Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.     

Coral growth with thermal stress and ocean acidification: lessons from the eastern tropical Pacific

The rapid growth of scleractinian corals is responsible for the persistence of coral reefs through time. Coral growth rates have declined over the past 30 years in the western Pacific, Indian, and North Atlantic Oceans. The spatial scale of this decline has led researchers to suggest that a global phenomenon like ocean acidification may be responsible. A multi-species inventory of coral growth from Pacific Panamá confirms that declines have occurred in some, but not all species. Linear extension declined significantly in the most important reef builder of the eastern tropical Pacific, Pocillopora damicornis, by nearly one-third from 1974 to 2006. The rate of decline in skeletal extension for P. damicornis from Pacific Panamá (0.9% year⁻¹) was nearly identical to massive Porites in the Indo-Pacific over the past 20–30 years (0.89–1.23% year⁻¹). The branching pocilloporid corals have shown an increased tolerance to recurrent thermal stress events in Panamá, but appear to be susceptible to acidification. In contrast, the massive pavonid corals have shown less tolerance to thermal stress, but may be less sensitive to acidification. These differing sensitivities will be a fundamental determinant of eastern tropical Pacific coral reef community structure with accelerating climate change that has implications for the future of reef communities worldwide.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Intragenic duplication and divergence in the spectrin superfamily of proteins

Many structural, signaling, and adhesion molecules contain tandemly repeated amino acid motifs. The alpha-actinin/spectrin/dystrophin superfamily of F-actin-crosslinking proteins contains an array of triple alpha-helical motifs (spectrin repeats). We present here the complete sequence of the novel beta-spectrin isoform beta(Heavy)- spectrin (beta H). The sequence of beta H supports the origin of alpha- and beta-spectrins from a common ancestor, and we present a novel model for the origin of the spectrins from a homodimeric actin-crosslinking precursor. The pattern of similarity between the spectrin repeat units indicates that they have evolved by a series of nested, nonuniform duplications. Furthermore, the spectrins and dystrophins clearly have common ancestry, yet the repeat unit is of a different length in each family. Together, these observations suggest a dynamic period of increase in repeat number accompanied by homogenization within each array by concerted evolution. However, today, there is greater similarity of homologous repeats between species than there is across repeats within species, suggesting that concerted evolution ceased some time before the arthropod/vertebrate split. We propose a two-phase model for the evolution of the spectrin repeat arrays in which an initial phase of concerted evolution is subsequently retarded as each new protein becomes constrained to a specific length and the repeats diverge at the DNA level. This evolutionary model has general applicability to the origins of the many other proteins that have tandemly repeated motifs.      

Species richness of motile cryptofauna across a gradient of reef framework erosion

Coral reef ecosystems contain exceptionally high concentrations of marine biodiversity, potentially encompassing millions of species. Similar to tropical rainforests and their insects, the majority of reef animal species are small and cryptic, living in the cracks and crevices of structural taxa (trees and corals). Although the cryptofauna make up the majority of a reef??s metazoan biodiversity, we know little about their basic ecology. We sampled motile cryptofaunal communities from both live corals and dead carbonate reef framework across a gradient of increasing erosion on a reef in Pacific Panamá. A total of 289 Operational Taxonomic Units (OTUs) from six phyla were identified. We used species-accumulation models fitted to individual- and sample-based rarefaction curves, as well as seven nonparametric richness estimators to estimate species richness among the different framework types. All procedures predicted the same trends in species richness across the differing framework types. Estimated species richness was higher in dead framework (261?C370 OTUs) than in live coral substrates (112?C219 OTUs). Surprisingly, richness increased as framework structure was eroded: coral rubble contained the greatest number of species (227?C320 OTUs) and the lowest estimated richness of 47?C115 OTUs was found in the zone where the reef framework had the greatest vertical relief. This contradicts the paradigm that abundant live coral indicates the apex of reef diversity.     

Patterns of resistance and resilience of the stress-tolerant coral Siderastrea radians (Pallas) to sub-optimal salinity and sediment burial

The coastal lagoons of south Florida, U.S., experience fluctuating levels of sedimentation and salinity and contain only a subset of the coral species found at the adjacent reefs of the Florida Reef Tract. The dominant species within these habitats is Siderastrea radians, which can reach densities of up to 68 colonies m^- 2 and is commonly exposed to salinity extremes (< 10 psu to > 37 psu) and chronic sediment burial. In this study, we document the patterns of resistance and resilience of S. radians to sub-optimal salinity levels and sediment burial in a series of short-term, long-term, acute, chronic, single-stressor, and sequential-stressor experiments.S. radians displayed remarkable patterns of resistance and resilience and mortality was documented only under prolonged (≥ 48 h) continuous exposure to salinity extremes (15 and 45 psu) and chronic sediment burial. A reduction in photosynthetic rates was documented for all salinity exposures and the decrease in photosynthesis was linearly related to exposure time. Negative impacts on photosynthetic rates were more severe under low salinity (15 psu) than under high salinity (45 psu). Corals exposed to intermediate, low-salinity levels (25 psu), exhibited initial declines in photosynthesis that were followed by temporary increases that may represent transient acclimatization patterns. The impacts of sediment burial were influenced by the duration of the burial period and ranged from a temporary reduction in photosynthesis to significant reductions in growth and tissue mortality. The maintenance of P/R ratios > 1 and the rapid (< 24 h) recovery of photosynthetic rates after burial periods of 2-24 h indicates that S. radians is able to resist short-term burial periods with minor physiological consequences. However, as burial periods increase and colonies become covered at multiple chronic intervals, sediment burial resulted in extended photosynthetic recovery periods, reduced growth, and mortality. Under normal conditions (i.e., no salinity stress), S. radians was very effective at clearing sediments, and > 50% of the colonies' surface area was cleared within 1 h. However, clearing rates were influenced by physiological status, and prior exposure to sub-optimal salinity significantly reduced the clearing rates of stressed colonies.The response of S. radians to disturbance documented in this study characterizes this species as highly stress-tolerant and provides an explanation for its present high abundance in both reef and marginal environments. Moreover, the key life-history attributes of S. radians, such as brooding reproductive strategy, small colony size, high stress-tolerance, and high recruitment rates, suggest the potential for this species to replace reef-building taxa under increased disturbance scenarios in Florida and elsewhere in the region.     

The Effect of Dosing Regimens on the Antimalarial Efficacy of Dihydroartemisinin-Piperaquine: A Pooled Analysis of Individual Patient Data

Background

Dihydroartemisinin-piperaquine (DP) is increasingly recommended for antimalarial treatment in many endemic countries; however, concerns have been raised over its potential under dosing in young children. We investigated the influence of different dosing schedules on DP''s clinical efficacy.

Methods and Findings

A systematic search of the literature was conducted to identify all studies published between 1960 and February 2013, in which patients were enrolled and treated with DP. Principal investigators were approached and invited to share individual patient data with the WorldWide Antimalarial Resistance Network (WWARN). Data were pooled using a standardised methodology. Univariable and multivariable risk factors for parasite recrudescence were identified using a Cox''s regression model with shared frailty across the study sites. Twenty-four published and two unpublished studies (n = 7,072 patients) were included in the analysis. After correcting for reinfection by parasite genotyping, Kaplan–Meier survival estimates were 97.7% (95% CI 97.3%–98.1%) at day 42 and 97.2% (95% CI 96.7%–97.7%) at day 63. Overall 28.6% (979/3,429) of children aged 1 to 5 years received a total dose of piperaquine below 48 mg/kg (the lower limit recommended by WHO); this risk was 2.3–2.9-fold greater compared to that in the other age groups and was associated with reduced efficacy at day 63 (94.4% [95% CI 92.6%–96.2%], p<0.001). After adjusting for confounding factors, the mg/kg dose of piperaquine was found to be a significant predictor for recrudescence, the risk increasing by 13% (95% CI 5.0%–21%) for every 5 mg/kg decrease in dose; p = 0.002. In a multivariable model increasing the target minimum total dose of piperaquine in children aged 1 to 5 years old from 48 mg/kg to 59 mg/kg would halve the risk of treatment failure and cure at least 95% of patients; such an increment was not associated with gastrointestinal toxicity in the ten studies in which this could be assessed.

Conclusions

DP demonstrates excellent efficacy in a wide range of transmission settings; however, treatment failure is associated with a lower dose of piperaquine, particularly in young children, suggesting potential for further dose optimisation. Please see later in the article for the Editors'' Summary     

Increasing coral calcification in Orbicella faveolata and Pseudodiploria strigosa at Flower Garden Banks,Gulf of Mexico

Coral Reefs - Coral reefs are globally in decline and western Atlantic reefs have experienced the greatest losses in live coral cover of any region. The Flower Garden Banks (FGB) in the Gulf of...