Development and characterisation of self-assembled graphene hydrogel-based anodes for bioelectrochemical systems.

In this work, we report a simple and scalable method to produce high efficiency 3D graphene-based electrodes (GH) for bioelectrochemical systems. GH were obtained by self-assembly of graphene oxide, through slow reduction with ascorbic acid over conductive mesh-works (carbon cloth and stainless-steel). The GH structure and composition were characterised by electron microscopy (SEM) and spectroscopy (FTIR and Raman), whereas the electrodes' performance was tested by chronoamperometry and cyclic voltammetry in a microbial electrolysis cell (MEC) inoculated with a pure culture of G. sulfurreducens. The hydrogel had a broad pore size distribution (>1 µm), which allowed bacterial colonisation within the framework. The macro-porous structure and chemical properties of the hydrogel rendered a higher bacterial loading capacity and substrate oxidation rate than other carbonaceous materials, including different reported graphene electrodes, which significantly increased MEC performance.

Computational Functional Analysis of Lipid Metabolic Enzymes.

The computational analysis of enzymes that participate in lipid metabolism has both common and unique challenges when compared to the whole protein universe. Some of the hurdles that interfere with the functional annotation of lipid metabolic enzymes that are common to other pathways include the definition of proper starting datasets, the construction of reliable multiple sequence alignments, the definition of appropriate evolutionary models, and the reconstruction of phylogenetic trees with high statistical support, particularly for large datasets. Most enzymes that take part in lipid metabolism belong to complex superfamilies with many members that are not involved in lipid metabolism. In addition, some enzymes that do not have sequence similarity catalyze similar or even identical reactions. Some of the challenges that, albeit not unique, are more specific to lipid metabolism refer to the high compartmentalization of the routes, the catalysis in hydrophobic environments and, related to this, the function near or in biological membranes.In this work, we provide guidelines intended to assist in the proper functional annotation of lipid metabolic enzymes, based on previous experiences related to the phospholipase D superfamily and the annotation of the triglyceride synthesis pathway in algae. We describe a pipeline that starts with the definition of an initial set of sequences to be used in similarity-based searches and ends in the reconstruction of phylogenies. We also mention the main issues that have to be taken into consideration when using tools to analyze subcellular localization, hydrophobicity patterns, or presence of transmembrane domains in lipid metabolic enzymes.

Analysis of triglyceride synthesis unveils a green algal soluble diacylglycerol acyltransferase and provides clues to potential enzymatic components of the chloroplast pathway.

BACKGROUND: Microalgal triglyceride (TAG) synthesis has attracted considerable attention. Particular emphasis has been put towards characterizing the algal homologs of the canonical rate-limiting enzymes, diacylglycerol acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT). Less work has been done to analyze homologs from a phylogenetic perspective. In this work, we used HMMER iterative profiling and phylogenetic and functional analyses to determine the number and sequence characteristics of algal DGAT and PDAT, as well as related sequences that constitute their corresponding superfamilies. We included most algae with available genomes, as well as representative eukaryotic and prokaryotic species. RESULTS: Amongst our main findings, we identified a novel clade of DGAT1-like proteins exclusive to red algae and glaucophyta and a previously uncharacterized subclade of DGAT2 proteins with an unusual number of transmembrane segments. Our analysis also revealed the existence of a novel DGAT exclusive to green algae with moderate similarity to plant soluble DGAT3. The DGAT3 clade shares a most recent ancestor with a group of uncharacterized proteins from cyanobacteria. Subcellular targeting prediction suggests that most green algal DGAT3 proteins are imported to the chloroplast, evidencing that the green algal chloroplast might have a soluble pathway for the de novo synthesis of TAGs. Heterologous expression of C. reinhardtii DGAT3 produces an increase in the accumulation of TAG, as evidenced by thin layer chromatography. CONCLUSIONS: Our analysis contributes to advance in the knowledge of complex superfamilies involved in lipid metabolism and provides clues to possible enzymatic players of chloroplast TAG synthesis.

Progesterone regulates the expression and activity of two mouse isoforms of the glycoprotein folding sensor UDP-Glc: glycoprotein glucosyltransferase (UGGT).

UDP-Glucose:glycoprotein glucosyltransferase (UGGT) is a central component of the endoplasmic reticulum (ER) glycoprotein-folding quality control system, which prevents the exit of partially folded species. UGGT activity can be regulated by the accumulation of misfolded proteins in the ER, a stimulus that triggers a complex signaling pathway known as unfolded protein response (UPR) which is closely associated with inflammation and disease. In this work, we investigated the effect of progesterone (P4) on the expression and activity of UGGT in a mouse hybridoma. We detected the expression of two UGGT isoforms, UGGT1 and UGGT2, and demonstrated that both isoforms are active in these cells. Interestingly, the expression of each isoform is regulated by high physiological P4 concentrations. This work provides the first evidence of a hormonal regulation of UGGT isoform expression and activity, which might influence the glycoprotein quality control mechanism. These findings could contribute to the study of pathologies triggered by the accumulation of misfolded proteins.

Efficacy, safety and cost-efficiency of using an alternative technique for automated exchange transfusion in pediatric patients with sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) patients suffer complications requiring simple and/or exchange transfusion. In 1999 we developed an automated exchange technique using infusion pumps and vascular catheters (IV Pump Method). OBJECTIVE: To prove that IV Pump Method is cost-efficient, and as safe and effective as automated cell separators. METHODS: Retrospective chart review of SCD patients requiring exchange transfusion admitted to PICU from 2003-2009. Evaluated method used, complications, costs, and Hemoglobin S% (HgS%) change, excluding patients not requiring exchange transfusion. RESULTS: Cost-reduction with IV Pump Method is around $1000. Average HgS% reduction using IV Pump Method was 30.3 vs. 28.8 in Blood Cell Separator group (p = 0.84). We had no complications or mortalities, with the majority of patients being male (p = 0.03) and on the oldest age group (11-19 y/o) for both methods. CONCLUSION: The IV Pump Method is a safe, effective, and cost-efficient alternative to perform exchange transfusion.

Multiparity increases trophoblast invasion and vascular endothelial growth factor expression at the maternal-fetal interface in mice.

To analyze immunomodulating effects related to parity status, we studied trophoblast invasion grade, placental expression and systemic concentration of VEGF and its receptor Flt-1 in normal fertile (CBA/JxBALB/c) mice and abortion-prone (CBA/JxDBA/2) H-2(d)xH-2(k) mice. BALB/c or DBA/2 mated CBA/J females were, respectively, divided into the following groups: primiparous young (3.0+/-0.5 months old); primiparous old (8.5+/-0.5 months old) and multiparous old (8.5+/-0.5 months old, with 4 pregnancies). Immunohistochemical analysis of term placentae from both multiparous groups revealed various layers of invasive trophoblast tissue, identified as cytokeratin+/vimentin- cells, in contrast to the single layer detected in the placentae of primiparous animals, indicating that multiparity increases trophoblast invasion regardless of the success of the pregnancy outcome. Invasive trophoblast tissue from primiparous CBA/JxDBA/2 placentae showed diminished VEGF expression in comparison with the normal fertile group, while both multiparous groups demonstrated high expression of VEGF in the invasive trophoblast tissue. Placental expression of Flt-1 was similar in all groups. However, the primiparous CBA/JxBALB/c group showed the highest plasma concentration of sFlt-1 at term, while both multiparous groups demonstrated low circulating levels. No differences in circulating VEGF levels were observed among the groups. These results demonstrate an increase in trophoblast invasion tissue and expression of VEGF in the maternal-fetal interface in multiparous mice compared to primiparous mice. Moreover, the placenta appears to be able to regulate the circulating levels of VEGF by releasing sFlt-1.