ALBERT

Description: DNA methylation is an important regulator of gene expression and chromatin structure. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is commonly used to identify regions of DNA methylation in eukary...

Description: Background: DNA methylation has essential roles in transcriptional regulation, imprinting, X chromosome inactivation and other cellular processes, and aberrant CpG methylation is directly involved in the pathogenesis of human imprinting disorders and many cancers. To address the need for a quantitative and highly multiplexed bisulfite sequencing method with long read lengths for targeted CpG methylation analysis, we developed single-molecule real-time bisulfite sequencing (SMRT-BS). Results: Optimized bisulfite conversion and PCR conditions enabled the amplification of DNA fragments up to ~1.5 kb, and subjecting overlapping 625–1491 bp amplicons to SMRT-BS indicated high reproducibility across all amplicon lengths (r = 0.972) and low standard deviations (≤0.10) between individual CpG sites sequenced in triplicate. Higher variability in CpG methylation quantitation was correlated with reduced sequencing depth, particularly for intermediately methylated regions. SMRT-BS was validated by orthogonal bisulfite-based microarray (r = 0.906; 42 CpG sites) and second generation sequencing (r = 0.933; 174 CpG sites); however, longer SMRT-BS amplicons (〉1.0 kb) had reduced, but very acceptable, correlation with both orthogonal methods (r = 0.836-0.897 and r = 0.892-0.927, respectively) compared to amplicons less than ~1.0 kb (r = 0.940-0.951 and r = 0.948-0.963, respectively). Multiplexing utility was assessed by simultaneously subjecting four distinct CpG island amplicons (702–866 bp; 325 CpGs) and 30 hematological malignancy cell lines to SMRT-BS (average depth of 110X), which identified a spectrum of highly quantitative methylation levels across all interrogated CpG sites and cell lines. Conclusions: SMRT-BS is a novel, accurate and cost-effective targeted CpG methylation method that is amenable to a high degree of multiplexing with minimal clonal PCR artifacts. Increased sequencing depth is necessary when interrogating longer amplicons (〉1.0 kb) and the previously reported bisulfite sequencing PCR bias towards unmethylated DNA should be considered when measuring intermediately methylated regions. Coupled with an optimized bisulfite PCR protocol, SMRT-BS is capable of interrogating ~1.5 kb amplicons, which theoretically can cover ~91% of CpG islands in the human genome.

Description: Androgens play an important role for the development of male fertility and gained interest as growth and survival factors for certain types of cancer. Androgens act via the androgen receptor (AR/Ar), which is inv...

Description: Background: Glaucoma is a leading cause of blindness. The participation of primary care physicians (PCPs) in glaucoma care may improve health outcomes for glaucoma patients.ObjectivesTo investigate PCPs’ attitudes towards their role in glaucoma care, perceived barriers, and self-reported performance in glaucoma management. Methods: PCPs working in the Haifa and Western Galilee District of Clalit Health Services, Israel’s largest Health Maintenance Organization (HMO) were asked to complete a self-administered structured questionnaire. Physicians were asked to rate their agreement with statements describing the PCP’s role in glaucoma care, and to state how often they behave accordingly in their practice. In addition, physicians were asked to rate the extent that factors such as time constraints and knowledge gaps impede their performance in glaucoma care. Results: Eighty-two physicians completed the questionnaire. The majority thought that PCPs have a major role in early detection of glaucoma (99 %), discussing the importance of adherence to treatment (93 %), and encouraging patients to make regular visits to their ophthalmologist (99 %). However, only 30 % reported asking patients about family history of glaucoma, 64 % reported discussing adherence to treatment, and only 35 % stated that they explain how to use eye drops, while most of respondents (87 %) regularly provide refill prescriptions for glaucoma medications. Sixty percent claimed that during their residency they had not acquired adequate knowledge and competence to allow them to take proper care of glaucoma patients. The main barriers reported were lack of time (43 %), lack of knowledge regarding treatment options and recommended follow-up (46 %), and not being familiar with glaucoma medications’ side effects (54 %). Conclusions: There is a gap between PCPs’ perceptions of their role in glaucoma care and their report on actual performance in early detection and management of glaucoma. Further research is needed to develop and assess interventions that aim at closing this gap.

Description: The relationship between net mixing and the estuarine exchange flow may be quantified using a salinity variance budget. Here “mixing” is defined as the rate of destruction of volume-integrated salinity variance, and the exchange flow is quantified using the total exchange flow. These concepts are explored using an idealized 3D model estuary. It is shown that in steady state (e.g., averaging over the spring–neap cycle) the volume-integrated mixing is approximately given by Mixing ≅ SinSoutQr, where Sin and Sout are the representative salinities of in- and outflowing layers at the mouth and Qr is the river volume flux. This relationship provides an extension of the familiar Knudsen relation, in which the exchange flow is diagnosed based on knowledge of these same three quantities, quantitatively linking mixing to the exchange flow.

Description: The roles of straining and dissipation in controlling stratification are derived analytically using a vertical salinity variance method. Stratification is produced by converting horizontal variance to vertical variance via straining, that is, differential advection of horizontal salinity gradients, and stratification is destroyed by the dissipation of vertical variance through turbulent mixing. A numerical model is applied to the Changjiang estuary in order to demonstrate the salinity variance balance and how it reveals the factors controlling stratification. The variance analysis reveals that dissipation reaches its maximum during spring tide in the Changjiang estuary, leading to the lowest stratification. Stratification increases from spring tide to neap tide because of the increasing excess of straining over dissipation. Throughout the spring–neap tidal cycle, straining is almost always larger than dissipation, indicating a net excess of production of vertical variance relative to dissipation. This excess is balanced on average by advection, which exports vertical variance out of the estuarine region into the plume. During neap tide, tidal straining shows a general tendency of destratification during the flood tide and restratification during ebb, consistent with the one-dimensional theory of tidal straining. During spring tide, however, positive straining occurs during flood because of the strong baroclinicity induced by the intensified horizontal salinity gradient. These results indicate that the salinity variance method provides a valuable approach for examining the spatial and temporal variability of stratification in estuaries and coastal environments.

Description: Observations and analyses of two tidally recurring, oblique, internal hydraulic jumps at a stratified estuary mouth (Columbia River, Oregon/Washington) are presented. These hydraulic features have not previously been studied due to the challenges of both horizontally resolving the sharp gradients and temporally resolving their evolution in numerical models and traditional observation platforms. The jumps, both of which recurred during ebb, formed adjacent to two engineered lateral channel constrictions and were identified in marine radar image time series. Jump occurrence was corroborated by (i) a collocated sharp gradient in the surface currents measured via airborne along-track interferometric synthetic aperture radar and (ii) the transition from supercritical to subcritical flow in the cross-jump direction via shipborne velocity and density measurements. Using a two-layer approximation, observed jump angles at both lateral constrictions are shown to lie within the theoretical bounds given by the critical internal long-wave (Froude) angle and the arrested maximum-amplitude internal bore angle, respectively. Also, intratidal and intertidal variability of the jump angles are shown to be consistent with that expected from the two-layer model, applied to varying stratification and current speed over a range of tidal and river discharge conditions. Intratidal variability of the upchannel jump angle is similar under all observed conditions, whereas the downchannel jump angle shows an additional association with stratification and ebb velocity during the low discharge periods. The observations additionally indicate that the upchannel jump achieves a stable position that is collocated with a similarly oblique bathymetric slope.

Description: High-resolution observations of velocity, salinity, and turbulence quantities were collected in a salt wedge estuary to quantify the efficiency of stratified mixing in a high-energy environment. During the ebb tide, a midwater column layer of strong shear and stratification developed, exhibiting near-critical gradient Richardson numbers and turbulent kinetic energy (TKE) dissipation rates greater than 10−4 m2 s−3, based on inertial subrange spectra. Collocated estimates of scalar variance dissipation from microconductivity sensors were used to estimate buoyancy flux and the flux Richardson number Rif. The majority of the samples were outside the boundary layer, based on the ratio of Ozmidov and boundary length scales, and had a mean Rif = 0.23 ± 0.01 (dissipation flux coefficient Γ = 0.30 ± 0.02) and a median gradient Richardson number Rig = 0.25. The boundary-influenced subset of the data had decreased efficiency, with Rif = 0.17 ± 0.02 (Γ = 0.20 ± 0.03) and median Rig = 0.16. The relationship between Rif and Rig was consistent with a turbulent Prandtl number of 1. Acoustic backscatter imagery revealed coherent braids in the mixing layer during the early ebb and a transition to more homogeneous turbulence in the midebb. A temporal trend in efficiency was also visible, with higher efficiency in the early ebb and lower efficiency in the late ebb when the bottom boundary layer had greater influence on the flow. These findings show that mixing efficiency of turbulence in a continuously forced, energetic, free shear layer can be significantly greater than the broadly cited upper bound from Osborn of 0.15–0.17.

Description: The linkage among total exchange flow, entrainment, and diffusive salt flux in estuaries is derived analytically using salinity coordinates, revealing the simple but important relationship between total exchange flow and mixing. Mixing is defined and quantified in this paper as the dissipation of salinity variance. The method uses the conservation of volume and salt to quantify and distinguish the diahaline transport of volume (i.e., entrainment) and diahaline diffusive salt flux. A numerical model of the Hudson estuary is used as an example of the application of the method in a realistic estuary with a persistent but temporally variable exchange flow. A notable finding of this analysis is that the total exchange flow and diahaline salt flux are out of phase with respect to the spring–neap cycle. Total exchange flow reaches its maximum near minimum neap tide, but diahaline salt transport reaches its maximum during the maximum spring tide. This phase shift explains the strong temporal variation of stratification and estuarine salt content through the spring–neap cycle. In addition to quantifying temporal variation, the method reveals the spatial variation of total exchange flow, entrainment, and diffusive salt flux through the estuary. For instance, the analysis of the Hudson estuary indicates that diffusive salt flux is intensified in the wider cross sections. The method also provides a simple means of quantifying numerical mixing in ocean models because it provides an estimate of the total dissipation of salinity variance, which is the sum of mixing due to the turbulence closure and numerical mixing.

Description: In this study, we investigate the skills of the regional climate model Consortium for Small-Scale Modeling in Climate Mode (CCLM) in reproducing historical climatic features and their added value to the driving global climate models (GCMs) of the Coordinated Regional Climate Downscaling Experiment—East Asia (CORDEX-EA) domain. An ensemble of climate simulations, with a resolution of 0.44°, was conducted by downscaling four GCMs: CNRM-CM5, EC-EARTH, HadGEM2, and MPI-ESM-LR. The CCLM outputs were compared with different observations and reanalysis datasets. Results showed strong seasonal variability of CCLM’s ability in reproducing climatological means, variability, and extremes. The bias of the simulated summer temperatures is generally smaller than that of the winter temperatures; in addition, areas where CCLM adds value to the driving GCMs in simulating temperature are larger in the winter than in the summer. CCLM outperforms GCMs in terms of generating climatological precipitation means and daily precipitation distributions for most regions in the winter, but this is not always the case for the summer. It was found that CCLM biases are partly inherited from GCMs and are significantly shaped by structural biases of CCLM. Furthermore, downscaled simulations show added value in capturing features of consecutive wet days for the tropics and of consecutive dry days for areas to the north of 30°N. We found considerable uncertainty from reanalysis and observation datasets in temperatures and precipitation climatological means for some regions that rival bias values of GCMs and CCLM simulations. We recommend carefully selecting reference datasets when evaluating modeled climate means.