Precipitation and recovery of metal sulfides from metal containing acidic wastewater in a sulfidogenic down-flow fluidized bed reactor

This study reports the feasibility of recovering metal precipitates from a synthetic acidic wastewater containing ethanol, Fe, Zn, and Cd at an organic loading rate of 2.5 g COD/L-day and a COD to sulfate ratio of 0.8 in a sulfate reducing down-flow fluidized bed reactor. The metals were added at increasing loading rates: Fe from 104 to 320 mg/L-day, Zn from 20 to 220 mg/L-day, and Cd from 5 to 20 mg/L-day. The maximum COD and sulfate removals attained were 54% and 41%, respectively. The biofilm reactor was operated at pH as low as 5.0 with stable performance, and no adverse effect over COD consumption or sulfide production was observed. The metals precipitation efficiencies obtained for Fe, Zn, and Cd exceeded 99.7%, 99.3%, and 99.4%, respectively. The total recovered precipitate was estimated to be 90% of the theoretical mass expected as metal sulfides. The precipitate was mainly recovered from the bottom of the reactor and the equalizer. The analysis of the precipitates showed the presence of pyrite (FeS2), sphalerite (ZnS) and greenockite (CdS); no metal hydroxides or carbonates in crystalline phases were identified. This study is the first in reporting the feasibility to recover metal sulfides separated from the biomass in a sulfate reducing process in one stage.     

Inhibitory activity against the fish pathogen Lactococcus garvieae produced by Lactococcus lactis TW34, a lactic acid bacterium isolated from the intestinal tract of a Patagonian fish

After enrichment of Odontesthes platensis intestinal contents, 53 lactic acid bacteria (LAB) were isolated. From the four isolates that showed inhibitory activity against Lactococcus garvieae 03/8460, strain TW34 was selected because it exerted the strongest inhibition. It also inhibited other Gram-positive bacteria, but not Gram-negative fish pathogens. Phenotypic and 16S rDNA phylogenetic analyses showed that TW34 belongs to Lactococcus lactis. In addition, TW34 showed to be sensitive to different antibiotics. The production of the inhibitory agent against L. garvieae was growth associated, and it was significantly influenced by the incubation temperature. The optimal temperature for the antimicrobial production was as low as 15°C. Both acidification and hydrogen peroxide production were ruled out as the source of inhibition. In contrast, the antimicrobial activity was completely lost by treatment with proteolytic enzymes, which confirmed that the inhibitory substance was a bacteriocin. The bacteriocin was highly thermostable (121°C for 15 min) and active between pH 3 and 11. It remained stable for up to 2 months when stored at 4°C and up to 6 months at −20°C. Our results suggest that the strain L. lactis TW34 could provide an alternative for lactococcosis control and therefore be considered for future challenge experiments with fish.     

Coevolution Predicts Direct Interactions between mtDNA-Encoded and nDNA-Encoded Subunits of Oxidative Phosphorylation Complex I

Despite years of research, the structure of the largest mammalian oxidative phosphorylation (OXPHOS) complex, NADH-ubiquinone oxidoreductase (complex I), and the interactions among its 45 subunits are not fully understood. Since complex I harbors subunits encoded by mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) genomes, with the former evolving ∼ 10 times faster than the latter, tight cytonuclear coevolution is expected and observed. Recently, we identified three nDNA-encoded complex I subunits that underwent accelerated amino acid replacement, suggesting their adjustment to the elevated mtDNA rate of change. Hence, they constitute excellent candidates for binding mtDNA-encoded subunits.Here, we further disentangle the network of physical cytonuclear interactions within complex I by analyzing subunits coevolution. Firstly, relying on the bioinformatic analysis of 10 protein complexes possessing solved structures, we show that signals of coevolution identified physically interacting subunits with nearly 90% accuracy, thus lending support to our approach. When applying this approach to cytonuclear interaction within complex I, we predict that the ‘rate-accelerated’ nDNA-encoded subunits of complex I, NDUFC2 and NDUFA1, likely interact with the mtDNA-encoded subunits ND5/ND4 and ND5/ND4/ND1, respectively. Furthermore, we predicted interactions among mtDNA-encoded complex I subunits. Using the yeast two-hybrid system, we experimentally confirmed the predicted interactions of human NDUFC2 with ND4, the interactions of human NDUFA1 with ND1 and ND4, and the lack of interaction of NDUFC2 with ND3 and NDUFA1, thus providing a proof of concept for our approach.Our study shows, for the first time, evidence for direct interactions between nDNA-encoded and mtDNA-encoded subunits of human OXPHOS complex I and paves the path towards deciphering subunit interactions within complexes lacking three-dimensional structures. Our subunit-interactions-predicting method, ComplexCorr, is available at http://webclu.bio.wzw.tum.de/complexcorr.     

Identifying Plasmodium falciparum EBA-175 homologue sequences that specifically bind to human erythrocytes

Erythrocyte binding antigen-160 (EBA-160) protein is a Plasmodium falciparum antigen homologue from the erythrocyte binding protein family (EBP). It has been shown that the EBP family plays a role in parasite binding to the erythrocyte surface. The EBA-160 sequence has been chemically synthesised in seventy 20-mer sequential peptides covering the entire 3D7 protein strain, each of which was tested in erythrocyte binding assays to identify possible EBA-160 functional regions. Five EBA-160 high activity binding peptides (HABPs) specifically binding to erythrocytes with high affinity were identified. Dissociation constants lay between 200 and 460 nM and Hill coefficients between 1.5 and 2.3. Erythrocyte membrane protein binding peptide cross-linking assays using SDS-PAGE showed that these peptides bound specifically to 12, 28, and 44 kDa erythrocyte membrane proteins. The nature of these receptor sites was studied in peptide binding assays using enzyme-treated erythrocytes. HABPs were able to block merozoite in vitro invasion of erythrocytes. HABPs’ potential as anti-malarial vaccine candidates is also discussed.     

Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions

The mechanism by which inhibition of Na+/H+ exchanger (NHE) reduces cell death in ischemic-reperfused myocardium remains controversial. This study investigated whether cariporide could inhibit mitochondrial NHE during ischemia, delaying H+ gradient dissipation and ATP exhaustion. Mouse cardiac myocytes (HL-1) were submitted to 1 h of simulated ischemia (SI) with NaCN/deoxyglucose (pH 6.4), with or without 7 microM cariporide, and mitochondrial concentration of Ca2+ (Rhod-2), 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and the charge difference across the mitochondrial membrane potential (Deltapsim, JC-1) were assessed. ATP content was measured by bioluminescence and mitochondrial swelling by spectrophotometry in isolated mitochondria. Cariporide significantly attenuated the acidification of the mitochondrial matrix induced by SI without modifying Deltapsim decay, and this effect was associated to a delayed ATP exhaustion and increased mitochondrial Ca2+ load. These effects were reproduced in sarcolemma-permeabilized cells exposed to SI. In these cells, cariporide markedly attenuated the fall in mitochondrial pH induced by removal of Na+ from the medium. In isolated mitochondria, cariporide significantly reduced the rate and magnitude of passive matrix swelling induced by Na+ acetate. In isolated rat hearts submitted to 40-min ischemia at different temperatures (35.5 degrees, 37 degrees, or 38.5 degrees C) pretreatment with cariporide limited ATP depletion during the first 10 min of ischemia and cell death (lactate dehydrogenase release) during reperfusion. These effects were mimicked when a similar ATP preservation was achieved by hypothermia and were abolished when the sparing effect of cariporide on ATP was suppressed by hyperthermia. We conclude that cariporide acts at the mitochondrial level, delaying mitochondrial matrix acidification and delaying ATP exhaustion during ischemia. These effects can contribute to reduce cell death secondary to ischemia-reperfusion.     

Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms

Neurofibrillary tangles are composed of insoluble aggregates of the microtubule-associated protein tau. In Alzheimer's disease the accumulation of neurofibrillary tangles occurs in the absence of tau mutations. Here we present mice that develop pathology from non-mutant human tau, in the absence of other exogenous factors, including beta-amyloid. The pathology in these mice is Alzheimer-like, with hyperphosphorylated tau accumulating as aggregated paired helical filaments. This pathologic tau accumulates in the cell bodies and dendrites of neurons in a spatiotemporally relevant distribution.     

Enzymes that counteract nitrosative stress promote fungal virulence

Enzymes that protect cells from reactive oxygen species (superoxide dismutase, catalase, peroxidase) have well-established roles in mammalian biology and microbial pathogenesis. Two recently identified enzymes detoxify nitric oxide (NO)-related molecules; flavohemoglobin denitrosylase consumes NO, and S-nitrosoglutathione (GSNO) reductase metabolizes GSNO. Although both enzymes protect microorganisms from nitrosative challenge in vitro, their relevance has not been established in physiological contexts. Here we studied their biological functions in Cryptococcus neoformans, an established human fungal pathogen that replicates in macrophages and whose growth in vitro and in infected animals is controlled by NO bioactivity. We show that both flavohemoglobin denitrosylase and GSNO reductase contribute to C. neoformans pathogenesis. FHB1 and GNO1 mutations abolished NO- and GSNO-consuming activity, respectively. Growth of fhb1 mutant cells was inhibited by nitrosative challenge, whereas that of gno1 mutants was not. fhb1 mutants showed attenuated virulence in a murine model, and virulence was restored in iNOS(-/-) animals. Survival of the fhb1 mutant was also reduced in activated macrophages and restored to wild-type by inhibition of NOS activity. Combining mutations in flavohemoglobin and GSNO reductase, or flavohemoglobin and superoxide dismutase, further attenuated virulence. These studies illustrate that fungal pathogens elaborate enzymatic defenses against nitrosative stress mounted by the host.     

Evaluation of the Malaria Evaluation Program in the national laboratory network in Colombia

In 1995, the Parasitology Group--National Reference Laboratory--at the Instituto Nacional de Salud (INS) started a national malaria diagnosis program with the Public Health Laboratory Network which included training, indirect quality control, external quality control, technical assistance, advisory, reference and counter-reference, together with an annual review of the program. The purpose of this study was to carry out a three year (1997-1999) analysis of the program. In the indirect quality control program, average positive and negative concordances of 98% and 97%, respectively, and a kappa index of 0.95 were obtained by the state public health laboratories. In the external quality control program, an average concordance of 74.2% was obtained with an 89.2% participation of the registered laboratories. At the municipal level, the indirect quality control had an average concordance of 91.4% in positivity, 92.5% concordance in negativity, and a kappa index of 0.84. On the other hand, indirect quality control has been scarcely implemented by the state public health laboratories in the municipalities under their jurisdiction. In general, the program shows a good performance, despite some economic and conflict-related difficulties in the country, because people responsible at all levels for the Malaria Program have permanently carried out all other activities of the network, either according to annual programming or upon request. However, it is important to improve its coverage and the participation in its activities.     

Bacterial biodiversity from anthropogenic extreme environments: a hyper-alkaline and hyper-saline industrial residue contaminated by chromium and iron

Anthropogenic extreme environments are among the most interesting sites for the bioprospection of extremophiles since the selection pressures may favor the presence of microorganisms of great interest for taxonomical and astrobiological research as well as for bioremediation technologies and industrial applications. In this work, T-RFLP and 16S rRNA gene library analyses were carried out to describe the autochthonous bacterial populations from an industrial waste characterized as hyper-alkaline (pH between 9 and 14), hyper-saline (around 100 PSU) and highly contaminated with metals, mainly chromium (from 5 to 18 g kg^?1) and iron (from 2 to 108 g kg^?1). Due to matrix interference with DNA extraction, a protocol optimization step was required in order to carry out molecular analyses. The most abundant populations, as evaluated by both T-RFLP and 16S rRNA gene library analyses, were affiliated to Bacillus and Lysobacter genera. Lysobacter related sequences were present in the three samples: solid residue and lixiviate sediments from both dry and wet seasons. Sequences related to Thiobacillus were also found; although strains affiliated to this genus are known to have tolerance to metals, they have not previously been detected in alkaline environments. Together with Bacillus (already described as a metal reducer), such organisms could be of use in bioremediation technologies for reducing chromium, as well as for the prospection of enzymes of biotechnological interest.