The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.     

Self-stacked submersible microbial fuel cell (SSMFC) for improved remote power generation from lake sediments

Electric energy can be harvested from aquatic sediments by utilizing microbial fuel cells (MFCs). A main challenge of this application is the limited voltage output. In this study, an innovative self-stacked submersible MFC (SSMFC) was developed to improve the voltage generation from lake sediments. The SSMFC successfully produced a maximum power density of 294 mW/m(2) and had an open circuit voltage (OCV) of 1.12 V. However, voltage reversal was observed in one cell at high current density. Investigation on the cause for voltage reversal revealed that voltage reversal was occurring only when low external resistance (≤ 400Ω in this study) was applied. In addition, the internal resistance and OCV were the most important parameters for predicting which cell unit had the highest probability to undergo voltage reversal. Use of a capacitor was found to be an effective way to prevent voltage reversal and at the same time store power. These results provide new insight into the development of effective MFC system, capable of extracting energy and promoting bioremediation of organic pollutants from sediments.     

Biohydrogen production in granular up-flow anaerobic sludge blanket (UASB) reactors with mixed cultures under hyper-thermophilic temperature (70 degrees C)

Hyper-thermophilic hydrogen production without methane was demonstrated for the first time in granular up-flow anaerobic sludge blanket (UASB) system fed with glucose using mixed cultures. The maximum hydrogen yield in this study was 2.47 +/- 0.15 mol H2/mol glucose. This high yield has never been previously reported in mixed culture systems and it was likely due to more favorable thermodynamic conditions at hyper-thermophilic temperatures. Different start-up strategies (bromoethanosulfonate (BES) addition and flow recycle) were evaluated. BES addition during start-up prevented the establishment of methanogenic cultures in granules. Flow recycle was important to achieve higher hydrogen yield through enriching better hydrogen-producing organisms and reduced the start-up period as well. This study indicated UASB system was a promising system for hydrogen production.     

Anammox for ammonia removal from pig manure effluents: Effect of organic matter content on process performance

The anammox process, under different organic loading rates (COD), was evaluated using a semi-continuous UASB reactor at 37 °C. Three different substrates were used: initially, synthetic wastewater, and later, two different pig manure effluents (after UASB-post-digestion and after partial oxidation) diluted with synthetic wastewater. High ammonium removal was achieved, up to 92.1 ± 4.9% for diluted UASB-post-digested effluent (95 mg COD L^?1) and up to 98.5 ± 0.8% for diluted partially oxidized effluent (121 mg COD L^?1). Mass balance clearly showed that an increase in organic loading (from 95 mg COD L^?1 to 237 mg COD L^?1 and from 121 mg COD L^?1 to 290 mg COD L^?1 for the UASB-post-digested effluent and the partially oxidized effluent, respectively) negatively affected the anammox process and facilitated heterotrophic denitrification. Partial oxidation as a pre-treatment method improved ammonium removal at high organic matter concentration. Up to threshold organic load concentration of 142 mg COD L^?1 of UASB-post-digested effluent and 242 mg COD L^?1 of partially oxidized effluent, no effect of organic loading on ammonia removal was registered (ammonium removal was above 80%). However, COD concentrations above 237 mg L^?1 (loading rate of 112 mg COD L^?1 day^?1) for post-digested effluent and above 290 mg L^?1 (loading rate of 136 mg COD L^?1 day^?1) for partially oxidized effluent resulted in complete cease of ammonium removal. Results obtained showed that, denitrification and anammox process were simultaneously occurring in the reactor. Denitrification became the dominant ammonium removal process when the COD loading was increased.     

Generation of Electricity and Analysis of Microbial Communities in Wheat Straw Biomass-Powered Microbial Fuel Cells

Electricity generation from wheat straw hydrolysate and the microbial ecology of electricity-producing microbial communities developed in two-chamber microbial fuel cells (MFCs) were investigated. The power density reached 123 mW/m² with an initial hydrolysate concentration of 1,000 mg chemical oxygen demand (COD)/liter, while coulombic efficiencies ranged from 37.1 to 15.5%, corresponding to the initial hydrolysate concentrations of 250 to 2,000 mg COD/liter. The suspended bacteria found were different from the bacteria immobilized in the biofilm, and they played different roles in electricity generation from the hydrolysate. The bacteria in the biofilm were consortia with sequences similar to those of Bacteroidetes (40% of sequences), Alphaproteobacteria (20%), Bacillus (20%), Deltaproteobacteria (10%), and Gammaproteobacteria (10%), while the suspended consortia were predominately Bacillus (22.2%). The results of this study can contribute to improving understanding of and optimizing electricity generation in microbial fuel cells.Wheat straw is one of the most abundant renewable resources. According to the Food and Agriculture Organization of the United Nations, approximately 1.9 × 10⁹ tons of wheat straw annually are produced worldwide, accompanied by 6.2 × 10⁸ tons of wheat production. Wheat straw is composed of 35 to 45% cellulose and 20 to 30% hemicelluloses with a relatively low lignin content (<20%) (42). The hemicellulose fraction of the straw is easily hydrolyzed to its constituent sugars by a hydrothermal treatment process, forming a carbohydrate-enriched liquid hydrolysate (46). Chemical and biological approaches to sustainable energy production from the liquefied hydrolysates to energy carriers, such as methane, ethanol, and H₂, have been developed. However, many of these approaches encounter technical and economical hurdles (10, 12, 15, 16). An alternative strategy is direct conversion of wheat straw biomass to electrical energy in microbial fuel cells (MFCs).MFCs are bioelectrochemical reactors in which microorganisms mediate the direct conversion of chemical energy stored in organic matter or bulk biomass into electrical energy (12, 15, 16, 40). Various substrates, such as simple carbohydrates, low-molecular-weight organic acids, starch, amino acids, chitin, cellulose, domestic wastewater, food-processing wastewater, recycled paper wastewater, and marine sediment organic matter, have been successfully utilized for power generation in MFCs (16-18, 27, 30, 33). To understand the microbial constraints on various fuel-powered MFCs, microbial communities have been characterized by several groups. Microbial communities from various systems are very different and often diverse, ranging from well-known metal- and anode-reducing bacteria to unknown exoelectrogens (1, 20, 21). It has been found that parameters such as the substrates used as fuels and the inocula used for starting up the MFCs can influence the anode bacterial communities in an MFC, which subsequently influence the efficiency of the MFCs (3, 14, 22, 38, 44). Different pure substrates, such as acetate, glucose, and lactate, were used as fuel to compare the microbial communities that developed in the MFCs. Regardless of the different substrates, all anode communities contained sequences closely affiliated with Geobacter sulfurreducens (>99% similarity) and an uncultured bacterium clone belonging to the family Bacteroidaceae (99% similarity). Firmicutes were only found in glucose-fed MFCs (20). Microbial-community analyses of MFCs powered with complex substrates have also been performed by several researchers, and their results were very diverse. The microbial community in starch wastewater-powered MFC was dominated by unidentified bacteria (35.9%), followed by Betaproteobacteria (25.0%), Alphaproteobacteria (20.1%), and the Cytophaga/Flexibacter/Bacteroides group (19.0%) (21). The anode-attached consortia in a cellulose-powered MFC were related to Clostridium spp., while Comamonas spp. were abundant in the suspended consortia (13). Although many studies have reported the microbial compositions of MFCs, it is still unclear which microbial communities develop as a function of the external parameters.Wheat straw biomass constitutes a large source for bioenergy production and shows promising prospects for electricity generation in MFCs. Therefore, wheat straw biomass was used to study the microbial communities that develop during the operation of an MFC in order to better understand the microbial electrochemical roles and potentially improve MFC performance.The objectives of this study were to (i) test wheat straw hydrolysate as a potential fuel in an MFC for electricity generation and (ii) study the microbial composition and evolution of electricity-producing communities in a two-chamber MFC system. Phylogenetic-diversity analysis of the enriched consortia was conducted to verify the presence of hydrolytic and respiratory anaerobes that could couple hydrolysate oxidation with proton reduction in the anode chamber. This is the first report of exploiting microbial communities for direct conversion of wheat straw hydrolysate to electrical energy in an MFC.     

Integrating In Silico and In Vitro Analysis of Peptide Binding Affinity to HLA-Cw*0102: A Bioinformatic Approach to the Prediction of New Epitopes

Background

Predictive models of peptide-Major Histocompatibility Complex (MHC) binding affinity are important components of modern computational immunovaccinology. Here, we describe the development and deployment of a reliable peptide-binding prediction method for a previously poorly-characterized human MHC class I allele, HLA-Cw*0102.

Methodology/Findings

Using an in-house, flow cytometry-based MHC stabilization assay we generated novel peptide binding data, from which we derived a precise two-dimensional quantitative structure-activity relationship (2D-QSAR) binding model. This allowed us to explore the peptide specificity of HLA-Cw*0102 molecule in detail. We used this model to design peptides optimized for HLA-Cw*0102-binding. Experimental analysis showed these peptides to have high binding affinities for the HLA-Cw*0102 molecule. As a functional validation of our approach, we also predicted HLA-Cw*0102-binding peptides within the HIV-1 genome, identifying a set of potent binding peptides. The most affine of these binding peptides was subsequently determined to be an epitope recognized in a subset of HLA-Cw*0102-positive individuals chronically infected with HIV-1.

Conclusions/Significance

A functionally-validated in silico-in vitro approach to the reliable and efficient prediction of peptide binding to a previously uncharacterized human MHC allele HLA-Cw*0102 was developed. This technique is generally applicable to all T cell epitope identification problems in immunology and vaccinology.     

Effect of operational and design parameters on removal efficiency of pilot-scale FWS constructed wetlands and comparison with HSF systems

In order to investigate the effect of temperature, hydraulic residence time (HRT), vegetation type, substrate material and wetland shape on the performance of free-water surface (FWS) constructed wetlands treating wastewater, 5 pilot-scale units were constructed and operated continuously from December 2004 until March 2007 in parallel experiments. Four of the units (A, B, C, D) were rectangular in plan view with dimensions 3.40 m in length and 0.85 m in width, and contained substrate material at a thickness of 0.45 m. The fifth unit (E) had a trapezoidal plan view shape, with a width at the inlet of 1.15 m and at the outlet of 0.55 m, while the length and the thickness of the substrate were the same as in the other four. All units operated at a water depth of 0.10 m. Units B–E contained clay substrate and unit A contained sand. The four units with clay were planted as follows: two with cattails (B and E), one with common reeds (C), and one with giant reeds (D). Unit A, containing sand, was planted with cattails. Planting and substrate material combinations were appropriate for comparison of the effect of vegetation and material type on the function of the system. Synthetic wastewater was introduced in the units. During the operation period four HRTs (i.e., 6 days, 8 days, 14 days and 20 days) were used, while wastewater temperatures varied from about 0.0 °C to 29.1 °C. The removal performance of the five constructed wetland units was good, since it reached on the average 77.5%, 67.9%, 60.4%, 53.9%, 56.0% and 51.7% for BOD, COD, TKN, ammonia (NH₄-N), ortho-phosphate (PO₄-P) and total phosphorus (TP), respectively. BOD and phosphorus removal efficiencies showed dependence on temperature in most units. The 14-day HRT was found adequate for acceptable removal of organic matter, nitrogen and phosphorus for most temperatures. A 20-day HRT is recommended for acceptable removal of BOD and PO₄-P in the cold season. The unit with the trapezoidal plan view shape showed the best performance, with mean removals of 80.1%, 73.5%, 70.4%, 68.6%, 64.7% and 63.5% for BOD, COD, TKN, NH₄-N, PO₄-P and TP, respectively. The cattail was found statistically more efficient than the other two plants in COD and PO₄-P removal. The unit that contained the clay substrate was found statistically more efficient in phosphorus removal than the unit containing sand. HSF CW units were found more efficient than FWS units in removal of most pollutant.     

Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish,Mice and Humans

Background

Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration.

Methods and Findings

We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-β resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus.

Conclusions

Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development.