Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

Dehalococcoides mccartyi strains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures of Dehalococcoides mccartyi strain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinct Dehalococcoides RDases and one Geobacter RDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.     

Dimerization of aurein 1.2: effects in structure, antimicrobial activity and aggregation of Cândida albicans cells

Antimicrobial peptides (AMPs) are a promising solution to face the antibiotic-resistant problem because they display little or no resistance effects. Dimeric analogues of select AMPs have shown pharmacotechnical advantages, making these molecules promising candidates for the development of novel antibiotic agents. Here, we evaluate the effects of dimerization on the structure and biological activity of the AMP aurein 1.2 (AU). AU and the C- and N-terminal dimers, (AU)₂K and E(AU)₂, respectively, were synthesized by solid-phase peptide synthesis. Circular dichroism spectra indicated that E(AU)₂ has a “coiled coil” structure in water while (AU)₂K has an α-helix structure. In contrast, AU displayed typical spectra for disordered structures. In LPC micelles, all peptides acquired a high amount of α-helix structure. Hemolytic and vesicle permeabilization assays showed that AU has a concentration dependence activity, while this effect was less pronounced for dimeric versions, suggesting that dimerization may change the mechanism of action of AU. Notably, the antimicrobial activity against bacteria and yeast decreased with dimerization. However, dimeric peptides promoted the aggregation of C. albicans. The ability to aggregate yeast cells makes dimeric versions of AU attractive candidates to inhibit the adhesion of C. albicans to biological targets and medical devices, preventing disease caused by this fungus.     

Enrichment of hexachlorobenzene and 1,3,5-trichlorobenzene transforming bacteria from sediments in Germany and Vietnam

Bacterial cultures were enriched from sediments in Germany and Vietnam reductively dechlorinating hexachlorobenzene and the highly persistent 1,3,5-trichlorobenzene to monochlorobenzene. The main products of the reductive dechlorination of hexachlorobenzene were monochlorobenzene and dichlorobenzenes (1,2-; 1,3- and 1,4-dichlorobenzene) while no trichlorobenzenes accumulated. For the reductive dechlorination of 1,3,5-trichlorobenzene with the mixed culture from Vietnam sediment, 1,3- dichlorobenzene and monochlorobenzene were produced as intermediate and final end-product, respectively. The pattern of dechlorination did not change when the cultures were repeatedly exposed to oxygen over seven transfers demonstrating oxygen tolerance of the dechlorinating bacteria. However, reductive dechlorination of 1,3,5-trichlorobenzene was inhibited by vancomycin at a concentration of 5 mg L^?1. Vancomycin delayed reductive dechlorination of hexachlorobenzene in mixed cultures by about 6 months. When repeatedly applied, vancomycin completely abolished the ability of the mixed culture to transform hexachlorobenzene. Sensitivity to vancomycin and insensitivity to brief exposure of oxygen indicates that the dechlorinating bacteria in the mixed cultures did not belong to the genus Dehalococcoides.     

The EBM-DPSER Conceptual Model: Integrating Ecosystem Services into the DPSIR Framework

There is a pressing need to integrate biophysical and human dimensions science to better inform holistic ecosystem management supporting the transition from single species or single-sector management to multi-sector ecosystem-based management. Ecosystem-based management should focus upon ecosystem services, since they reflect societal goals, values, desires, and benefits. The inclusion of ecosystem services into holistic management strategies improves management by better capturing the diversity of positive and negative human-natural interactions and making explicit the benefits to society. To facilitate this inclusion, we propose a conceptual model that merges the broadly applied Driver, Pressure, State, Impact, and Response (DPSIR) conceptual model with ecosystem services yielding a Driver, Pressure, State, Ecosystem service, and Response (EBM-DPSER) conceptual model. The impact module in traditional DPSIR models focuses attention upon negative anthropomorphic impacts on the ecosystem; by replacing impacts with ecosystem services the EBM-DPSER model incorporates not only negative, but also positive changes in the ecosystem. Responses occur as a result of changes in ecosystem services and include inter alia management actions directed at proactively altering human population or individual behavior and infrastructure to meet societal goals. The EBM-DPSER conceptual model was applied to the Florida Keys and Dry Tortugas marine ecosystem as a case study to illustrate how it can inform management decisions. This case study captures our system-level understanding and results in a more holistic representation of ecosystem and human society interactions, thus improving our ability to identify trade-offs. The EBM-DPSER model should be a useful operational tool for implementing EBM, in that it fully integrates our knowledge of all ecosystem components while focusing management attention upon those aspects of the ecosystem most important to human society and does so within a framework already familiar to resource managers.     

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.     

Caregivers’ Attitudes towards HIV Testing and Disclosure of HIV Status to At-Risk Children in Rural Uganda

Caregivers of HIV-positive children were interviewed in the Mbarara and Isingiro districts of Uganda to identify current trends in practices related to HIV testing and the disclosure of HIV status to the child. A total of 28 caregivers of at least one HIV-positive child participated in semi-structured interviews exploring when and why they tested the child for HIV, when the child was informed of their positive status, and what the caregiver did to prepare themselves and the child for status disclosure. For a majority (96%) of respondents, the decision to test the child for HIV was due to existing illness in either the child or a relative. Other common themes identified included the existence of stigma in the caregivers’ communities and doubt that the children truly understood what was being explained to them when their status was disclosed. Most (65%) children were informed of their HIV status between the ages of 5 and 9, with the mean age of disclosure occurring at the age of 7. General provision of HIV information typically began at the same age as disclosure, and as many as two thirds (64%) of the caregivers sought advice from an HIV counsellor prior to disclosure. How a caregiver chose to prepare themselves and the child did not affect the caregiver’s perception of whether the disclosure experience was beneficial or not. These findings suggest that the HIV disclosure experience in Mbarara and Isingiro districts differs from current guidelines, especially with respect to age of disclosure, how caregivers prepare themselves and the child, and approaching disclosure as an ongoing process. The doubts expressed by caregivers regarding the child’s level of HIV understanding following the disclosure experience suggest the children may be insufficiently prepared at the time of the initial disclosure event. The findings also suggest that examining the content of pre-disclosure counselling and HIV education, and how health care professionals are trained to facilitate the disclosure process as important avenues for further research.     

Habitat heterogeneity drives bird species richness,nestedness and habitat selection by individual species in fluvial wetlands of the Paraná River,Argentina

We assessed the relationship between habitat heterogeneity and bird species richness and composition within wetlands of the floodplain of the Middle Paraná River, Argentina. Given the high habitat heterogeneity in these wetland systems, we sought to determine whether (i) there was a positive relationship between bird species richness and habitat heterogeneity; (ii) whether bird species richness was associated with certain types of individual habitat types; (iii) whether there was a pattern of species nestedness and turnover between sites as a function of habitat heterogeneity and composition, respectively; and (iv) whether individual species exhibited associations with habitat heterogeneity. Point counts were used to survey birds at 60 sites. We estimated the area of eight habitat types found within a 200‐m radius from the centre of each site and calculated number and Pielou's evenness of habitat types. These indices, together with area proportion of each habitat type, were used as explanatory factors of bird species richness in linear regression models. Habitat heterogeneity per se rather than area of individual habitat types was a more important predictor of species richness in these fluvial wetlands. Sites with more habitat types supported more bird species. Results showed that individual bird species were associated with different habitat types and, therefore, sites that contained more habitat types contained more species. Number of habitat types accounted for species nestedness between sites whereas composition of habitat types accounted for species turnover between sites. Results suggest that selection of heterogeneous sites by individual species could help explain the positive heterogeneity–species richness relationship. Our findings highlight the importance of habitat heterogeneity per se resulting from flood disturbances in maintaining bird richness in fluvial systems.