Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury

Previous results on nitric oxide (NO) metabolism after traumatic brain injury (TBI) show variations in NO availability and controversial effects of exogenous nitric oxide synthase (NOS)-inhibitors. Furthermore, elevated levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) were reported in cerebro-spinal fluid (CSF) after traumatic subarachnoid hemorrhage (SAH). Therefore, we examined whether ADMA and the enzymes involved in NO- and ADMA-metabolism are expressed in brain tissue after TBI and if time-dependent changes occur. TBI was induced by controlled cortical impact injury (CCII) and neurological performance was monitored. Expression of NOS, ADMA, dimethylarginine dimethylaminohydrolases (DDAH) and protein-arginine methyltransferase 1 (PRMT1) was determined by immunostaining in different brain regions and at various time-points after CCII. ADMA and PRMT1 expression decreased in all animals after TBI compared to the control group, while DDAH1 and DDAH2 expression increased in comparison to controls. Furthermore, perilesionally ADMA is positively correlated with neuroscore performance, while DDAH1 and DDAH2 are negatively correlated. ADMA and its metabolizing enzymes show significant temporal changes after TBI and may be new targets in TBI treatment.     

Isolation and Structural Elucidation of Antiproliferative Compounds of Lipidic Fractions from White Shrimp Muscle (Litopenaeus vannamei)

Shrimp is one of the most popular seafood items worldwide, and has been reported as a source of chemopreventive compounds. In this study, shrimp lipids were separated by solvent partition and further fractionated by semi-preparative RP-HPLC and finally by open column chromatography in order to obtain isolated antiproliferative compounds. Antiproliferative activity was assessed by inhibition of M12.C3.F6 murine cell growth using the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay. The methanolic fraction showed the highest antiproliferative activity; this fraction was separated into 15 different sub-fractions (M1–M15). Fractions M8, M9, M10, M12, and M13 were antiproliferative at 100 µg/mL and they were further tested at lower concentrations. Fractions M12 and M13 exerted the highest growth inhibition with an IC₅₀ of 19.5 ± 8.6 and 34.9 ± 7.3 µg/mL, respectively. Fraction M12 was further fractionated in three sub-fractions M12a, M12b, and M12c. Fraction M12a was identified as di-ethyl-hexyl-phthalate, fraction M12b as a triglyceride substituted by at least two fatty acids (predominantly oleic acid accompanied with eicosapentaenoic acid) and fraction M12c as another triglyceride substituted with eicosapentaenoic acid and saturated fatty acids. Bioactive triglyceride contained in M12c exerted the highest antiproliferative activity with an IC₅₀ of 11.33 ± 5.6 µg/mL. Biological activity in shrimp had been previously attributed to astaxanthin; this study demonstrated that polyunsaturated fatty acids are the main compounds responsible for antiproliferative activity.     

Specificity of a UDP‐GalNAc Pyranose–Furanose Mutase: A Potential Therapeutic Target for Campylobacter jejuni Infections

Pyranose–furanose mutases are essential enzymes in the life cycle of a number of microorganisms, but are absent in mammalian systems, and hence represent novel targets for drug development. To date, all such mutases show preferential recognition of a single substrate (e.g., UDP‐Gal). We report here the detailed structural characterization of the first bifunctional pyranose–furanose mutase, which recognizes both UDP‐Gal and UDP‐GalNAc. The enzyme under investigation (cjUNGM) is involved in the biosynthesis of capsular polysaccharides (CPSs) in Campylobacter jejuni 11168. These CPSs are known virulence factors that are required for adhesion and invasion of human epithelial cells. Using a combination of UV/visible spectroscopy, X‐ray crystallography, saturation transfer difference NMR spectroscopy, molecular dynamics and CORCEMA‐ST calculations, we have characterized the binding of the enzyme to both UDP‐Galp and UDP‐GalpNAc, and compared these interactions with those of a homologous monofunctional mutase enzyme from E. coli (ecUGM). These studies reveal that two arginines in cjUNGM, Arg59 and Arg168, play critical roles in the catalytic mechanism of the enzyme and in controlling its specificity to ultimately lead to a GalfNAc‐containing CPS. In ecUGM, these arginines are replaced with histidine and lysine, respectively, and this results in an enzyme that is selective for UDP‐Gal. We propose that these changes in amino acids allow C. jejuni 11168 to produce suitable quantities of the sugar nucleotide substrate required for the assembly of a CPS containing GalfNAc, which is essential for viability.     

Combined Mutagenesis and Kinetics Characterization of the Bilin‐Binding GAF Domain of the Protein Slr1393 from the Cyanobacterium Synechocystis PCC6803

The gene slr1393 from Synechocystis sp. PCC6803 encodes a protein composed of three GAF domains, a PAS domain, and a histidine kinase domain. GAF3 is the sole domain able to bind phycocyanobilin (PCB) as chromophore and to accomplish photochemistry: switching between a red‐absorbing parental and a green‐absorbing photoproduct state (λ_max=649 and 536 nm, respectively). Conversions in both directions were followed by time‐resolved absorption spectroscopy with the separately expressed GAF3 domain of Slr1393. Global fit analysis of the recorded absorbance changes yielded three lifetimes (3.2 μs, 390 μs, and 1.5 ms) for the red‐to‐green conversion, and 1.2 μs, 340 μs, and 1 ms for the green‐to‐red conversion. In addition to the wild‐type (WT) protein, 24 mutated proteins were studied spectroscopically. The design of these site‐directed mutations was based on sequence alignments with related proteins and by employing the crystal structure of AnPixJg2 (PDB ID: 3W2Z), a Slr1393 orthologous from Anabaena sp. PCC7120. The structure of AnPixJg2 was also used as template for model building, thus confirming the strong structural similarity between the proteins, and for identifying amino acids to target for mutagenesis. Only amino acids in close proximity to the chromophore were exchanged, as these were considered likely to have an impact on the spectral and dynamic properties. Three groups of mutants were found: some showed absorption features similar to the WT protein, a second group showed modified absorbance properties, and the third group had lost the ability to bind the chromophore. The most unexpected result was obtained for the exchange at residue 532 (N532Y). In vivo assembly yielded a red‐absorbing, WT‐like protein. Irradiation, however, not only converted it into the green‐absorbing form, but also produced a 660 nm, further‐red‐shifted absorbance band. This photoproduct was fully reversible to the parental form upon green light irradiation.     

Dynamic-MFA examination of Chilean housing stock: long-term changes and earthquake damage

The dynamics and metabolism of the built environment are important for understanding the construction, renovation and demolition activities that characterize its long-term physical development. Previous research on building stock development is limited, particularly for developing countries. As one of the most seismically active countries in the world, Chile's built environment is constantly subjected to losses. This study included earthquake vulnerability and damage in a dynamic material flow analysis (MFA) of the Chilean housing stock. Stock and flows of residential floor area were modelled using population and life style parameters. Demolition was modelled assuming a constant demolition rate. By characterizing the building stock according to vulnerability classes and establishing a mass balance per class, the effects of different typology distributions on the overall damage to the stock caused by earthquakes could be assessed. Scenarios with different trends in typology distribution for new construction and renovation suggest that significant potential exists for reducing future earthquake damage in the Chilean housing stock. This is best achieved by gradually reducing the vulnerability of buildings to earthquakes. Legislation, building standards and financial instruments could be used to ensure such reductions in earthquake damage and thereby provide socio-economic benefits in the future.     

Springtime Monitoring of Passive Cool Roofs in Subtropical Climate

Passive strategies for acclimatization of buildings have been studied by several authors in many countries, especially the evaporative and radiant cooling techniques. Fiber cement tiles are very common in popular constructions due to their low cost. However, they have over twice of the value thermal transmittance indicated to this bioclimatic zone according to Brazilian guidelines. The objective is to present an alternative to reduce high temperatures on fiber cement tiles. In this paper, the monitoring of passive cooling of roofing during the spring season in a city with subtropical climate is described. Single and combined reflective and evaporative cooling systems were studied in different environmental conditions. Internal surface temperatures of tiles were monitored together with weather variables. Results show a decrease of about 6 ℃, 9 ℃, 10 ℃ and 11 ℃ as compared to the original tiles according to environment conditions and the combined passive cooling techniques. These results allow for the conclusion that the use of passive cooling techniques opens up new possibilities to attenuate the internal surface temperatures of tiles and to consequently decrease the roofing solar heat gain into buildings, thus, providing less air cooling energy consumption.     

Dissecting Anion Effects in Gold(I)‐Catalyzed Intermolecular Cycloadditions

From a series of gold complexes of the type [t‐BuXPhosAu(MeCN)]X (X=anion), the best results in intermolecular gold(I)‐catalyzed reactions are obtained with the complex with the bulky and soft anion BAr₄^F− [BAr₄^F−=3,5‐bis(trifluoromethyl)phenylborate] improving the original protocols by 10–30% yield. A kinetic study on the [2+2] cycloaddition reaction of alkynes with alkenes is consistent with an scenario in which the rate‐determining step is the ligand exchange to generate the (η²‐phenylacetylene)gold(I) complex. We have studied in detail the subtle differences that can be attributed to the anion in this formation, which result in a substantial decrease in the formation of unproductive σ,π‐(alkyne)digold(I) complexes by destabilizing the conjugated acid formed.

     

Ageing of V2O5 thin films induced by Li intercalation multi-cycling

Cyclic voltammetry, XPS, RBS and AFM have been combined to study the ageing mechanism of Li intercalation in V₂O₅ thin films prepared by thermal oxidation of vanadium metal. Multi-cycling tests were performed in 1 M LiClO₄-PC in the potential range E ∈ [3.8, 2.8 V] versus Li/Li⁺, corresponding to the α-to-δ phase transition. XPS and AFM were performed using direct anaerobic and anhydrous transfer. Capacity fading remains inferior to 20% during ∼2500 cycles. XPS shows slight modifications of the oxide composition with a V⁴⁺ concentration increasing from ∼5% prior to cycling to ∼16–27% after cycling, due to Li trapped in the oxide film and to the loss of V₂O₅ active material. The presence of lithium carbonate and lithium-alkyl carbonate species evidences the formation of the so-called SEI layer. AFM evidences the loss of crystalline material by grain exfoliation from the outer V₂O₅ layer of the oxide film. By further exfoliation, the inner VO₂ layer of the oxide film is reached and pits are formed, occupying ∼9–13% of the surface. This de-cohesion at grain boundaries is attributed to the strain generated by repeated lattice distortions. After 3300 cycles, the disappearance of lithium carbonates, whereas Li-alkyl carbonates and/or Li-alkoxides remain on the surface, indicates the dissolution and/or conversion of the SEI layer. After 4500 cycles, the oxide film became very labile and could be stripped away by rinsing to reveal the vanadium metal substrate.