects on proteoglycan content, cellularity and disc cell viability after 28 days in culture. However, hyperosmolarity caused increased cell death in the early culture phase and counteracted up-regulation of type I collagen gene expression in nucleus and annulus cells. Moreover, the initially decreased cellular dehydrogenase activity recovered with osmotic stimulation after 4 weeks and aggrecan gene down-regulation was delayed, although the latter was not significant according to our statistical criteria. In contrast, collagen type II did not respond to the osmotic changes and was down-regulated in both groups. In conclusion, diurnal hyper-osmotic stimulation of a whole-organ disc/endplate culture partially inhibits a matrix gene expression profile as encountered in degenerative disc disease and counteracts cellular metabolic hypo-activity.

Effects of a divided high loading dose of caffeine on circulatory variables in preterm infants

BACKGROUND: A single high loading dose of 25 mg/kg caffeine has been shown to be effective for the prevention of apnoea, but may result in considerable reductions in blood flow velocity (BFV) in cerebral and intestinal arteries. OBJECTIVE: To assess the effects of two loading doses of 12.5 mg/kg caffeine given four hours apart on BFV in cerebral and intestinal arteries, left ventricular output (LVO), and plasma caffeine concentrations in preterm infants. DESIGN: Sixteen preterm neonates of <34 weeks gestation were investigated one hour after the first oral dose and one, two, and 20 hours after the second dose by Doppler sonography. RESULTS: The mean (SD) plasma caffeine concentrations were 31 (7) and 29 (7) mg/l at two and 20 hours respectively after the second dose. One hour after the first dose, none of the circulatory variables had changed significantly. One hour after the second caffeine dose, mean BFV in the internal carotid artery and anterior cerebral artery showed significant reductions of 17% and 19% (p = 0.01 and p = 0.003 respectively). BFV in the coeliac artery and superior mesenteric artery, LVO, PCO2, and respiratory rate had not changed significantly. Total vascular resistance, calculated as the ratio of mean blood pressure to LVO, had increased significantly one and two hours after the second dose (p = 0.049 and p = 0.023 respectively). CONCLUSION: A divided high loading dose of 25 mg/kg caffeine given four hours apart had decreased BFV in cerebral arteries after the second dose, whereas BFV in intestinal arteries and LVO were not affected.

Immediate loading of implants with 3-unit fixed partial dentures: a 12-month clinical study

PURPOSE: The aim of the present clinical trial was to evaluate the 12-month success rate of titanium dental implants placed in the posterior mandible and immediately loaded with 3-unit fixed partial dentures. MATERIALS AND METHODS: Patients with missing mandibular premolars and molars were enrolled in this study. To be included in the study, the implants had to show good primary stability. Implant stability was measured with resonance frequency analysis using the Osstell device (Integration Diagnostics). Implants were included in the study when the stability quotient (ISQ) exceeded 62. Clinical measurements, such as width of keratinized tissue, ISQ, and radiographic assessment of peri-implant bone crest levels, were performed at baseline and at the 12-month follow-up. The comparison between the baseline and the 12-month visits was performed with the Student t test for paired data (statistically significant at a level of alpha = 0.05). RESULTS: Forty implants with a sandblasted, large grit, acid-etched (SLA) surface (Straumann) were placed in 20 patients. At 12 months, only 1 implant had been lost because of an acute infection. The remaining 39 implants were successful, resulting in a 1-year success rate of 97.5%. Neither peri-implant bone levels, measured radiographically, nor implant stability changed significantly from baseline to the 12-month follow-up (P > .05). DISCUSSION: The immediate functional loading of implants placed in this case series study resulted in a satisfactory success rate. CONCLUSION: The findings from this clinical study showed that the placement of SLA transmucosal implants in the mandibular area and their immediate loading with 3-unit fixed partial dentures may be a safe and successful procedure.

Posterior Simulation in the Generalized Linear Model with Semiparmetric Random Effects

Generalized linear mixed models with semiparametric random effects are useful in a wide variety of Bayesian applications. When the random effects arise from a mixture of Dirichlet process (MDP) model, normal base measures and Gibbs sampling procedures based on the Pólya urn scheme are often used to simulate posterior draws. These algorithms are applicable in the conjugate case when (for a normal base measure) the likelihood is normal. In the non-conjugate case, the algorithms proposed by MacEachern and Müller (1998) and Neal (2000) are often applied to generate posterior samples. Some common problems associated with simulation algorithms for non-conjugate MDP models include convergence and mixing difficulties. This paper proposes an algorithm based on the Pólya urn scheme that extends the Gibbs sampling algorithms to non-conjugate models with normal base measures and exponential family likelihoods. The algorithm proceeds by making Laplace approximations to the likelihood function, thereby reducing the procedure to that of conjugate normal MDP models. To ensure the validity of the stationary distribution in the non-conjugate case, the proposals are accepted or rejected by a Metropolis-Hastings step. In the special case where the data are normally distributed, the algorithm is identical to the Gibbs sampler.

The Optimal Confidence Region for a Random Parameter

Under a two-level hierarchical model, suppose that the distribution of the random parameter is known or can be estimated well. Data are generated via a fixed, but unobservable realization of this parameter. In this paper, we derive the smallest confidence region of the random parameter under a joint Bayesian/frequentist paradigm. On average this optimal region can be much smaller than the corresponding Bayesian highest posterior density region. The new estimation procedure is appealing when one deals with data generated under a highly parallel structure, for example, data from a trial with a large number of clinical centers involved or genome-wide gene-expession data for estimating individual gene- or center-specific parameters simultaneously. The new proposal is illustrated with a typical microarray data set and its performance is examined via a small simulation study.

RANDOM EFFECTS MODELS IN A META-ANALYSIS OF THE ACCURACY OF DIAGNOSTIC TESTS WITHIN A GOLD STANDARD IN THE PRESENCE OF MISSING DATA

In evaluating the accuracy of diagnosis tests, it is common to apply two imperfect tests jointly or sequentially to a study population. In a recent meta-analysis of the accuracy of microsatellite instability testing (MSI) and traditional mutation analysis (MUT) in predicting germline mutations of the mismatch repair (MMR) genes, a Bayesian approach (Chen, Watson, and Parmigiani 2005) was proposed to handle missing data resulting from partial testing and the lack of a gold standard. In this paper, we demonstrate an improved estimation of the sensitivities and specificities of MSI and MUT by using a nonlinear mixed model and a Bayesian hierarchical model, both of which account for the heterogeneity across studies through study-specific random effects. The methods can be used to estimate the accuracy of two imperfect diagnostic tests in other meta-analyses when the prevalence of disease, the sensitivities and/or the specificities of diagnostic tests are heterogeneous among studies. Furthermore, simulation studies have demonstrated the importance of carefully selecting appropriate random effects on the estimation of diagnostic accuracy measurements in this scenario.

Calcium indicator loading of neurons using single-cell electroporation

Studies of subcellular Ca(2+) signaling rely on methods for labeling cells with fluorescent Ca(2+) indicator dyes. In this study, we demonstrate the use of single-cell electroporation for Ca(2+) indicator loading of individual neurons and small neuronal networks in rat neocortex in vitro and in vivo. Brief voltage pulses were delivered through glass pipettes positioned close to target cells. This approach resulted in reliable and rapid (within seconds) loading of somata and subsequent complete labeling of dendritic and axonal arborizations. By using simultaneous whole-cell recordings in brain slices, we directly addressed the effect of electroporation on neurons. Cell viability was high (about 85%) with recovery from the membrane permeabilization occurring within a minute. Electrical properties of recovered cells were indistinguishable before and after electroporation. In addition, Ca(2+) transients with normal appearance could be evoked in dendrites, spines, and axonal boutons of electroporated cells. Using negative-stains of somata, targeted single-cell electroporation was equally applicable in vivo. We conclude that electroporation is a simple approach that permits Ca(2+) indicator loading of multiple cells with low background staining within a short amount of time, which makes it especially well suited for functional imaging of subcellular Ca(2+) dynamics in small neuronal networks.