Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (μM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.     

A strategy for modelling dynamic responses in metabolic samples characterized by GC/MS

A multivariate strategy for studying the metabolic response over time in urinary GC/MS data is presented and exemplified by a study of drug-induced liver toxicity in the rat. The strategy includes the generation of representative data through hierarchical multivariate curve resolution (H-MCR), highlighting the importance of obtaining resolved metabolite profiles for quantification and identification of exogenous (drug related) and endogenous compounds (potential biomarkers) and for allowing reliable comparisons of multiple samples through multivariate projections. Batch modelling was used to monitor and characterize the normal (control) metabolic variation over time as well as to map the dynamic response of the drug treated animals in relation to the control. In this way treatment related metabolic responses over time could be detected and classified as being drug related or being potential biomarkers. In summary the proposed strategy uses the relatively high sensitivity and reproducibility of GC/MS in combination with efficient multivariate curve resolution and data analysis to discover individual markers of drug metabolism and drug toxicity. The presented results imply that the strategy can be of great value in drug toxicity studies for classifying metabolic markers in relation to their dynamic responses as well as for biomarker identification.     

Functional Real-Time Optoacoustic Imaging of Middle Cerebral Artery Occlusion in Mice

Background and Purpose

Longitudinal functional imaging studies of stroke are key in identifying the disease progression and possible therapeutic interventions. Here we investigate the applicability of real-time functional optoacoustic imaging for monitoring of stroke progression in the whole brain of living animals.

Materials and Methods

The middle cerebral artery occlusion (MCAO) was used to model stroke in mice, which were imaged preoperatively and the occlusion was kept in place for 60 minutes, after which optoacoustic scans were taken at several time points.

Results

Post ischemia an asymmetry of deoxygenated hemoglobin in the brain was observed as a region of hypoxia in the hemisphere affected by the ischemic event. Furthermore, we were able to visualize the penumbra in-vivo as a localized hemodynamically-compromised area adjacent to the region of stroke-induced perfusion deficit.

Conclusion

The intrinsic sensitivity of the new imaging approach to functional blood parameters, in combination with real time operation and high spatial resolution in deep living tissues, may see it become a valuable and unique tool in the development and monitoring of treatments aimed at suspending the spread of an infarct area.     

Hydrogen and oxygen trapping at the H-cluster of [FeFe]-hydrogenase revealed by site-selective spectroscopy and QM/MM calculations

[FeFe]-hydrogenases are superior hydrogen conversion catalysts. They bind a cofactor (H-cluster) comprising a four-iron and a diiron unit with three carbon monoxide (CO) and two cyanide (CN^?) ligands. Hydrogen (H₂) and oxygen (O₂) binding at the H-cluster was studied in the C169A variant of [FeFe]-hydrogenase HYDA1, in comparison to the active oxidized (Hox) and CO-inhibited (Hox-CO) species in wildtype enzyme. ⁵⁷Fe labeling of the diiron site was achieved by in vitro maturation with a synthetic cofactor analogue. Site-selective X-ray absorption, emission, and nuclear inelastic/forward scattering methods and infrared spectroscopy were combined with quantum chemical calculations to determine the molecular and electronic structure and vibrational dynamics of detected cofactor species. Hox reveals an apical vacancy at Fe_d in a [4Fe4S-2Fe]^3 ? complex with the net spin on Fe_d whereas Hox-CO shows an apical CN^? at Fe_d in a [4Fe4S-2Fe(CO)]^3 ? complex with net spin sharing among Fe_p and Fe_d (proximal or distal iron ions in [2Fe]). At ambient O₂ pressure, a novel H-cluster species (Hox-O₂) accumulated in C169A, assigned to a [4Fe4S-2Fe(O₂)]^3 ? complex with an apical superoxide (O₂^?) carrying the net spin bound at Fe_d. H₂ exposure populated the two-electron reduced Hhyd species in C169A, assigned as a [(H)4Fe4S-2Fe(H)]^3 ? complex with the net spin on the reduced cubane, an apical hydride at Fe_d, and a proton at a cysteine ligand. Hox-O₂ and Hhyd are stabilized by impaired O₂^– protonation or proton release after H₂ cleavage due to interruption of the proton path towards and out of the active site.     

Structural Properties of the Human Protease-Activated Receptor 1 Changing by a Strong Antagonist

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In-vitro analysis of the expression of TGFbeta -superfamily-members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture

Traditional surgical methods for the reconstruction of cartilage defects rely on the transplantation of autologous and allogenous tissues. The disadvantages of these techniques are the limited availability of suitable tissues and the donor site morbidity of transplants. In addition, in cultured chondrocytes, the dedifferentiation of cells seems unavoidable during multiplication. In this study, we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) and human mesenchymal stem cells (MSC) in cell culture using microarray technique, immunohistochemistry and RT-PCR. Transforming growth factor beta (TGFbeta) is a multifunctional peptide that plays play a crucial role in inducing and maintaining chondrogenic differentiation. In dedifferentiating chondrocytes, the gene for TGFbeta1 was constantly expressed, while the gene for TGFbeta2 was never expressed. The genes for TGFalpha, TGFbeta4 and TGFbetai were activated with ongoing dedifferentiation. TGFbeta-receptor 3 was constantly expressed, while the genes for the TGFbeta-receptors 1 and 2 were never expressed. Immunohistochemical staining for TGFbeta beta 3 revealed upregulation in the course of dedifferentiation. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation, whereas the gene for LTBP3 was constantly expressed, and negative results were obtained for the gene for LTBP4. The genes for LTBP1 and LTBP2 were activated with ongoing dedifferentiation. During chondrogenic differentiation, the MSCs constantly expressed TGFbeta1, beta2, beta3 and beta4. LTBP1, LTBP2 and TGFbeta-R3 were downregulated. In conclusion, TGFbeta3, TGFbeta4, TGFbetai, LTBP1 and LTBP2 may assist the process of dedifferentiation, while TGFbeta1 and beta2 might not be involved in this process. Of the TGFbeta-receptors, only type 3 might be involved in dedifferentiation.     

MITOCHONDRIAL-DNA ANALYSES AND THE ORIGIN AND RELATIVE AGE OF PARTHENOGENETIC LIZARDS (GENUS CNEMIDOPHORUS). III. C. VELOX AND C. EXSANGUIS

Mitochondrial DNAs (mtDNAs) of two unisexual, parthenogenetically reproducing species of whiptail lizards (Cnemidophorus velox and C. exsanguis) and their bisexual relatives were compared by restriction-enzyme analysis to assess levels of mtDNA variation and to establish the maternal ancestry of the unisexuals. No cleavage-site differences were found to be diagnostic between C. velox and C. exsanguis mtDNAs, suggesting an ancestry rooted in the same maternal lineage. The mtDNA of the unisexuals is relatively homogeneous, indicating that these lineages are of recent origin. Phylogenetic analysis revealed that the maternal ancestor of both C. velox and C. exsanguis was most probably C. burti stictogrammus, C. costatus barrancorum, or an unidentified taxon closely related to them. In addition, the mtDNA analyses demonstrate conclusively that the triploid species C. velox could not have been formed by the fertilization of an unreduced (diploid) C. inornatus egg, further strengthening the hypothesis that parthenogenesis in Cnemidophorus results from hybridization.     

Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

Background

Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy.

Methodology/Principal Findings

EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2±2.9% and 83.7±3.0% vs. 53.5±2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62±0.03 and 1.68±0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6±0.3 and 8.1±0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7±44.1 vs. 340.0±29.1 CD34⁺/CD45⁻ cells/1×10⁵ mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9±0.7 vs. 2.6±0.4 CD34⁺ cells/HPF, P<0.001) 3 days after the last injection.

Conclusions/Significance

Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases.     

Sequentially Deposited versus Conventional Nonfullerene Organic Solar Cells: Interfacial Trap States,Vertical Stratification,and Exciton Dissociation

Bulk heterojunction (BHJ) nonfullerene organic solar cells prepared from sequentially deposited donor and acceptor layers (sq‐BHJ) have recently been shown to be highly efficient, environmentally friendly, and compatible with large area and roll‐to‐roll fabrication. However, the related photophysics at donor‐acceptor interface and the vertical heterogeneity of donor‐acceptor distribution, critical for exciton dissociation and device performance, have been largely unexplored. Herein, steady‐state and time‐resolved optical and electrical techniques are employed to characterize the interfacial trap states. Correlating with the luminescent efficiency of interfacial states and its nonradiative recombination, interfacial trap states are characterized to be about 40% more populated in the sq‐BHJ devices than the as‐cast BHJ (c‐BHJ), which probably limits the device voltage output. Cross‐sectional energy‐dispersive X‐ray spectroscopy and ultraviolet photoemission spectroscopy depth profiling directly visualize the donor–acceptor vertical stratification with a precision of 1–2 nm. From the proposed “needle” model, the high exciton dissociation efficiency is rationalized. This study highlights the promise of sequential deposition to fabricate efficient solar cells, and points toward improving the voltage output and overall device performance via eliminating interfacial trap states.     

A gene for resistance to the Varroa mite (Acari) in honey bee (Apis mellifera) pupae

Social insect colonies possess a range of defences which protect them against highly virulent parasites and colony collapse. The host–parasite interaction between honey bees (Apis mellifera) and the mite Varroa destructor is unusual, as honey bee colonies are relatively poorly defended against this parasite. The interaction has existed since the mid‐20th Century, when Varroa switched host to parasitize A. mellifera. The combination of a virulent parasite and relatively naïve host means that, without acaricides, honey bee colonies typically die within 3 years of Varroa infestation. A consequence of acaricide use has been a reduced selective pressure for the evolution of Varroa resistance in honey bee colonies. However, in the past 20 years, several natural‐selection‐based breeding programmes have resulted in the evolution of Varroa‐resistant populations. In these populations, the inhibition of Varroa's reproduction is a common trait. Using a high‐density genome‐wide association analysis in a Varroa‐resistant honey bee population, we identify an ecdysone‐induced gene significantly linked to resistance. Ecdysone both initiates metamorphosis in insects and reproduction in Varroa. Previously, using a less dense genetic map and a quantitative trait loci analysis, we have identified Ecdysone‐related genes at resistance loci in an independently evolved resistant population. Varroa cannot biosynthesize ecdysone but can acquire it from its diet. Using qPCR, we are able to link the expression of ecdysone‐linked resistance genes to Varroa's meals and reproduction. If Varroa co‐opts pupal compounds to initiate and time its own reproduction, mutations in the host's ecdysone pathway may represent a key selection tool for honey bee resistance and breeding.