VKORC1L1, an Enzyme Rescuing the Vitamin K 2,3-Epoxide Reductase Activity in Some Extrahepatic Tissues during Anticoagulation Therapy

Vitamin K is involved in the γ-carboxylation of the vitamin K-dependent proteins, and vitamin K epoxide is a by-product of this reaction. Due to the limited intake of vitamin K, its regeneration is necessary and involves vitamin K 2,3-epoxide reductase (VKOR) activity. This activity is known to be supported by VKORC1 protein, but recently a second gene, VKORC1L1, appears to be able to support this activity when the encoded protein is expressed in HEK293T cells. Nevertheless, this protein was described as being responsible for driving the vitamin K-mediated antioxidation pathways. In this paper we precisely analyzed the catalytic properties of VKORC1L1 when expressed in Pichia pastoris and more particularly its susceptibility to vitamin K antagonists. Vitamin K antagonists are also inhibitors of VKORC1L1, but this enzyme appears to be 50-fold more resistant to vitamin K antagonists than VKORC1. The expression of Vkorc1l1 mRNA was observed in all tissues assayed, i.e. in C57BL/6 wild type and VKORC1-deficient mouse liver, lung, and testis and rat liver, lung, brain, kidney, testis, and osteoblastic cells. The characterization of VKOR activity in extrahepatic tissues demonstrated that a part of the VKOR activity, more or less important according to the tissue, may be supported by VKORC1L1 enzyme especially in testis, lung, and osteoblasts. Therefore, the involvement of VKORC1L1 in VKOR activity partly explains the low susceptibility of some extrahepatic tissues to vitamin K antagonists and the lack of effects of vitamin K antagonists on the functionality of the vitamin K-dependent protein produced by extrahepatic tissues such as matrix Gla protein or osteocalcin.     

Identification of Pauses during Formation of HIV-1 Virus Like Particles

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.     

Surface functionalization of carbon nanomaterials by self‐assembling hydrophobin proteins

Class I fungal hydrophobins are small surface‐active proteins that self‐assemble to form amphipathic monolayers composed of amyloid‐like rodlets. The monolayers are extremely robust and can adsorb onto both hydrophobic and hydrophilic surfaces to reverse their wettability. This adherence is particularly strong for hydrophobic materials. In this report, we show that the class I hydrophobins EAS and HYD3 can self‐assemble to form a single‐molecule thick coating on a range of nanomaterials, including single‐walled carbon nanotubes (SWCNTs), graphene sheets, highly oriented pyrolytic graphite, and mica. Moreover, coating by class I hydrophobin results in a stable, dispersed preparation of SWCNTs in aqueous solutions. No cytotoxicity is detected when hydrophobin or hydrophobin‐coated SWCNTs are incubated with Caco‐2 cells in vitro. In addition, we are able to specifically introduce covalently linked chemical moieties to the hydrophilic side of the rodlet monolayer. Hence, class I hydrophobins provide a simple and effective strategy for controlling the surfaces of a range of materials at a molecular level and exhibit strong potential for biomedical applications. © 2012 Wiley Periodicals, Inc.     

Mathematical Modeling of Respiratory System Mechanics in the Newborn Lamb

In this paper, a mathematical model of the respiratory mechanics is used to reproduce experimental signal waveforms acquired from three newborn lambs. As the main challenge is to determine specific lamb parameters, a sensitivity analysis has been realized to find the most influent parameters, which are identified using an evolutionary algorithm. Results show a close match between experimental and simulated pressure and flow waveforms obtained during spontaneous ventilation and pleural pressure variations acquired during the application of positive pressure, since root mean square errors equal to 0.0119, 0.0052 and 0.0094. The identified parameters were discussed in light of previous knowledge of respiratory mechanics in the newborn.     

C-terminal truncation of Noxa1 greatly enhances its ability to activate Nox2 in a pure reconstitution system

Noxa1 activates Nox2 together with Noxo1 and Rac in a pure reconstitution system, but the resulting activity is considerably lower than that induced by p67^phox and p47^phox. In this study, we found that C-terminal-truncated forms of Noxa1 exhibited higher activities than full-length Noxa1. Of the truncations examined, Noxa1(1-225) showed the highest ability for activation. Kinetic studies revealed that Noxa1(1-225) had a threefold higher V_max value than full-length Noxa1 with a similar EC₅₀ value. The affinities of Noxo1 and RacQ61L were not much altered by the truncation. Conversely, the affinity of FAD for the Nox2 complex was enhanced after the truncation. In the absence of Noxo1, Noxa1(1-225) showed much higher activity with a lower EC₅₀ than full-length Noxa1. Noxa1(1-225) showed comparable activity to that of p67^phox with either Noxo1 or p47^phox, although the stability was lower than that with p67^phox and p47^phox. These findings indicate that the role of the C-terminal half of Noxa1 is autoinhibition. The data suggest a two-step autoinhibition mechanism, comprising self-masking to interrupt the binding to the oxidase, and holding of the activation domain in a suboptimal position to the oxidase. This study reveals that when both types of inhibition are released, Noxa1 achieves high-level superoxide production.     

PIK3R1 Mutations Cause Syndromic Insulin Resistance with Lipoatrophy

Short stature, hyperextensibility of joints and/or inguinal hernia, ocular depression, Rieger anomaly, and teething delay (SHORT) syndrome is a developmental disorder with an unknown genetic cause and hallmarks that include insulin resistance and lack of subcutaneous fat. We ascertained two unrelated individuals with SHORT syndrome, hypothesized that the observed phenotype was most likely due to de novo mutations in the same gene, and performed whole-exome sequencing in the two probands and their unaffected parents. We then confirmed our initial observations in four other subjects with SHORT syndrome from three families, as well as 14 unrelated subjects presenting with syndromic insulin resistance and/or generalized lipoatrophy associated with dysmorphic features and growth retardation. Overall, we identified in nine affected individuals from eight families de novo or inherited PIK3R1 mutations, including a mutational hotspot (c.1945C>T [p.Arg649Trp]) present in four families. PIK3R1 encodes the p85α, p55α, and p50α regulatory subunits of class IA phosphatidylinositol 3 kinases (PI3Ks), which are known to play a key role in insulin signaling. Functional data from fibroblasts derived from individuals with PIK3R1 mutations showed severe insulin resistance for both proximal and distal PI3K-dependent signaling. Our findings extend the genetic causes of severe insulin-resistance syndromes and provide important information with respect to the function of PIK3R1 in normal development and its role in human diseases, including growth delay, Rieger anomaly and other ocular affections, insulin resistance, diabetes, paucity of fat, and ovarian cysts.     

Benthic indicators of sediment quality associated with run-of-river reservoirs

Freshwater ecosystems have been fragmented by the construction of large numbers of dams. In addition to disruption of ecological continuity and physical disturbance downstream, accumulation of large amounts of sediment within run-of-river reservoirs constitutes a latent ecotoxic risk to aquatic communities. To date, run-of-river reservoirs and ecotoxic risks associated with contaminated sediment to the biodiversity and functioning of such systems are little studied. Therefore, the main objective of this study was to describe macroinvertebrate assemblages, and the functioning of these systems, and to propose indicators of sediment contamination to integrate in in-situ risk assessment methodology. To identify specific assemblages of run-of-river reservoirs, we first compared macroinvertebrate assemblages and their biotrait profiles (i.e. from a database of biological and ecological traits) in reservoirs (n = 6) and associated river sites (upstream and downstream of dams). Then, we compared responses of assemblages and biotrait profiles to sediment contamination of the banks and channels of reservoirs to select the most useful spatial scale to identify sediment contamination. Nineteen indicator taxa were observed to be specifically associated with channel habitats of reservoirs. Among these, the abundance of three taxa (Tanypodinae (Diptera), Ephemerella (Ephemeroptera) and Atherix (Diptera)) revealed the effect of metal sediment contamination. “Between-reservoirs” differences in their biotrait profile were found along the contamination gradient, with a shift of communities’ composition and functionality, and an increase in functional similarity. Many traits (response traits), for example “maximum size”, “transverse distribution”, “substrate preferences”, “saprobity”, “temperature”, “resistance forms”, and “locomotion”, were specifically linked to contamination of sediments by metals. This study indicates how sediment contamination can change the structural and functional composition of run-of-river reservoir assemblages. Indicator taxa and response traits identified in this study could improve current risk assessment methodology and potentially enable prediction of the risks of contaminated sediments stored in reservoirs in downstream ecosystems.