Diversity of Bacteria in the Marine Sponge Aplysina fulva in Brazilian Coastal Waters

Microorganisms can account for up to 60% of the fresh weight of marine sponges. Marine sponges have been hypothesized to serve as accumulation spots of particular microbial communities, but it is unknown to what extent these communities are directed by the organism or the site or occur randomly. To address this question, we assessed the composition of specific bacterial communities associated with Aplysina fulva, one of the prevalent sponge species inhabiting Brazilian waters. Specimens of A. fulva and surrounding seawater were collected in triplicate in shallow water at two sites, Caboclo Island and Tartaruga beach, Búzios, Brazil. Total community DNA was extracted from the samples using “direct” and “indirect” approaches. 16S rRNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the total bacterial community and of specific bacterial groups—Pseudomonas and Actinobacteria—revealed that the structure of these assemblages in A. fulva differed drastically from that observed in seawater. The DNA extraction methodology and sampling site were determinative for the composition of actinobacterial communities in A. fulva. However, no such effects could be gleaned from total bacterial and Pseudomonas PCR-DGGE profiles. Bacterial 16S rRNA gene clone libraries constructed from directly and indirectly extracted DNA did not differ significantly with respect to diversity and composition. Altogether, the libraries encompassed 15 bacterial phyla and the candidate division TM7. Clone sequences affiliated with the Cyanobacteria, Chloroflexi, Gamma- and Alphaproteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria were, in this order, most abundant. The bacterial communities associated with the A. fulva specimens were distinct and differed from those described in studies of sponge-associated microbiota performed with other sponge species.The phylum Porifera (sponges) consists of benthic (sessile) organisms that occur primarily in marine environments at different depths (26). Sponges are classified into three groups, namely, the Calcarea (calcareous sponges), Hexactinellida (glass sponges), and Demospongiae (5, 26). The group Demospongiae, also called demosponges, encompasses 95% of the ca. 5,500 living sponge species described thus far (5). As typical filter feeders, demosponges are the prime bacterial filters of the sea. They are capable of pumping thousands of liters of water per day (23), using prokaryotic microorganisms as the main source of food (1, 43, 47). In addition to demosponges feeding on microorganisms, the presence of bacteria in high density in internal sponge layers (mesohyl) indicates that a selective process favoring particular prokaryotes, involving microbe-sponge interactions, is likely to occur (64). Furthermore, the dawn of the interactions between Prokarya and higher organisms may actually lie in the demosponges, whose origin is estimated to date back to 550 million years ago (5, 33).Putative interactions between demosponges and microorganisms, presumably mostly consisting of Bacteria and Archaea, were first demonstrated by transmission electron microscopy (TEM), where high amounts of microorganisms were observed in the mesohyl (1, 14, 16, 64). Hence, these bacterium-rich sponges have been termed “bacteriosponges” (46). While investigating 11 taxonomically different demosponges using TEM, Vacelet and Donadey (64) identified two different sponge types in respect of their association with bacteria. Sponges with thick mesohyl contained abundant, dense, and morphologically diverse microbial communities (i.e., bacteriosponge), while those with a well-developed aquiferous system and low-density mesohyl contained few bacterial cells and typically only single bacterial morphotypes. The two types have recently been called “high-microbial-abundance” (HMA) and “low-microbial-abundance” (LMA) sponges, respectively (23). In HMA sponges, bacterial densities may reach 10⁸ to 10¹⁰ bacterial cells per g (wet weight) of sponge, exceeding seawater concentrations by 2 to 4 orders of magnitude (15, 23). Based on the analysis of 16S rRNA genes, over 15 bacterial phyla have thus far been reported to occur in association with marine sponges (11, 23, 56). Among these are typical sponge-associated bacteria such as members of the Cyanobacteria, Chloroflexi, Proteobacteria, Acidobacteria, Verrucomicrobia, and the candidate phyla “Poribacteria” and TM6 (14, 30, 51, 56, 60, 68).Increasing research interest in the sponge-associated microbiota has emerged in the past few years, mainly due to the in spongium production of an enormous diversity of biologically active secondary metabolites (56). Recent studies suggest that certain bioactive compounds retrieved from marine sponges—such as complex polypeptides and nonribosomal peptides—are likely to be synthesized by the symbiont bacteria (27, 41, 42). Such bioactive secondary metabolites offer great promise for use in biotechnology and medicine (3, 22, 27, 41, 42, 51, 59). In particular, cytotoxic compounds, i.e., antitumoral substances and polyketides, may find application in anticancer therapies (13, 42, 51). Recent investigations revealed the presence of dibromotyrosine-derived metabolites in Aplysina fulva (Pallas, 1766) specimens collected along the Brazilian shore (39). However, a putative role of microbial symbionts in the production of such metabolites, commonly found to display biological activity, remains to be evaluated.Despite the global-scale occurrence of sponges in Earth''s marine ecosystems, the investigation of their associated bacterial communities has thus far been restricted only to certain areas (1, 11, 13, 14, 27, 54, 58, 68). To our knowledge, no studies have been conducted, to date, on sponge-associated microbes in subtropical, South Atlantic open shore waters. In the present study, we assess the diversity and composition of the bacterial community associated with the demosponge A. fulva collected at two different sites at the Brazilian shore. A suite of tools, ranging from plate count estimations and TEM to sponge DNA-based analyses of bacterial 16S rRNA genes, was used. We hypothesized that a distinct bacterial community occurs in A. fulva, which is different from that in the surrounding bulk water, as well as from those in other sponge species.     

Interaction between the CBM of Cel9A from Thermobifida fusca and cellulose fibers

Molecular docking and molecular dynamics (MD) simulations were used to investigate the binding of a cellodextrin chain in a crystal-like conformation to the carbohydrate-binding module (CBM) of Cel9A from Thermobifida fusca. The fiber was found to bind to the CBM in a single and well-defined configuration in-line with the catalytic cleft, supporting the hypothesis that this CBM plays a role in the catalysis by feeding the catalytic domain (CD) with a polysaccharide chain. The results also expand the current known list of residues involved in the binding. The polysaccharide-protein attachment is shown to be mediated by five amine/amide-containing residues. E478 and E559 were found not to interact directly with the sugar chain; instead they seem to be responsible to stabilize the binding motif via hydrogen bonds.     

Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis

The objective of this work is to investigate the utilization of two abundant agricultural residues in Brazil for the production and application of cellulolytic enzymes. Different materials obtained after pretreatment of sugarcane bagasse, as well as pure synthetic substrates, were considered for cellulase production by Penicillium funiculosum. The best results for FPase (354 U L^?1) and β-glucosidase (1,835 U L^?1) production were observed when sugarcane bagasse partially delignified cellulignin (PDC) was used. The crude extract obtained from PDC fermentation was then partially characterized. Optimal temperatures for cellulase action ranged from 52 to 58°C and pH values of around 4.9 contributed to maximum enzyme activity. At 37°C, the cellulases were highly stable, losing less than 15% of their initial activity after 23 h of incubation. There was no detection of proteases in the P. funiculosum extract, but other hydrolases, such as endoxylanases, were identified (147 U L^?1). Finally, when compared to commercial preparations, the cellulolytic complex from P. funiculosum showed more well-balanced amounts of β-glucosidase, endo- and exoglucanase, resulting in the desired performance in the presence of a lignocellulosic material. Cellulases from this filamentous fungus had a higher glucose production rate (470 mg L^?1 h^?1) when incubated with corn cob than with Celluclast^®, GC 220^® and Spezyme^® (312, 454 and 400 mg L^?1 h^?1, respectively).     

Fine litterfall in the Brazilian Atlantic Forest

Litterfall is an essential component of tropical forest productivity, transferring nutrients from the vegetation back to soils. Here, we summarize the data from 105 estimates of fine litterfall production from 45 sites in the Atlantic Forest domain, including two types of forests, evergreen and seasonal, and two successional stages, secondary and old growth. The overall litterfall average was 8.0 ± 2.5 Mg/ha. Litterfall was significantly in higher seasonal forests than in evergreen forests and in old growth versus secondary forests. Leaves were the major component of litterfall, contributing 68 percent to the total. The second most important component was branches, contributing 22 percent, followed by reproductive organs (flowers and fruits), at 6 percent. Accurate measurements of tropical forest productivity are crucial for estimating their role in sequestering atmospheric carbon, and we suggest some ways to standardize litterfall sampling to obtain better estimates.     

Chitosan molecular structure as a function of N-acetylation

Molecular dynamics simulations have been carried out to characterize the structure and solubility of chitosan nanoparticle‐like structures as a function of the deacetylation level (0, 40, 60, and 100%) and the spatial distribution of the N‐acetyl groups in the particles. The polysaccharide chains of highly N‐deacetylated particles where the N‐acetyl groups are uniformly distributed present a high flexibility and preference for the relaxed two‐fold helix and five‐fold helix motifs. When these groups are confined to a given region of the particle, the chains adopt preferentially a two‐fold helix with ? and ψ values close to crystalline chitin. Nanoparticles with up to 40% acetylation are moderately soluble, forming stable aggregates when the N‐acetyl groups are unevenly distributed. Systems with 60% or higher N‐acetylation levels are insoluble and present similar degrees of swelling regardless the distribution of their N‐acetyl groups. Overall particle solvation is highly affected by electrostatic forces resulting from the degree of acetylation. The water mobility and orientation around the polysaccharide chains affects the stability of the intramolecular O3‐HO3_(n)···O5_{(n +1)} hydrogen bond, which in turn controls particle aggregation. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 448–460, 2011.     

Response of the zoophytophagous predators Macrolophus pygmaeus and Nesidiocoris tenuis to volatiles of uninfested plants and to plants infested by prey or conspecifics

Knowledge about the orientation mechanisms used by two important predaceous mirids (Macrolophus pygmaeus Rambour and Nesidiocoris tenuis (Reuter)) in finding their prey (whitefly Bemisia tabaci (Gennadius) and the tomato borer Tuta absoluta (Meyrick)) is limited. In a Y-tube olfactometer, we tested the behavioral responses of naïve and experienced predators to uninfested plants, herbivore-induced plant volatiles (HIPVs) from plants infested with T. absoluta and/or B. tabaci, the sex pheromone of T. absoluta, and volatiles produced by plants injured by the predators. Nesidiocoris tenuis responds to volatiles produced by uninfested plants only after experience with the plant, whereas naïve and experienced M. pygmaeus show positive chemotaxis. Both predators are attracted to volatiles from prey-infested plants, and we provide the first evidence that experience affects this response in M. pygmaeus. Infestation of the same plant by both prey species elicited similar responses by the two predators as plants infested by either herbivore singly. Neither predator responded to sex pheromones of T. absoluta. Macrolophus pygmaeus avoided plants injured by conspecifics, while N. tenuis females were attracted by such plants. The implications of these results for augmentative biological control are discussed.     

Effect of Time of Day on Performance,Hormonal and Metabolic Response during a 1000-M Cycling Time Trial

The aim of this study was to determine the effect of time of day on performance, pacing, and hormonal and metabolic responses during a 1000-m cycling time-trial. Nine male, recreational cyclists visited the laboratory four times. During the 1^st visit the participants performed an incremental test and during the 2^nd visit they performed a 1000-m cycling familiarization trial. On the 3^rd and 4^th visits, the participants performed a 1000-m TT at either 8 am or 6 pm, in randomized, repeated-measures, crossover design. The time to complete the time trial was lower in the evening than in the morning (88.2±8.7 versus 94.7±10.9 s, respectively, p<0.05), but there was no significant different in pacing. However, oxygen uptake and aerobic mechanical power output at 600 and 1000 m tended to be higher in the evening (p<0.07 and 0.09, respectively). There was also a main effect of time of day for insulin, cortisol, and total and free testosterone concentration, which were all higher in the morning (+60%, +26%, +31% and +22%, respectively, p<0.05). The growth hormone, was twofold higher in the evening (p<0.05). The plasma glucose was ∼11% lower in the morning (p<0.05). Glucagon, norepinephrine, epinephrine and lactate were similar for the morning and evening trials (p>0.05), but the norepinephrine response to the exercise was increased in the morning (+46%, p<0.05), and it was accompanied by a 5-fold increase in the response of glucose. Muscle recruitment, as measured by electromyography, was similar between morning and evening trials (p>0.05). Our findings suggest that performance was improved in the evening, and it was accompanied by an improved hormonal and metabolic milieu.     

Effects of Ezetimibe on Endothelial Progenitor Cells and Microparticles in High-Risk Patients

Imbalance on endothelial turnover can predict cardiovascular outcomes. We aimed at evaluating the effects of lipid-modifying therapies on circulating endothelial progenitor cells (EPCs), endothelial microparticles (EMPs), and platelet microparticles (PMPs) in high cardiovascular risk subjects with elevated C-reactive protein (CRP). Sixty-three individuals with coronary heart disease (CHD) or CHD risk equivalent on stable statin therapy, with LDL-cholesterol <100 mg/dL and CRP ≥2.0 mg/L were selected. After a 4-week run-in period with atorvastatin 10 mg, those with persistent CRP ≥2.0 mg/L were randomized to another 4-week treatment period with atorvastatin 40 mg, ezetimibe 10 mg or atorvastatin 40 mg/ezetimibe 10 mg. EPC (CD34⁺/CD133⁺/KDR⁺), EMP (CD51⁺), and PMP (CD42⁺/CD31⁺) were quantified by flow cytometry. Atorvastatin 40 mg and atorvastatin 40 mg/ezetimibe 10 mg reduced LDL-cholesterol (P < 0.001, paired T test, vs. baseline). Combined therapy, but not ezetimibe reduced CRP. CD34⁺/KDR⁺ EPC were reduced after ezetimibe alone (P = 0.011 vs. baseline, Wilcoxon test) or combined with atorvastatin (P = 0.016 vs. baseline, Wilcoxon test). In addition, ezetimibe increased CD51⁺ EMP (P = 0.017 vs. baseline, Wilcoxon test). No correlations between these markers and LDL-cholesterol or CRP were observed. These results contribute to understand the link between inflammation and vascular homeostasis and highlight the broader benefit of statins decreasing inflammation and preventing microparticles release, an effect not observed with ezetimibe alone.     

Hepatocellular transport of cyclosomatostatins: evidence for a carrier system related to the multispecific bile acid transporter.

The uptake of the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, was studied in isolated hepatocytes. 008 is taken up by hepatocytes in a concentration-, time-, energy- and temperature- dependent manner. Since 008 is hydrophobic, it binds rapidly to liver cells. This is evident by the positive intercept at the gamma-axis in the uptake curves. At higher concentrations, a minor part of the transport occurs by diffusion at a rate of 8.307.10(-6) cm/s. This part of diffusion is measured at 4 degrees C and can be subtracted from the uptake at 37 degrees C resulting in the carrier mediated part of uptake which is saturable. Kinetic parameters for the saturable part of uptake are Km 1.5 microM and Vmax 40.0 pmol/mg per min. The transport is decreased in the absence of oxygen and in the presence of metabolic inhibitors. Uptake is accelerated at temperatures above 20 degrees C. The activation energy was determined to be 30.77 kJ/mol. The membrane potential and not a sodium gradient is the main driving force for 008 transport. Cholate (a typical substrate of the multispecific bile acid transporter) and taurocholate are mutual competitive inhibitors of 008 uptake. Phalloidin, antamanide and iodipamide, typical foreign substrates of the transporter, interfere with the uptake of 008. AS 30D ascites hepatoma cells, known to be unable to transport bile acids, phalloidin and iodipamide, are also unfit to transport 008. Interestingly, sulfobromophthalein (BSP) but not rifampicin, both foreign substrates of the bilirubin carrier, inhibits the transport of 008 in a competitive manner.