Morphological and transcriptional effects of crude oil and dispersant exposure on the marine sponge Cinachyrella alloclada.

Marine sponges play important roles in benthic ecosystems. More than providing shelter and food to other species, they help maintain water quality by regulating nitrogen and ammonium levels in the water, and bioaccumulate heavy metals. This system, however, is particularly sensitive to sudden environmental changes including catastrophic pollution event such as oil spills. Hundreds of oil platforms are currently actively extracting oil and gas in the Gulf of Mexico. To test the vulnerability of the benthic ecosystems to oil spills, we utilized the Caribbean reef sponge, Cinachyrella alloclada, as a novel experimental indicator. We have exposed organisms to crude oil and oil dispersant for up to 24 h and measured resultant gene expression changes. Our findings indicate that 1-hour exposure to water accommodated fractions (WAF) was enough to elicit massive shifts in gene expression in sponges and host bacterial communities (8052 differentially expressed transcripts) with the up-regulation of stress related pathways, cancer related pathways, and cell integrity pathways. Genes that were upregulated included heat shock proteins, apoptosis, oncogenes (Rab/Ras, Src, CMYC), and several E3 ubiquitin ligases. 24-hour exposure of chemically enhanced WAF (CE-WAF) had the greatest impact to benthic communities, resulting in mostly downregulation of gene expression (4248 differentially expressed transcripts). Gene deregulation from 1-hour treatments follow this decreasing trend of toxicity: WAF > CE-WAF > Dispersant, while the 24-hour treatment showed a shift to CE-WAF > Dispersant > WAF in our experiments. Thus, this study supports the development of Cinachyrella alloclada as a research model organism and bioindicator species for Florida reefs and underscores the importance of developing more efficient and safer ways to remove oil in the event of a spill catastrophe.

Increasing knowledge in IGF1R defects: lessons from 35 new patients.

BACKGROUND: The type 1 insulin-like growth factor receptor (IGF1R) is a keystone of fetal growth regulation by mediating the effects of IGF-I and IGF-II. Recently, a cohort of patients carrying an IGF1R defect was described, from which a clinical score was established for diagnosis. We assessed this score in a large cohort of patients with identified IGF1R defects, as no external validation was available. Furthermore, we aimed to develop a functional test to allow the classification of variants of unknown significance (VUS) in vitro. METHODS: DNA was tested for either deletions or single nucleotide variant (SNV) and the phosphorylation of downstream pathways studied after stimulation with IGF-I by western blot analysis of fibroblast of nine patients. RESULTS: We detected 21 IGF1R defects in 35 patients, including 8 deletions and 10 heterozygous, 1 homozygous and 1 compound-heterozygous SNVs. The main clinical characteristics of these patients were being born small for gestational age (90.9%), short stature (88.2%) and microcephaly (74.1%). Feeding difficulties and varying degrees of developmental delay were highly prevalent (54.5%). There were no differences in phenotypes between patients with deletions and SNVs of IGF1R. Functional studies showed that the SNVs tested were associated with decreased AKT phosphorylation. CONCLUSION: We report eight new pathogenic variants of IGF1R and an original case with a homozygous SNV. We found the recently proposed clinical score to be accurate for the diagnosis of IGF1R defects with a sensitivity of 95.2%. We developed an efficient functional test to assess the pathogenicity of SNVs, which is useful, especially for VUS.

A stoichio-kinetic model for a DPPH∙ -ferulic acid reaction.

Estimates of the activity of antioxidants in the literature often appear inconsistent. In the specific case of the DPPH∙ test, the diversity of measurements may arise from variations in the protocols followed. This paper proposes an unbiased method which models the reduction mechanism. This method is applied to the reduction of DPPH∙ by ferulic acid. A scheme with eight coupled reactions is proposed and has been validated on different solvents and using a wide range of DPPH ̇, ferulic acid, and 5,5'-diferulic acid concentrations, and verified using data from the literature on ferulic acid activity. This modeling approach permits a correction to the bias of the 8th reaction (spontaneous reduction of DPPH ̇), because of its sensitivity to solvent, which in most cases is not taken into account. The best experimental strategy to determine the Efficient Concentration of ferulic acid to reduce 20% (EC20) and 50% (EC50) of DPPH∙ is then detailed in terms of initial DPPH∙ concentrations and the duration of the experiment.

Responses of the picoprasinophyte Micromonas commoda to light and ultraviolet stress.

Micromonas is a unicellular marine green alga that thrives from tropical to polar ecosystems. We investigated the growth and cellular characteristics of acclimated mid-exponential phase Micromonas commoda RCC299 over multiple light levels and over the diel cycle (14:10 hour light:dark). We also exposed the light:dark acclimated M. commoda to experimental shifts from moderate to high light (HL), and to HL plus ultraviolet radiation (HL+UV), 4.5 hours into the light period. Cellular responses of this prasinophyte were quantified by flow cytometry and changes in gene expression by qPCR and RNA-seq. While proxies for chlorophyll a content and cell size exhibited similar diel variations in HL and controls, with progressive increases during day and decreases at night, both parameters sharply decreased after the HL+UV shift. Two distinct transcriptional responses were observed among chloroplast genes in the light shift experiments: i) expression of transcription and translation-related genes decreased over the time course, and this transition occurred earlier in treatments than controls; ii) expression of several photosystem I and II genes increased in HL relative to controls, as did the growth rate within the same diel period. However, expression of these genes decreased in HL+UV, likely as a photoprotective mechanism. RNA-seq also revealed two genes in the chloroplast genome, ycf2-like and ycf1-like, that had not previously been reported. The latter encodes the second largest chloroplast protein in Micromonas and has weak homology to plant Ycf1, an essential component of the plant protein translocon. Analysis of several nuclear genes showed that the expression of LHCSR2, which is involved in non-photochemical quenching, and five light-harvesting-like genes, increased 30 to >50-fold in HL+UV, but was largely unchanged in HL and controls. Under HL alone, a gene encoding a novel nitrite reductase fusion protein (NIRFU) increased, possibly reflecting enhanced N-assimilation under the 625 µmol photons m-2 s-1 supplied in the HL treatment. NIRFU's domain structure suggests it may have more efficient electron transfer than plant NIR proteins. Our analyses indicate that Micromonas can readily respond to abrupt environmental changes, such that strong photoinhibition was provoked by combined exposure to HL and UV, but a ca. 6-fold increase in light was stimulatory.

Effect of α-tocopherol on oxidative stability of oil during spray drying and storage of dried emulsions.

Lipophilic compounds such as polyunsaturated fatty acids (PUFAs) and antioxidants can be encapsulated by spray drying in order to protect and prolong their functionalities and get new handling properties. The aim of this work was to study the effect of both the spray drying stage and storage (60°C - 50% RH) on the oxidation of lipophilic compounds encapsulated in spray dried oil-in-water emulsions (10% w/w oil in dry matter) using maltodextrin as matrix and Tween® 20 as emulsifier. Emulsions were prepared with oil containing or not containing α-tocopherol (482ppm in oil) in order to also demonstrate the influence of the antioxidant. Results showed that there is a beginning of oxidation during spray drying, evidenced by a slight increase of markers of rancidity, i.e. conjugated dienes and volatile organic compounds. During storage, the oxidative degradation of PUFAs and α-tocopherol started quickly under the conditions of aging. This was shown to be due to the negative effect of the process and to the porosity of the solid matrix to oxygen, associated with the hollow structure of the particles. An inhibitory action of maltodextrin on α-tocopherol was also hypothesized, but it has to be confirmed.

Partitioning of vanillic acid in oil-in-water emulsions: Impact of the Tween®40 emulsifier.

Tween®40, a non-ionic emulsifier, was studied regarding its influence on the partitioning of vanillic acid, chosen as a model of phenolic antioxidants, in oil/water emulsions. Three oil-in-water (30:70) systems at pH3.5 were compared: an oil/water two-phase system, a system with oil/water/Tween just mixed, and an emulsified oil/water/Tween system. The partitioning of vanillic acid in the aqueous and oily phases was studied by 2D front-face fluorescence spectroscopy and UV spectroscopy. Tween®40 used at two concentrations 0.9% and 3.6% was only partitioned in the aqueous phase and at the interface between oil and water. Vanillic acid in the oil/water two-phase system reflected its amphiphilic nature: 3/4 was partitioned in the aqueous phase and 1/4 in the oily phase. In the presence of Tween®40, the major part of vanillic acid (90%) was found in the aqueous phase of the non-emulsified system, where a high proportion was associated with Tween®40 micelles. In the emulsion system, vanillic acid moved with Tween®40 to the o/w interface of the oil droplets. Fluorescence analyses demonstrated that the interactions between vanillic acid and Tween®40 were dependent on pH.

Two distinct microbial communities revealed in the sponge Cinachyrella.

Marine sponges are vital components of benthic and coral reef ecosystems, providing shelter and nutrition for many organisms. In addition, sponges act as an essential carbon and nutrient link between the pelagic and benthic environment by filtering large quantities of seawater. Many sponge species harbor a diverse microbial community (including Archaea, Bacteria and Eukaryotes), which can constitute up to 50% of the sponge biomass. Sponges of the genus Cinachyrella are common in Caribbean and Floridian reefs and their archaeal and bacterial microbiomes were explored here using 16S rRNA gene tag pyrosequencing. Cinachyrella specimens and seawater samples were collected from the same South Florida reef at two different times of year. In total, 639 OTUs (12 archaeal and 627 bacterial) belonging to 2 archaeal and 21 bacterial phyla were detected in the sponges. Based on their microbiomes, the six sponge samples formed two distinct groups, namely sponge group 1 (SG1) with lower diversity (Shannon-Weiner index: 3.73 ± 0.22) and SG2 with higher diversity (Shannon-Weiner index: 5.95 ± 0.25). Hosts' 28S rRNA gene sequences further confirmed that the sponge specimens were composed of two taxa closely related to Cinachyrella kuekenthalli. Both sponge groups were dominated by Proteobacteria, but Alphaproteobacteria were significantly more abundant in SG1. SG2 harbored many bacterial phyla (>1% of sequences) present in low abundance or below detection limits (<0.07%) in SG1 including: Acidobacteria, Chloroflexi, Gemmatimonadetes, Nitrospirae, PAUC34f, Poribacteria, and Verrucomicrobia. Furthermore, SG1 and SG2 only had 95 OTUs in common, representing 30.5 and 22.4% of SG1 and SG2's total OTUs, respectively. These results suggest that the sponge host may exert a pivotal influence on the nature and structure of the microbial community and may only be marginally affected by external environment parameters.

Thermal degradation of folates under varying oxygen conditions.

Folate losses in thermally treated foods are mainly due to oxidation. Other mechanisms and folate vitamers behaviour are poorly described. Our study evaluated oxygen impact on total folate degradation and derivatives' evolution during thermal treatments. Spinach and green bean purees were heated, in an instrumented reactor, in anaerobic conditions, under an oxygen partial pressure of 40 kPa. Folates were stable in the absence of oxygen, whilst they were degraded under 40 kPa of oxygen. Total folate showed a sharp decrease in the first hour driven by the degradation of 5-CH3-H4folate, followed by a plateau due to the formyl derivatives and minor compounds stability. The different evolution of the main derivatives was confirmed by the degradation of 5-CH3-H4folate and folic acid in solution, under the same conditions of oxygen concentrations. The stability of folic acid and the high susceptibility of 5-CH3-H4folate to degradation in the presence of oxygen were confirmed.

Lipid oxidation in baked products: impact of formula and process on the generation of volatile compounds.

This paper investigates the effect of ingredients on the reactions occurring during the making of sponge cake and leading to the generation of volatile compounds related to flavour quality. To obtain systems sensitive to lipid oxidation (LO), a formulation design was applied varying the composition of fatty matter and eggs. Oxidation of polyunsaturated fatty acids (PUFA) and formation of related volatile compounds were followed at the different steps of cake-making. Optimised dynamic Solid Phase Micro Extraction was applied to selectively extract either volatile or semi-volatile compounds directly from the baking vapours. We show for the first time that in the case of alveolar baked products, lipid oxidation occurs very early during the step of dough preparation and to a minor extent during the baking process. The generation of lipid oxidation compounds depends on PUFA content and on the presence of endogenous antioxidants in the raw matter. Egg yolk seemed to play a double role on reactivity: protecting unsaturated lipids from oxidation and being necessary to generate a broad class of compounds of the Maillard reaction during baking and linked to the typical flavour of sponge cake.

Global distribution patterns of distinct clades of the photosynthetic picoeukaryote Ostreococcus.

Ostreococcus is a marine picophytoeukaryote for which culture studies indicate there are 'high-light' and 'low-light' adapted ecotypes. Representatives of these ecotypes fall within two to three 18S ribosomal DNA (rDNA) clades for the former and one for the latter. However, clade distributions and relationships to this form of niche partitioning are unknown in nature. We developed two quantitative PCR primer-probe sets and enumerated the proposed ecotypes in the Pacific Ocean as well as the subtropical and tropical North Atlantic. Statistical differences in factors such as salinity, temperature and NO(3) indicated the ecophysiological parameters behind clade distributions are more complex than irradiance alone. Clade OII, containing the putatively low-light adapted strains, was detected at warm oligotrophic sites. In contrast, Clade OI, containing high-light adapted strains, was present in cooler mesotrophic and coastal waters. Maximal OI abundance (19 555±37 18S rDNA copies per ml) was detected in mesotrophic waters at 40 m depth, approaching the nutricline. OII was often more abundant at the deep chlorophyll maximum, when nutrient concentrations were significantly higher than at the surface (stratified euphotic zone waters). However, in mixed euphotic-zone water columns, relatively high numbers (for example, 891±107 18S rDNA copies per ml, Sargasso Sea, springtime) were detected at the surface. Both Clades OI and OII were found at multiple euphotic zone depths, but co-occurrence at the same geographical location appeared rare and was detected only in continental slope waters. In situ growth rate estimates using these primer-probes and better comprehension of physiology will enhance ecological understanding of Ostreococcus Clades OII and OI which appear to be oceanic and coastal clades, respectively.

Targeted metagenomics and ecology of globally important uncultured eukaryotic phytoplankton.

Among eukaryotes, four major phytoplankton lineages are responsible for marine photosynthesis; prymnesiophytes, alveolates, stramenopiles, and prasinophytes. Contributions by individual taxa, however, are not well known, and genomes have been analyzed from only the latter two lineages. Tiny "picoplanktonic" members of the prymnesiophyte lineage have long been inferred to be ecologically important but remain poorly characterized. Here, we examine pico-prymnesiophyte evolutionary history and ecology using cultivation-independent methods. 18S rRNA gene analysis showed pico-prymnesiophytes belonged to broadly distributed uncultivated taxa. Therefore, we used targeted metagenomics to analyze uncultured pico-prymnesiophytes sorted by flow cytometry from subtropical North Atlantic waters. The data reveal a composite nuclear-encoded gene repertoire with strong green-lineage affiliations, which contrasts with the evolutionary history indicated by the plastid genome. Measured pico-prymnesiophyte growth rates were rapid in this region, resulting in primary production contributions similar to the cyanobacterium Prochlorococcus. On average, pico-prymnesiophytes formed 25% of global picophytoplankton biomass, with differing contributions in five biogeographical provinces spanning tropical to subpolar systems. Elements likely contributing to success include high gene density and genes potentially involved in defense and nutrient uptake. Our findings have implications reaching beyond pico-prymnesiophytes, to the prasinophytes and stramenopiles. For example, prevalence of putative Ni-containing superoxide dismutases (SODs), instead of Fe-containing SODs, seems to be a common adaptation among eukaryotic phytoplankton for reducing Fe quotas in low-Fe modern oceans. Moreover, highly mosaic gene repertoires, although compositionally distinct for each major eukaryotic lineage, now seem to be an underlying facet of successful marine phytoplankton.

Green evolution and dynamic adaptations revealed by genomes of the marine picoeukaryotes Micromonas.

Picoeukaryotes are a taxonomically diverse group of organisms less than 2 micrometers in diameter. Photosynthetic marine picoeukaryotes in the genus Micromonas thrive in ecosystems ranging from tropical to polar and could serve as sentinel organisms for biogeochemical fluxes of modern oceans during climate change. These broadly distributed primary producers belong to an anciently diverged sister clade to land plants. Although Micromonas isolates have high 18S ribosomal RNA gene identity, we found that genomes from two isolates shared only 90% of their predicted genes. Their independent evolutionary paths were emphasized by distinct riboswitch arrangements as well as the discovery of intronic repeat elements in one isolate, and in metagenomic data, but not in other genomes. Divergence appears to have been facilitated by selection and acquisition processes that actively shape the repertoire of genes that are mutually exclusive between the two isolates differently than the core genes. Analyses of the Micromonas genomes offer valuable insights into ecological differentiation and the dynamic nature of early plant evolution.

Widespread distribution of a unique marine protistan lineage.

Unicellular eukaryotes (protists) are key components of marine food webs, yet knowledge of their diversity, distributions and respective ecologies is limited. We investigated uncultured protists using 18S rRNA gene sequencing, phylogenetic analyses, specific fluorescence in situ hybridization (FISH) probes and other methods. Because few studies have been conducted in warm water systems, we focused on two Atlantic subtropical regions, the Sargasso Sea and the Florida Current. Cold temperate waters were also sampled. Gene sequences comprising a unique eukaryotic lineage, herein termed 'biliphytes', were identified in most samples, whether from high- (30 degrees C) or from low- (5 degrees C) temperature waters. Sequences within this uncultured group have previously been retrieved from high latitudes. Phylogenetic analyses suggest biliphytes are a sister group to the cryptophytes and katablepharids, although the relationship is not statistically supported. Bootstrap-supported subclades were delineated but coherence was not obvious with respect to geography or physicochemical parameters. Unlike results from the initial publication on these organisms (therein 'picobiliphytes'), we could not detect a nucleomorph, either visually, or by targeted primers. Phycobilin-like fluorescence associated with biliphyte-specific FISH-probed cells supports the hypothesis that they are photosynthetic. Our data indicate the biliphytes are nanoplanktonic in size, averaging 4.1 +/- 1.0 x 3.5 +/- 0.8 microm (+/-SD) for one probed group, and 3.5 +/- 0.9 x 3.0 +/- 0.9 microm (+/-SD) for another. We estimate biliphytes contributed 28 (+/-6)% of the phytoplanktonic biomass in tropical eddy-influenced surface waters. Given their broad thermal and geographic distribution, understanding the role these protists play in biogeochemical cycling within different habitats is essential.

Prevalence and distribution of fecal indicator organisms in South Florida beach sand and preliminary assessment of health effects associated with beach sand exposure.

Fecal indicator levels in nearshore waters of South Florida are routinely monitored to assess microbial contamination at recreational beaches. However, samples of sand from the surf zone and upper beach are not monitored which is surprising since sand may accumulate and harbor fecal-derived organisms. This study examined the prevalence of fecal indicator organisms in tidally-affected beach sand and in upper beach sand and compared these counts to levels in the water. Since indicator organisms were statistically elevated in sand relative to water, the study also considered the potential health risks associated with beach use and exposure to sand. Fecal coliforms, Escherichia coli, enterococci, somatic coliphages, and F(+)-specific coliphages were enumerated from sand and water at three South Florida beaches (Ft. Lauderdale Beach, Hollywood Beach, and Hobie Beach) over a 2-year period. Bacteria were consistently more concentrated in 100g samples of beach sand (2-23 fold in wet sand and 30-460 fold in dry sand) compared to 100ml samples of water. Somatic coliphages were commonly recovered from both sand and water while F(+)-specific coliphages were less commonly detected. Seeding experiments revealed that a single specimen of gull feces significantly influenced enterococci levels in some 3.1m(2) of beach sand. Examination of beach sand on a micro-spatial scale demonstrated that the variation in enterococci density over short distances was considerable. Results of multiple linear regression analysis showed that the physical and chemical parameters monitored in this study could only minimally account for the variation observed in indicator densities. A pilot epidemiological study was conducted to examine whether the length of exposure to beach water and sand could be correlated with health risk. Logistic regression analysis results provided preliminary evidence that time spent in the wet sand and time spent in the water were associated with a dose-dependent increase in gastrointestinal illness.

In-depth analyses of marine microbial community genomics.

Marine microbes have evolved to live along extreme environmental gradients, whether at the microscale, in proximity to particles or over the entire water column. Using community genomics, DeLong et al. highlight deduced biological differences that result from open-ocean depth gradients. The power of the large-insert libraries used is that both phylogeny and function can be inferred from the genetic material obtained--even for uncultured microbes. Together with complete genomes of marine isolates and advances in physiology and ecology, this study paves the way for ecosystems biology approaches to dynamics and controls of marine microbial populations.