High-diversity biofilm for the oxidation of sulfide-containing effluents

In the present work, we describe for the first time the utilization of a complex microbial biofilm for the treatment of sulfide-containing effluents. A non-aerated packed-column reactor was inoculated with anoxic lake sediment and exposed to light. A biofilm developed in the column and showed a stable oxidation performance for several weeks. Microbial species composition was analyzed by microscopy, pigment analysis and a bacterial 16S rRNA gene clone library. Colorless sulfur bacteria, green algae and purple sulfur bacteria were observed microscopically. Pigment composition confirmed the presence of algae and purple sulfur bacteria. The clone library was dominated by alpha-Proteobacteria (mostly Rhodobacter group), followed by gamma-Proteobacteria (Chromatiaceae-like and Thiothrix-like aerobic sulfur oxidizers) and the Cytophaga-Flavobacterium-Bacteroides group. Plastid signatures from algae were also present and a few clones belonged to both the beta- (Rhodoferax sp., Thiobacillus sp.) and delta-Proteobacteria (Desulfocapsa sp.) and to the low G+C Gram-positive bacteria (Firmicutes group). The coexistence of aerobic, anaerobic, phototrophic and chemotrophic microorganisms in the biofilm, the species richness found within these metabolic groups (42 operational taxonomic units) and the microdiversity observed within some species could be very important for the long-term functioning and versatility of the reactor.     

Phylogenetic and functional diversity of bacterioplankton during Alexandrium spp. blooms

The phylogenetic and functional diversity of the bacterioplankton assemblage associated with blooms of toxic Alexandrium spp. was studied in three harbours of the NW Mediterranean. Denaturing gradient gel electrophoresis and DNA sequence analysis revealed the presence of a bacterium within the Roseobacter clade related to the presence of Alexandrium cells. Phylogenetic diversity was affected by the presence of Alexandrium spp., geographic situation and seasonality. In contrast, functional diversity, assessed with Biolog plates, was clearly affected by seasonality, but not by the presence of Alexandrium, indicating that the presence of the bacterium associated with the blooms was not enough to modify the metabolic pattern of the bacterioplankton assemblage.     

In vitro characterization and in vivo functionality of erythropoietin-secreting cells immobilized in alginate-poly-L-lysine-alginate microcapsules

The in vitro and in vivo characterization of cell-loaded immobilization devices is an important challenge in cell encapsulation technology for the long-term efficacy of this approach. In the present paper, alginate-poly-l-lysine-alginate (APA) microcapsules containing erythropoietin (Epo)-secreting C2C12 myoblasts have been elaborated, characterized, and tested both in vitro and in vivo. High mechanical and chemical resistance of the elaborated microcapsules was observed. Moreover, the in vitro cultured encapsulated cells released 81.9 +/- 8.2 mIU/mL/24 h (by 100 cell-loaded microcapsules) by day 7, reaching the highest peak at day 21 (161.7 +/- 0.9 mIU/mL/24 h). High and constant hematocrit levels were maintained over 120 days after a single subcutaneous administration of microcapsules and lacking immunosuppressive protocols. No major host reaction was observed. On the basis of the results obtained in our study, cell encapsulation technology might be considered a suitable therapeutic strategy for the long-term delivery of biologically active products, such as Epo.     

Clofibric and ethacrynic acids prevent experimental pyelonephritis by Escherichia coli in mice

Interfering Escherichia coli attachment to the urinary tract, using P-fimbriation inhibitors, can prevent pyelonephritis. Clofibric and ethacrynic acids are organic compounds structurally related, but with different pharmacological uses. These agents are potentially active in the urinary tract due to its elimination in an unaltered form by the renal route. This study described a pyelonephritogenic E. coli strain, grown in the presence of sub-inhibitory concentrations of clofibric or ethacrynic acids (0.1 and 1 mM, respectively), which exhibits inhibition of P1 erythrocytes agglutination and a drastic decrease in fimbriation, using electron microscopy and quantitative analyses of superficial proteins (decrease to a 17-25% in comparison with the control). In vivo assays were performed using ascending urinary tract infection in mice. The treatment with therapeutic doses of the drugs, administered 2 days before the bacterial challenge and daily until the end of the experiment (22 days), abolished renal infection after 7-10 days of drug exposure. Within this period clofibric acid did not produce adverse effects on the renal parenchyma. However, ethacrynic acid caused pyelitis and tubular cellular desquamation. These results suggested that clofibric acid might be useful in the short-term prophylaxis of urinary tract infection.     

Kv7 channels can function without constitutive calmodulin tethering

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function.     

Factors Controlling the Year-Round Variability in Carbon Flux Through Bacteria in a Coastal Marine System

Data from several years of monthly samplings are combined with a 1-year detailed study of carbon flux through bacteria at a NW Mediterranean coastal site to delineate the bacterial role in carbon use and to assess whether environmental factors or bacterial assemblage composition affected the in situ rates of bacterial carbon processing. Leucine (Leu) uptake rates [as an estimate of bacterial heterotrophic production (BHP)] showed high interannual variability but, on average, lower values were found in winter (around 50 pM Leu⁻¹ h⁻¹) as compared to summer (around 150 pM Leu⁻¹ h⁻¹). Leu-to-carbon conversion factors ranged from 0.9 to 3.6 kgC mol Leu⁻¹, with generally higher values in winter. Leu uptake was only weakly correlated to temperature, and over a full-year cycle (in 2003), Leu uptake peaked concomitantly with winter chlorophyll a (Chl a) maxima, and in periods of high ectoenzyme activities in spring and summer. This suggests that both low molecular weight dissolved organic matter (DOM) released by phytoplankton, and high molecular weight DOM in periods of low Chl a, can enhance BHP. Bacterial respiration (BR, range 7–48 μg C l⁻¹ d⁻¹) was not correlated to BHP or temperature, but was significantly correlated to DOC concentration. Total bacterial carbon demand (BHP plus BR) was only met by dissolved organic carbon produced by phytoplankton during the winter period. We measured bacterial growth efficiencies by the short-term and the long-term methods and they ranged from 3 to 42%, increasing during the phytoplankton blooms in winter (during the Chl a peaks), and in spring. Changes in bacterioplankton assemblage structure (as depicted by denaturing gradient gel electrophoresis fingerprinting) were not coupled to changes in ecosystem functioning, at least in bacterial carbon use.     

Evaluation of DNA extraction methods from complex phototrophic biofilms

Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 ± 12 to 1893 ± 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol–chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol–chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms.     

Atheromatous plaque from human carotid artery: Potential involvement of the endothelin-1 and their receptors

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that can modulate the behaviour of vascular smooth muscle cells and thus impact on the development of human atherosclerosis. Circulating plasma levels of ET-1 were measured from 82 patients with ischemic cardiomyopathy (ICM) and 42 healthy controls. A significant increase was found in plasma levels of ET-1 in the patients compared to the controls. These circulating levels of ET-1 were greater in patients with diabetes or involvement of several territories. Gene expression of pre-proET-1 and its receptors ET_A and ET_B was analyzed in the atheromatous plaques from carotid arteries (n = 8) and the internal mammary artery (IMA) (n = 8). Our group observed an increase in pre-proET-1 and ET_A in IMA compared with the atheromatous plaques. Immunohistochemical studies in the atherosclerotic plaque showed that the expression of ET-1 was greater in the areas where the macrophages and lipid nucleus were located.Our findings in this group of patients with symptomatic vascular disease suggest that the endothelin system may play an important role in atherothrombosis.     

Regulation of brain anandamide by acute administration of ethanol

FgFtr1 and FgFtr2 are putative iron permeases, and FgFet1 and FgFet2 are putative ferroxidases of Fusarium graminearum. They have high homologies with iron permease ScFtr1 and ferroxidase ScFet3 of Saccharomyces cerevisiae at the amino acid level. The genes encoding iron permease and ferroxidase were localized to the same chromosome in the manner of FgFtr1/FgFet1 and FgFtr2/FgFet2. The GFP (green fluorescent protein)-fused versions of FgFtr1 and FgFtr2 showed normal functions when compared with FgFtr1 and FgFtr2 in an S. cerevisiae system, and the cellular localizations of FgFtr1 and FgFtr2 in S. cerevisiae depended on the expression of their putative ferroxidase partners FgFet1 and FgFet2 respectively. Although FgFtr1 was found on the plasma membrane when FgFet1 and FgFtr1 were co-transformed in S. cerevisiae, most of the FgFtr1 was found in the endoplasmic reticulum compartment when co-expressed with FgFet2. Furthermore, FgFtr2 was found on the vacuolar membrane when FgFet2 was co-expressed. From the two-hybrid analysis, we confirmed the interaction of FgFtr1 and FgFet1, and the same result was found between FgFtr2 and FgFet2. Iron-uptake activity also depended on the existence of the respective partner. Finally, the FgFtr1 and FgFtr2 were found on the plasma and vacuolar membrane respectively, in F. graminearum. Taken together, these results strongly suggest that FgFtr1 and FgFtr2 from F. graminearum encode the iron permeases of the plasma membrane and vacuolar membrane respectively, and require their specific ferroxidases to carry out normal function. Furthermore, the present study suggests that the reductive iron-uptake system is conserved from yeast to filamentous fungi.