Use of DNA melting simulation software for in silico diagnostic assay design: targeting regions with complex melting curves and confirmation by real-time PCR using intercalating dyes

BACKGROUND: DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium amplicons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria. RESULTS: The SYTO9 melting curve profiles of three species of Naegleria and two species of Cryptosporidium were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated in vitro. Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts in vitro. CONCLUSION: Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for different species of Naegleria and Cryptosporidium and the complex melting profiles generated in vitro using SYTO9 verified that the complex melting profile of PCR amplicons was solely the result of DNA dissociation. Other data outputs from these simulations were also used to identify the melting domains that contributed to the observed melting peaks for each of the different PCR amplicons

How I do it: Minimally invasive Cox-Maze IV procedure

Clinical vignette Our patient is a 66-year-old female with a 2-year history of atrial fibrillation (AF) and mitral valve prolapse who presented with dyspnea on exertion. She was found to be in AF upon her admission electrocardiogram. A transthoracic echocardiogram was performed demonstrating moderate-to-severe mitral regurgitation (MR) with a left atrial (LA) diameter of 5.1 cm and normal left ventricular (LV) function. After completion of her workup, it was decided that the patient would best be treated by a minimally invasive Cox-Maze IV (CMIV) and concomitant mitral valve procedure given her significant MR and symptoms. This article and accompanying video will discuss how the minimally invasive CMIV procedure is performed. Surgical technique

First Detection of NaI D lines in High-Redshift Damped Lyman-alpha Systems

A Near-infrared (1.18-1.35 micron) high-resolution spectrum of the gravitationally-lensed QSO APM 08279+5255 was obtained with the IRCS mounted on the Subaru Telescope using the AO system. We detected strong NaI D 5891,5897 doublet absorption in high-redshift DLAs at z=1.062 and 1.181, confirming the presence of NaI, which was first reported for the rest-frame UV NaI 3303.3,3303.9 doublet by Petitjean et al. This is the first detection of NaI D absorption in a high-redshift (z>1) DLA. In addition, we detected a new NaI component in the z=1.062 DLA and four new components in the z=1.181 DLA. Using an empirical relationship between NaI and HI column density, we found that all "components" have large HI column density, so that each component is classified as DLA absorption. We also detected strong NaI D absorption associated with a MgII system at z=1.173. Because no other metal absorption lines were detected in this system at the velocity of the NaI absorption in previously reported optical spectra (observed 3.6 years ago), we interpret this NaI absorption cloud probably appeared in the line of sight toward the QSO after the optical observation. This newly found cloud is likely to be a DLA based upon its large estimated HI column density. We found that the N(NaI)/N(CaII) ratios in these DLAs are systematically smaller than those observed in the Galaxy; they are more consistent with the ratios seen in the Large Magellanic Cloud. This is consistent with dust depletion generally being smaller in lower metallicity environments. However, all five clouds of the z=1.181 system have a high N(NaI)/N(CaII) ratio, which is characteristic of cold dense gas. We tentatively suggest that the host galaxy of this system may be the most significant contributor to the gravitational-lens toward APM 08279+5255.Comment: 22 pages, 6 Postscript figures, 3 tables, ApJ in press (Vol.643, 2 June 2006

Opportunities for improving recognition of coastal wetlands in global ecosystem assessment frameworks

Vegetated coastal wetlands, including seagrass, saltmarsh and mangroves, are threatened globally, yet the need to avert these losses is poorly recognized in international policy, such as in the Convention on Biological Diversity and the United Nations (UN) Sustainable Development Goals. Identifying the impact of overlooking coastal wetlands in ecosystem assessment frameworks could help prioritize research efforts to fill these gaps. Here, we examine gaps in the recognition of coastal wetlands in globally applicable ecosystem assessments. We address both shortfalls in assessment frameworks when it comes to assessing wetlands, and gaps in data that limit widespread application of assessments. We examine five assessment frameworks that track fisheries, greenhouse gas emissions, ecosystem threats, and ecosystem services. We found that these assessments inform management decisions, but that the functions provided by coastal wetlands are incompletely represented. Most frameworks had sufficient complexity to measure wetland status, but limitations in data meant they were incompletely informed about wetland functions and services. Incomplete representation of coastal wetlands may lead to them being overlooked by research and management. Improving the coverage of coastal wetlands in ecosystem assessments requires improving global scale mapping of wetland trends, developing global-scale indicators of wetland function and synthesis to quantitatively link animal population dynamics to wetland trends. Filling these gaps will help ensure coastal wetland conservation is properly informed to manage them for the outstanding benefits they bring humanity

Recombinant Acid Ceramidase Reduces Inflammation and Infection in Cystic Fibrosis.

Rationale: In cystic fibrosis the major cause of morbidity and mortality is lung disease characterized by inflammation and infection. The influence of sphingolipid metabolism is poorly understood with a lack of studies using human airway model systems. Objectives: To investigate sphingolipid metabolism in cystic fibrosis and the effects of treatment with recombinant human acid ceramidase on inflammation and infection. Methods: Sphingolipids were measured using mass spectrometry in fully-differentiated cultures of primary human airway epithelial cells and co-cultures with Pseudomonas aeruginosa. In situ activity assays, Western blotting and quantitative polymerase chain reaction were used to investigate function and expression of ceramidase and sphingomyelinase. Effects of treatment with recombinant human acid ceramidase on sphingolipid profile and inflammatory mediator production were assessed in cell cultures and murine models. Measurements and Main Results: Ceramide is increased in cystic fibrosis airway epithelium due to differential function of enzymes regulating sphingolipid metabolism. Sphingosine, a metabolite of ceramide with antimicrobial properties, is not upregulated in response to Pseudomonas aeruginosa by cystic fibrosis airway epithelia. Tumor necrosis factor receptor 1 is increased in the apical membrane of cystic fibrosis epithelia and activates NF-κB signaling, generating inflammation. Treatment with recombinant human acid ceramidase, to decrease ceramide, reduced both inflammatory mediator production and susceptibility to infection. Conclusions: Sphingolipid metabolism is altered in airway epithelial cells cultured from people with cystic fibrosis. Treatment with recombinant acid ceramidase ameliorates the two pivotal features of cystic fibrosis lung disease, inflammation and infection, and thus represents a therapeutic approach worthy of further exploration. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Sphingopyxis Species Isolated from Sand Filter Biofilm at an Australian Drinking Water Treatment Works

Three strains isolated by geosmin enrichment from a sand filter in an Australian drinking water treatment works were genome sequenced to identify their taxonomic placement, and a bench-scale batch experiment confirmed their geosmin-degrading capability. Using the average nucleotide identity based on the MUMmer algorithm (ANIm), pairwise digital DNA-DNA hybridization (dDDH), and phylogenomic analyses, the strains were identified as Sphingopyxis species

Hypercapnia modulates cAMP signalling and cystic fibrosis transmembrane conductance regulator-dependent anion and fluid secretion in airway epithelia

Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist-stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP-regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin-stimulated elevations in intracellular cAMP as well as both adenosine and forskolin-stimulated increases in CFTR-dependent transepithelial short-circuit current, in polarised cultures of Calu-3 human airway cells. This CO2-induced reduction in anion secretion was not due to a decrease in HCO3− transport given that neither a change in CFTR-dependent HCO3− efflux, nor Na+/HCO3− cotransporter-dependent HCO3− influx were CO2-sensitive. Hypercapnia also reduced the volume of forskolin-stimulated fluid secretion over 24 h, yet had no effect on the HCO3− content of the secreted fluid. Our data reveal that hypercapnia reduces CFTR-dependent, electrogenic Cl− and fluid secretion, but not CFTR-dependent HCO3− secretion, which highlights a differential sensitivity of Cl− and HCO3− transporters to raised CO2 in Calu-3 cells. Hypercapnia also reduced forskolin-stimulated CFTR-dependent anion secretion in primary human airway epithelia. Based on current models of airways biology, a reduction in fluid secretion, associated with hypercapnia, would be predicted to have important consequences for airways hydration and the innate defence mechanisms of the lungs

The association of academic tracking to depressive symptoms among adolescents in three Caribbean countries

<p>Abstract</p> <p>Background</p> <p>Students who are tracked into low performing schools or classrooms that limit their life chances may report increased depressive symptoms. Limited research has been conducted on academic tracking and its association with depressive symptoms among high school students in the Caribbean. This project examines levels of depressive symptoms among tenth grade students tracked within and between high schools in Jamaica, St. Vincent and St. Kitts and Nevis.</p> <p>Methods</p> <p>Students enrolled in grade ten of the 2006/2007 academic year in Jamaica, St. Kitts and Nevis and St. Vincent were administered the Beck Depression Inventory II (BDI-II). In Jamaica and St. Vincent, academic tracking was operationalized using data provided by the local Ministries of Education. These Ministries ranked ordered schools according to students' performance on Caribbean school leaving examinations. In St. Kitts and Nevis tracking was operationalized by classroom assignments within schools whereby students were grouped into classrooms according to their levels of academic achievement. Multiple regression analyses were conducted to examine the relationships between academic tracking and BDI-II depression scores.</p> <p>Results</p> <p>A wide cross-section of 4^thform students in each nation was sampled (n = 1738; 278 from Jamaica, 737 St. Kitts and Nevis, 716 from St. Vincent; 52% females, 46.2% males and 1.8% no gender reported; age 12 to 19 years, mean = 15.4 yrs, sd = .9 yr). Roughly half (53%) of the students reported some symptoms of depression with 19.2% reporting moderate and 10.7% reporting severe symptoms of depression. Students in Jamaica reported significantly higher depression scores than those in either St. Kitts and Nevis or St. Vincent (p < .01). Students assigned to a higher academic track reported significantly lower BDI-II scores than students who were assigned to the lower academic track (p < .01).</p> <p>Conclusions</p> <p>There appears to be an association between academic tracking and depressive symptoms that is differentially manifested across the islands of Jamaica, St. Kitts and Nevis and St. Vincent.</p

The need for focused, hard X-ray investigations of the Sun

Understanding the nature of energetic particles in the solar atmosphere is one of the most important outstanding problems in heliophysics. Flare-accelerated particles compose a huge fraction of the flare energy budget; they have large influences on how events develop; they are an important source of high-energy particles found in the heliosphere; and they are the single most important corollary to other areas of high-energy astrophysics. Despite the importance of this area of study, this topic has in the past decade received only a small fraction of the resources necessary for a full investigation. For example, NASA has selected no new Explorer-class instrument in the past two decades that is capable of examining this topic. The advances that are currently being made in understanding flare-accelerated electrons are largely undertaken with data from EOVSA (NSF), STIX (ESA), and NuSTAR (NASA Astrophysics). This is despite the inclusion in the previous Heliophysics decadal survey of the FOXSI concept as part of the SEE2020 mission, and also despite NASA's having invested heavily in readying the technology for such an instrument via four flights of the FOXSI sounding rocket experiment. Due to that investment, the instrumentation stands ready to implement a hard X-ray mission to investigate flare-accelerated electrons. This white paper describes the scientific motivation for why this venture should be undertaken soon.Comment: White paper submitted to the Decadal Survey for Solar and Space Physics (Heliophysics) 2024-2033; 15 pages, 5 figure

Radial Artery Versus Right Internal Thoracic Artery Versus Saphenous Vein as the Second Conduit for Coronary Artery Bypass Surgery:A Network Meta-Analysis of Clinical Outcomes

Background-There remains uncertainty regarding the second-best conduit after the internal thoracic artery in coronary artery bypass grafting. Few studies directly compared the clinical results of the radial artery (RA), right internal thoracic artery (RITA), and saphenous vein (SV). No network meta-analysis has compared these 3 strategies.Methods and Results-MEDLINE and EMBASE were searched for adjusted observational studies and randomized controlled trials comparing the RA, SV, and/or RITA as the second conduit for coronary artery bypass grafting. The primary end point was all-cause long-term mortality. Secondary end points were operative mortality, perioperative stroke, perioperative myocardial infarction, and deep sternal wound infection (DSWI). Pairwise and network meta-analyses were performed. A total of 149 902 patients (4 randomized, 31 observational studies) were included (RA, 16 201, SV, 112 018, RITA, 21 683). At NMA, the use of SV was associated with higher long-term mortality compared with the RA (incidence rate ratio, 1.23; 95% CI, 1.12-1.34) and RITA (incidence rate ratio, 1.26; 95% CI, 1.17-1.35). The risk of DSWI for SV was similar to RA but lower than RITA (odds ratio, 0.71; 95% CI, 0.55-0.91). There were no differences for any outcome between RITA and RA, although DSWI trended higher with RITA (odds ratio, 1.39; 95% CI, 0.92-2.1). The risk of DSWI in bilateral internal thoracic artery studies was higher when the skeletonization technique was not used.Conclusions-The use of the RA or the RITA is associated with a similar and statistically significant long-term clinical benefit compared with the SV. There are no differences in operative risk or complications between the 2 arterial conduits, but DSWI remains a concern with bilateral ITA when skeletonization is not used.</p