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Find video protocols related to scientific articles indexed in Pubmed.
Human fascia lata ECM scaffold augmented with immobilized hyaluronan: inflammatory response and remodeling in the canine body wall and shoulder implantation sites.
J Biomater Sci Polym Ed
PUBLISHED: 11-18-2014
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We postulate that immobilization of tyramine-substituted hyaluronan (THA) into an extracellular matrix (ECM) scaffold may be a strategy to promote an anti-inflammatory response to the ECM. Further, we posit that the implantation site could influence the inflammatory response and remodeling of an ECM scaffold. Eight beagles underwent implantation of fascia ECM grafts, treated with either immobilized low molecular weight (57 kDa) THA or water only, in both the shoulder injury and body wall sites. Dogs were euthanized at 12 weeks and fascia grafts harvested en bloc for histology. Grafts implanted at the body wall had significantly higher inflammatory cell infiltrate and vascularity, and significantly lower retardance (collagen density), than grafts at the shoulder, suggestive of a more intense, persistent, and perhaps degradative inflammatory and remodeling response at the body wall than shoulder injury site in the canine model. However, the presence of immobilized low MW THA had no effect on the inflammation response or remodeling of fascia ECM compared to water-treated controls. Importantly, these results suggest that the inflammatory response and remodeling of biomaterial implants depends on the location of implantation and therefore our animal models need to be carefully chosen. Further, the potential anti-inflammatory advantages of hyaluronan (HA) in wound healing do not appear to be realized when presenting it to the host as non-degradable hydrogel even if its capacity for binding HA binding protein is maintained. Further study treating ECM with uncross-linked (free) HA or immobilized low MW THA as a means to deliver free HA or other biomolecules to a surgical repair site is warranted.
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The future of decision-making in critical care after Cuthbertson v. Rasouli.
Can J Anaesth
PUBLISHED: 08-28-2014
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The Supreme Court of Canada (SCC) ruling on Cuthbertson v. Rasouli has implications for all acute healthcare providers. This well-publicized case involved a disagreement between healthcare providers and a patient's family regarding the principles surrounding withdrawal of life support, which the physicians involved considered no longer of medical benefit and outside the standard of care, and whether consent was required for such withdrawals. Our objective in writing this article is to clarify the implications of this ruling on the care of critically ill patients.
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Cervical internal carotid Artery pseudoaneurysm complicating malignant otitis externa: First case report.
Laryngoscope
PUBLISHED: 08-14-2014
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Pseudoaneurysm of the internal carotid artery (ICA) is a rare complication of head and neck infections. To date, three cases of petrous ICA pseudoaneurysm have been described as a complication of otogenic infection, including only one secondary to malignant otitis externa. We present here the first case of cervical ICA pseudoaneurysm as a complication of malignant otitis externa, and stress the importance of timely diagnosis to avoid fatal outcomes.
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Not just who, but how many: the importance of partner abundance in reef coral symbioses.
Front Microbiol
PUBLISHED: 08-04-2014
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The performance and function of reef corals depends on the genetic identity of their symbiotic algal partners, with some symbionts providing greater benefits (e.g., photosynthate, thermotolerance) than others. However, these interaction outcomes may also depend on partner abundance, with differences in the total number of symbionts changing the net benefit to the coral host, depending on the particular environmental conditions. We suggest that symbiont abundance is a fundamental aspect of the dynamic interface between reef corals and the abiotic environment that ultimately determines the benefits, costs, and functional responses of these symbioses. This density-dependent framework suggests that corals may regulate the size of their symbiont pool to match microhabitat-specific optima, which may contribute to the high spatiotemporal variability in symbiont abundance observed within and among colonies and reefs. Differences in symbiont standing stock may subsequently explain variation in energetics, growth, reproduction, and stress susceptibility, and may mediate the impacts of environmental change on these outcomes. However, the importance of symbiont abundance has received relatively little recognition, possibly because commonly-used metrics based on surface area (e.g., symbiont cells cm(-2)) may be only weakly linked to biological phenomena and are difficult to compare across studies. We suggest that normalizing symbionts to biological host parameters, such as units of protein or numbers of host cells, will more clearly elucidate the functional role of symbiont abundance in reef coral symbioses. In this article, we generate testable hypotheses regarding the importance of symbiont abundance by first discussing different metrics and their potential links to symbiosis performance and breakdown, and then describing how natural variability and dynamics of symbiont communities may help explain ecological patterns on coral reefs and predict responses to environmental change.
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The importance of coagulation factors binding to adenovirus: historical perspectives and implications for gene delivery.
Expert Opin Drug Deliv
PUBLISHED: 07-18-2014
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The interaction of human adenovirus (HAdV) serotype 5 (HAdV-5) with the blood coagulation factor X (FX) results in a high liver transduction after AdV intravascular administration, causing toxicity and limiting AdV delivery to the target tissue. However, FX also protects adenoviral vectors from neutralization by the complement system and natural antibodies, potentially benefiting adenoviral gene delivery, as neutralization results in the reduction of HAdV-5 binding to host cells.
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Phase segregation of sulfonate groups in Nafion interface lamellae, quantified via neutron reflectometry fitting techniques for multi-layered structures.
Soft Matter
PUBLISHED: 07-01-2014
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Neutron reflectometry analysis methods for under-determined, multi-layered structures are developed and used to determine the composition depth profile in cases where the structure is not known a priori. These methods, including statistical methods, sophisticated fitting routines, and coupling multiple data sets, are applied to hydrated and dehydrated Nafion nano-scaled films with thicknesses comparable to those found coating electrode particles in fuel cell catalyst layers. These results confirm the lamellar structure previously observed on hydrophilic substrates, and demonstrate that for hydrated films they can accurately be described as layers rich in both water and sulfonate groups alternating with water-poor layers containing an excess of fluorocarbon groups. The thickness of these layers increases slightly and the amplitude of the water volume fraction oscillation exponentially decreases away from the hydrophilic interface. For dehydrated films, the composition oscillations die out more rapidly. The Nafion-SiO2 substrate interface contains a partial monolayer of sulfonate groups bonded to the substrate and a large excess of water compared to that expected by the water-to-sulfonate ratio, ?, observed throughout the rest of the film. Films that were made thin enough to truncate this lamellar region showed a depth profile nearly identical to thicker films, indicating that there are no confinement or surface effects altering the structure. Comparing the SLD profile measured for films dried at 60 °C to modeled composition profiles derived by removing water from the hydrated lamellae suggests incomplete re-mixing of the polymer groups upon dehydration, indicated limited polymer mobility in these Nafion thin films.
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Deferred consent in a minimal-risk study involving critically ill subarachnoid hemorrhage patients.
Can. Respir. J.
PUBLISHED: 06-10-2014
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Alterations from first-party and surrogate decision-maker consent can enhance the feasibility of research involving critically ill patients.
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Vital signs after cardiac arrest following withdrawal of life-sustaining therapy: a multicenter prospective observational study*.
Crit. Care Med.
PUBLISHED: 05-10-2014
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Controversies regarding the process and timing of the determination of death for controlled organ donation after circulatory death persist. This study assessed the feasibility of conducting a prospective, observational study of continuous monitoring of vital signs for 30 minutes after the clinical determination of death in five Canadian ICUs. Waveform data were analyzed.
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Capsid modification strategies for detargeting adenoviral vectors.
Methods Mol. Biol.
PUBLISHED: 05-09-2014
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Adenoviral vectors hold immense potential for a wide variety of gene therapy based applications; however, their efficacy and toxicity is dictated by "off target" interactions that preclude cell specific targeting to sites of disease. A number of "off target" interactions have been described in the literature that occur between the three major capsid proteins (hexon, penton, and fiber) and components of the circulatory system, including cells such as erythrocytes, white blood cells, and platelets, as well as circulatory proteins including complement proteins, coagulation factors, von Willebrand Factor, p-selectin as well as neutralizing antibodies. Thus, to improve efficacious targeting to sites of disease and limit nonspecific uptake of virus to non-target tissues, specifically the liver and the spleen, it is necessary to develop suitable strategies for genetically modifying the capsid proteins to preclude these interactions. To this end we have developed versatile systems based on homologous recombination for modification of each of the major capsid proteins, which are described herein.
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Change in algal symbiont communities after bleaching, not prior heat exposure, increases heat tolerance of reef corals.
Glob Chang Biol
PUBLISHED: 05-07-2014
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Mutualistic organisms can be particularly susceptible to climate change stress, as their survivorship is often limited by the most vulnerable partner. However, symbiotic plasticity can also help organisms in changing environments by expanding their realized niche space. Coral-algal (Symbiodinium spp.) symbiosis exemplifies this dichotomy: the partnership is highly susceptible to 'bleaching' (stress-induced symbiosis breakdown), but stress-tolerant symbionts can also sometimes mitigate bleaching. Here, we investigate the role of diverse and mutable symbiotic partnerships in increasing corals' ability to thrive in high temperature conditions. We conducted repeat bleaching and recovery experiments on the coral Montastraea cavernosa, and used quantitative PCR and chlorophyll fluorometry to assess the structure and function of Symbiodinium communities within coral hosts. During an initial heat exposure (32 °C for 10 days), corals hosting only stress-sensitive symbionts (Symbiodinium C3) bleached, but recovered (at either 24 °C or 29 °C) with predominantly (>90%) stress-tolerant symbionts (Symbiodinium D1a), which were not detected before bleaching (either due to absence or extreme low abundance). When a second heat stress (also 32 °C for 10 days) was applied 3 months later, corals that previously bleached and were now dominated by D1a Symbiodinium experienced less photodamage and symbiont loss compared to control corals that had not been previously bleached, and were therefore still dominated by Symbiodinium C3. Additional corals that were initially bleached without heat by a herbicide (DCMU, at 24 °C) also recovered predominantly with D1a symbionts, and similarly lost fewer symbionts during subsequent thermal stress. Increased thermotolerance was also not observed in C3-dominated corals that were acclimated for 3 months to warmer temperatures (29 °C) before heat stress. These findings indicate that increased thermotolerance post-bleaching resulted from symbiont community composition changes, not prior heat exposure. Moreover, initially undetectable D1a symbionts became dominant only after bleaching, and were critical to corals' resilience after stress and resistance to future stress.
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Platelet-mediated changes to neuronal glutamate receptor expression at sites of microthrombosis following experimental subarachnoid hemorrhage.
J. Neurosurg.
PUBLISHED: 04-22-2014
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Object Glutamate is important in the pathogenesis of brain damage after cerebral ischemia and traumatic brain injury. Notably, brain extracellular and cerebrospinal fluid as well as blood glutamate concentrations increase after experimental and clinical trauma. While neurons are one potential source of glutamate, platelets also release glutamate as part of their recruitment and might mediate neuronal damage. This study investigates the hypothesis that platelet microthrombi release glutamate that mediates excitotoxic brain injury and neuron dysfunction after subarachnoid hemorrhage (SAH). Methods The authors used two models, primary neuronal cultures exposed to activated platelets, as well as a whole-animal SAH preparation. Propidium iodide was used to evaluate neuronal viability, and surface glutamate receptor staining was used to evaluate the phenotype of platelet-exposed neurons. Results The authors demonstrate that thrombin-activated platelet-rich plasma releases glutamate, at concentrations that can exceed 300 ?M. When applied to neuronal cultures, this activated plasma is neurotoxic, and the toxicity is attenuated in part by glutamate receptor antagonists. The authors also demonstrate that exposure to thrombin-activated platelets induces marked downregulation of the surface glutamate receptor glutamate receptor 2, a marker of excitotoxicity exposure and a possible mechanism of neuronal dysfunction. Linear regression demonstrated that 7 days after SAH in rats there was a strong correlation between proximity to microthrombi and reduction of surface glutamate receptors. Conclusions The authors conclude that platelet-mediated microthrombosis contributes to neuronal glutamate receptor dysfunction and might mediate brain injury after SAH.
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Biophilosophical basis for identifying the death of a person.
J Crit Care
PUBLISHED: 04-14-2014
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The capacity for consciousness and self-awareness is uniquely synonymous with human life and personhood, and its absence is necessary and sufficient to identify that death has occurred. The presence of these functions is uniquely synonymous with human life. Ongoing organ function, response to infection, growth, wound healing, or the ability to sustain an unborn fetus do not alone constitute the unique experience of life and personhood. Death occurs after the loss of the ability to use oxygen by the brain, which occurs because of either raised intracranial pressure preventing any cerebral blood flow or, more commonly, the absence of systemic blood flow following abrupt or hypoxic circulatory cessation. The abrupt cessation of circulation leads to loss of consciousness and brain electrical activity; and when it becomes truly permanent and then irreversible, this becomes an operational definition of death. One must infer and decide that sufficient ischemic hypoxic injury has rendered the potential for reinstating any consciousness and brain stem function irreversibly lost. Progressive hypoxia that is seen in many patients after withdrawal of advanced physiologic support leads to apnea and then circulatory arrest. The outward sign of apnea that is then followed by circulatory arrest is the basis for inferring that irreversible loss of capacity for consciousness and self-awareness has occurred and that death can be identified has having occurred. The capacity for consciousness and self-awareness is the only irreplaceable emergent phenomenon—arising from physiologic function of the brain—that is necessary and sufficient to define the life of a person.
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Endothelial apoptosis in pulmonary hypertension is controlled by a microRNA/programmed cell death 4/caspase-3 axis.
Hypertension
PUBLISHED: 04-14-2014
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Pulmonary endothelial cell apoptosis is a transient, yet defining pathogenic event integral to the onset of many pulmonary vascular diseases such as pulmonary hypertension (PH). However, there is a paucity of information concerning the molecular pathway(s) that control pulmonary arterial endothelial cell apoptosis. Here, we introduce a molecular axis that when functionally active seems to induce pulmonary arterial endothelial cell apoptosis in vitro and PH in vivo. In response to apoptotic stimuli, human pulmonary arterial endothelial cells exhibited robust induction of a programmed cell death 4 (PDCD4)/caspase-3/apoptotic pathway that was reversible by direct PDCD4 silencing. Indirectly, this pathway was also repressed by delivery of a microRNA-21 mimic. In vivo, genetic deletion of microRNA-21 in mice (miR-21(-/-) mice) resulted in functional activation of the PDCD4/caspase-3 axis in the pulmonary tissues, leading to the onset of progressive PH. Conversely, microRNA-21-overexpressing mice (CAG-microRNA-21 mice) exhibited reduced PDCD4 expression in pulmonary tissues and were partially resistant to PH in response to chronic hypoxia plus SU 5416 injury. Furthermore, direct PDCD4 knockout in mice (PDCD4(-/-) mice) potently blocked pulmonary caspase-3 activation and the development of chronic hypoxia plus SU 5416 PH, confirming its importance in disease onset. Broadly, these findings support the existence of a microRNA-21-responsive PDCD4/caspase-3 pathway in the pulmonary tissues that when active serves to promote endothelial apoptosis in vitro and PH in vivo.
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Retargeting adenovirus serotype 48 fiber knob domain by peptide incorporation.
Hum. Gene Ther.
PUBLISHED: 04-11-2014
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Adenovirus type 5 (Ad5) is a commonly used vector for gene therapy, but its efficacy is limited by high seroprevalence and off-target hepatic and splenic sequestration. In order to circumvent these limitations, the use of vectors derived from rare species adenoviruses is appealing. The opportunity to retarget rare species vectors to defined cell types through the incorporation of peptide ligands would be advantageous, particularly in targeting tumors and disseminated metastases. We used predictive structural modeling to assess the CD, DG, HI, and IJ loops of the Ad48 fiber knob and identify optimal incorporation locales for the 20-mer peptide, A20FMDV2 (A20). A20FMDV2 targets ???6 integrin, which is overexpressed in human carcinomas. Recombinant Ad48 fiber knob proteins Knob48, Knob48-CD-A20, Knob48-DG-A20, Knob48-HI-A20, and Knob48-IJ-A20 were engineered and purified after expression in Escherichia coli. We confirmed that Knob48, Knob48-CD-A20, and Knob48-IJ-A20 formed stable homotrimers. However, Knob48-DG-A20 and Knob-HI-A20 failed to form a trimer. All A20-modified knob proteins blocked the transduction of Ad5-EGFPA20 via ???6, demonstrating that the inserted A20 peptide was functional. In conclusion, we show that the CD and IJ loops of Ad48 represent suitable sites for targeting peptide incorporation. Interestingly, in vitro gene transfer mediated by the non-factor-X-binding Ad48 vector was not sensitive to immunoglobulins and complement when incubated in the presence of mouse serum, unlike Ad5. These data support the future generation of the corresponding Ad48 viral vectors, Ad48-CD-A20 and Ad48-IJ-A20, which may offer favorable characteristics for targeted delivery in vivo.
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Distribution models for koalas in South Australia using citizen science-collected data.
Ecol Evol
PUBLISHED: 04-03-2014
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The koala (Phascolarctos cinereus) occurs in the eucalypt forests of eastern and southern Australia and is currently threatened by habitat fragmentation, climate change, sexually transmitted diseases, and low genetic variability throughout most of its range. Using data collected during the Great Koala Count (a 1-day citizen science project in the state of South Australia), we developed generalized linear mixed-effects models to predict habitat suitability across South Australia accounting for potential errors associated with the dataset. We derived spatial environmental predictors for vegetation (based on dominant species of Eucalyptus or other vegetation), topographic water features, rain, elevation, and temperature range. We also included predictors accounting for human disturbance based on transport infrastructure (sealed and unsealed roads). We generated random pseudo-absences to account for the high prevalence bias typical of citizen-collected data. We accounted for biased sampling effort along sealed and unsealed roads by including an offset for distance to transport infrastructures. The model with the highest statistical support (wAIC c ? 1) included all variables except rain, which was highly correlated with elevation. The same model also explained the highest deviance (61.6%), resulted in high R (2)(m) (76.4) and R (2)(c) (81.0), and had a good performance according to Cohen's ? (0.46). Cross-validation error was low (? 0.1). Temperature range, elevation, and rain were the best predictors of koala occurrence. Our models predict high habitat suitability in Kangaroo Island, along the Mount Lofty Ranges, and at the tips of the Eyre, Yorke and Fleurieu Peninsulas. In the highest-density region (5576 km(2)) of the Adelaide-Mount Lofty Ranges, a density-suitability relationship predicts a population of 113,704 (95% confidence interval: 27,685-199,723; average density = 5.0-35.8 km(-2)). We demonstrate the power of citizen science data for predicting species' distributions provided that the statistical approaches applied account for some uncertainties and potential biases. A future improvement to citizen science surveys to provide better data on search effort is that smartphone apps could be activated at the start of the search. The results of our models provide preliminary ranges of habitat suitability and population size for a species for which previous data have been difficult or impossible to gather otherwise.
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Molecular evolution of calcification genes in morphologically similar but phylogenetically unrelated scleractinian corals.
Mol. Phylogenet. Evol.
PUBLISHED: 03-31-2014
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Molecular phylogenies of scleractinian corals often fail to agree with traditional phylogenies derived from morphological characters. These discrepancies are generally attributed to non-homologous or morphologically plastic characters used in taxonomic descriptions. Consequently, morphological convergence of coral skeletons among phylogenetically unrelated groups is considered to be the major evolutionary process confounding molecular and morphological hypotheses. A strategy that may help identify cases of convergence and/or diversification in coral morphology is to compare phylogenies of existing "neutral" genetic markers used to estimate genealogic phylogenetic history with phylogenies generated from non-neutral genes involved in calcification (biomineralization). We tested the hypothesis that differences among calcification gene phylogenies with respect to the "neutral" trees may represent convergent or divergent functional strategies among calcification gene proteins that may correlate to aspects of coral skeletal morphology. Partial sequences of two nuclear genes previously determined to be involved in the calcification process in corals, "Cnidaria-III" membrane-bound/secreted ?-carbonic anhydrase (CIII-MBS?-CA) and bone morphogenic protein (BMP) 2/4, were PCR-amplified, cloned and sequenced from 31 scleractinian coral species in 26 genera and 9 families. For comparison, "neutral" gene phylogenies were generated from sequences from two protein-coding "non-calcification" genes, one nuclear (?-tubulin) and one mitochondrial (cytochrome b), from the same individuals. Cloned CIII-MBS?-CA sequences were found to be non-neutral, and phylogenetic analyses revealed CIII-MBS?-CAs to exhibit a complex evolutionary history with clones distributed between at least 2 putative gene copies. However, for several coral taxa only one gene copy was recovered. With CIII-MBS?-CA, several recovered clades grouped taxa that differed from the "non-calcification" loci. In some cases, these taxa shared aspects of their skeletal morphology (i.e., convergence or diversification relative to the "non-calcification" loci), but in other cases they did not. For example, the "non-calcification" loci recovered Atlantic and Pacific mussids as separate evolutionary lineages, whereas with CIII-MBS?-CA, clones of two species of Atlantic mussids (Isophyllia sinuosa and Mycetophyllia sp.) and two species of Pacific mussids (Acanthastrea echinata and Lobophyllia hemprichii) were united in a distinct clade (except for one individual of Mycetophyllia). However, this clade also contained other taxa which were not unambiguously correlated with morphological features. BMP2/4 also contained clones that likely represent different gene copies. However, many of the sequences showed no significant deviation from neutrality, and reconstructed phylogenies were similar to the "non-calcification" tree topologies with a few exceptions. Although individual calcification genes are unlikely to precisely explain the diverse morphological features exhibited by scleractinian corals, this study demonstrates an approach for identifying cases where morphological taxonomy may have been misled by convergent and/or divergent molecular evolutionary processes in corals. Studies such as this may help illuminate our understanding of the likely complex evolution of genes involved in the calcification process, and enhance our knowledge of the natural history and biodiversity within this central ecological group.
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Acidification of floodplains due to river level decline during drought.
J. Contam. Hydrol.
PUBLISHED: 03-17-2014
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A severe drought from 2007 to 2010 resulted in the lowest river levels (1.75 m decline from average) in over 90 years of records at the end of the Murray-Darling Basin in South Australia. Due to the low river level and inability to apply irrigation, the groundwater depth on the adjacent agricultural flood plain also declined substantially (1-1.5 m) and the alluvial clay subsoils dried and cracked. Sulfidic material (pH>4, predominantly in the form of pyrite, FeS2) in these subsoils oxidised to form sulfuric material (pH<4) over an estimated 3300 ha on 13 floodplains. Much of the acidity in the deeply cracked contaminated soil layers was in available form (in pore water and on cation exchange sites), with some layers having retained acidity (iron oxyhydroxysulfate mineral jarosite). Post drought, the rapid raising of surface and ground water levels mobilised acidity in acid sulfate soil profiles to the floodplain drainage channels and this was transported back to the river via pumping. The drainage water exhibited low pH (2-5) with high soluble metal (Al, Co, Mn, Fe, Mn, Ni, and Zn) concentrations, in exceedance of guidelines for ecosystem protection. Irrigation increased the short-term transport of acidity, however loads were generally greater in the non-irrigation (winter) season when rainfall is highest (0.0026 tonnes acidity/ha/day) than in the irrigation (spring-summer) season (0.0013 tonnes acidity/ha/day). Measured reductions in groundwater acidity and increases in pH have been observed over time but severe acidification persisted in floodplain sediments and waters for over two years post-drought. Results from 2-dimensional modelling of the river-floodplain hydrological processes were consistent with field measurements during the drying phase and illustrated how the declining river levels led to floodplain acidification. A modelled management scenario demonstrated how river level stabilisation and limited irrigation could have prevented, or greatly lessened the severity of the acidification.
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Investigating muscle regeneration with a dermis/small intestinal submucosa scaffold in a rat full-thickness abdominal wall defect model.
J. Biomed. Mater. Res. Part B Appl. Biomater.
PUBLISHED: 03-04-2014
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Repair of large complex ventral wall hernias is challenging and outcomes are often poor due to hernia recurrence and compromised abdominal wall function. Currently, biological grafts are used to assist in repairing these complex hernias. Dermis grafts are often chosen because of their mechanical characteristics; however, dermis does not have the ability to promote the muscle regeneration needed to regain abdominal wall function. In contrast, small intestinal submucosa (SIS) grafts have been shown to promote muscle generation in volumetric muscle loss (VML) models. Hence, the objective of this study is to investigate the extent to which SIS grafts can be used together with dermis grafts to repair and promote muscle regeneration in a full-thickness abdominal wall defect in a rat model. The dermis layer is intended to mechanically bridge the defect and support constructive tissue remodeling while the SIS is intended to degrade and promote neo-muscle formation. After 16 weeks of implantation, we found only a small amount of vascularized muscle (<10% of the defect area) in the repaired defects. No significant difference in defect muscle area was found between the groups receiving the dermis?+?SIS scaffolds and the control (dermis alone) group. Our findings indicate that the SIS constructions investigated could not promote appreciable muscle regeneration in this rigorous animal model of VML and incomplete abdominal closure. Future investigation into combination scaffold, cell and molecular therapies would be warranted to address the need for functional muscle regeneration in challenging clinical conditions such as complex abdominal wall repair. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2014.
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Thermally tolerant corals have limited capacity to acclimatize to future warming.
Glob Chang Biol
PUBLISHED: 02-25-2014
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Thermal stress affects organism performance differently depending on the ambient temperature to which they are acclimatized, which varies along latitudinal gradients. This study investigated whether differences in physiological responses to temperature are consistent with regional differences in temperature regimes for the stony coral Oculina patagonica. To resolve this question, we experimentally assessed how colonies originating from four different locations characterized by >3 °C variation in mean maximum annual temperature responded to warming from 20 to 32 °C. We assessed plasticity in symbiont identity, density, and photosynthetic properties, together with changes in host tissue biomass. Results show that, without changes in the type of symbiont hosted by coral colonies, O. patagonica has limited capacity to acclimatize to future warming. We found little evidence of variation in overall thermal tolerance, or in thermal optima, in response to spatial variation in ambient temperature. Given that the invader O. patagonica is a relatively new member of the Mediterranean coral fauna, our results also suggest that coral populations may need to remain isolated for a long period of time for thermal adaptation to potentially take place. Our study indicates that for O. patagonica, mortality associated with thermal stress manifests primarily through tissue breakdown under moderate but prolonged warming (which does not impair symbiont photosynthesis and, therefore, does not lead to bleaching). Consequently, projected global warming is likely to cause repeat incidents of partial and whole colony mortality and might drive a gradual range contraction of Mediterranean corals.
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The Black-tailed Antechinus, Antechinus arktos sp. nov.: a new species of carnivorous marsupial from montane regions of the Tweed Volcano caldera, eastern Australia.
Zootaxa
PUBLISHED: 02-14-2014
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We describe a new species of dasyurid marsupial within the genus Antechinus that was previously known as a northern outlier of Dusky Antechinus (A. swainsonii). The Black-tailed Antechinus, Antechinus arktos sp. nov., is known only from areas of high altitude and high rainfall on the Tweed Volcano caldera of far south-east Queensland and north-east New South Wales, Australia. Antechinus arktos formerly sheltered under the taxonomic umbrella of A. swainsonii mimetes, the widespread mainland form of Dusky Antechinus. With the benefit of genetic hindsight, some striking morphological differences are herein resolved: A. s. mimetes is more uniformly deep brown-black to grizzled grey-brown from head to rump, with brownish (clove brown-raw umber) hair on the upper surface of the hindfoot and tail, whereas A. arktos is more vibrantly coloured, with a marked change from greyish-brown head to orange-brown rump, fuscous black on the upper surface of the hindfoot and dense, short fur on the evenly black tail. Further, A. arktos has marked orange-brown fur on the upper and lower eyelid, cheek and in front of the ear and very long guard hairs all over the body; these characters are more subtle in A. s. mimetes. There are striking genetic differences between the two species: at mtDNA, A. s. mimetes from north-east New South Wales is 10% divergent to A. arktos from its type locality at Springbrook NP, Queensland. In contrast, the Ebor A. s. mimetes clades closely with conspecifics from ACT and Victoria. A. arktos skulls are strikingly different to all subspecies of A. swainsonii. A. arktos are markedly larger than A. s. mimetes and A. s. swainsonii (Tasmania) for a range of craniodental measures. Antechinus arktos were historically found at a few proximate mountainous sites in south-east Queensland, and have only recently been recorded from or near the type locality. Even there, the species is likely in low abundance. The Black-tailed Antechinus has plausibly been detrimentally affected by climate change in recent decades, and will be at further risk with increasing warming trends. 
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International guideline development for the determination of death.
Intensive Care Med
PUBLISHED: 01-07-2014
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This report summarizes the results of the first phase in the development of international guidelines for death determination, focusing on the biology of death and the dying process, developed by an invitational forum of international content experts and representatives of a number of professional societies.
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Growth dynamics of the threatened Caribbean staghorn coral Acropora cervicornis: influence of host genotype, symbiont identity, colony size, and environmental setting.
PLoS ONE
PUBLISHED: 01-01-2014
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The drastic decline in the abundance of Caribbean acroporid corals (Acropora cervicornis, A. palmata) has prompted the listing of this genus as threatened as well as the development of a regional propagation and restoration program. Using in situ underwater nurseries, we documented the influence of coral genotype and symbiont identity, colony size, and propagation method on the growth and branching patterns of staghorn corals in Florida and the Dominican Republic.
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The use of automated bioacoustic recorders to replace human wildlife surveys: an example using nightjars.
PLoS ONE
PUBLISHED: 01-01-2014
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To be able to monitor and protect endangered species, we need accurate information on their numbers and where they live. Survey methods using automated bioacoustic recorders offer significant promise, especially for species whose behaviour or ecology reduces their detectability during traditional surveys, such as the European nightjar. In this study we examined the utility of automated bioacoustic recorders and the associated classification software as a way to survey for wildlife, using the nightjar as an example. We compared traditional human surveys with results obtained from bioacoustic recorders. When we compared these two methods using the recordings made at the same time as the human surveys, we found that recorders were better at detecting nightjars. However, in practice fieldworkers are likely to deploy recorders for extended periods to make best use of them. Our comparison of this practical approach with human surveys revealed that recorders were significantly better at detecting nightjars than human surveyors: recorders detected nightjars during 19 of 22 survey periods, while surveyors detected nightjars on only six of these occasions. In addition, there was no correlation between the amount of vocalisation captured by the acoustic recorders and the abundance of nightjars as recorded by human surveyors. The data obtained from the recorders revealed that nightjars were most active just before dawn and just after dusk, and least active during the middle of the night. As a result, we found that recording at both dusk and dawn or only at dawn would give reasonably high levels of detection while significantly reducing recording time, preserving battery life. Our analyses suggest that automated bioacoustic recorders could increase the detection of other species, particularly those that are known to be difficult to detect using traditional survey methods. The accuracy of detection is especially important when the data are used to inform conservation.
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A protective lipidomic biosignature associated with a balanced omega-6/omega-3 ratio in fat-1 transgenic mice.
PLoS ONE
PUBLISHED: 01-01-2014
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A balanced omega-6/omega-3 polyunsaturated fatty acid (PUFA) ratio has been linked to health benefits and the prevention of many chronic diseases. Current dietary intervention studies with different sources of omega-3 fatty acids (omega-3) lack appropriate control diets and carry many other confounding factors derived from genetic and environmental variability. In our study, we used the fat-1 transgenic mouse model as a proxy for long-term omega-3 supplementation to determine, in a well-controlled manner, the molecular phenotype associated with a balanced omega-6/omega-3 ratio. The fat-1 mouse can convert omega-6 to omega-3 PUFAs, which protect against a wide variety of diseases including chronic inflammatory diseases and cancer. Both wild-type (WT) and fat-1 mice were subjected to an identical diet containing 10% corn oil, which has a high omega-6 content similar to that of the Western diet, for a six-month duration. We used a multi-platform lipidomic approach to compare the plasma lipidome between fat-1 and WT mice. In fat-1 mice, an unbiased profiling showed a significant increase in the levels of unesterified eicosapentaenoic acid (EPA), EPA-containing cholesteryl ester, and omega-3 lysophosphospholipids. The increase in omega-3 lipids is accompanied by a significant reduction in omega-6 unesterified docosapentaenoic acid (omega-6 DPA) and DPA-containing cholesteryl ester as well as omega-6 phospholipids and triacylglycerides. Targeted lipidomics profiling highlighted a remarkable increase in EPA-derived diols and epoxides formed via the cytochrome P450 (CYP450) pathway in the plasma of fat-1 mice compared with WT mice. Integration of the results of untargeted and targeted analyses has identified a lipidomic biosignature that may underlie the healthful phenotype associated with a balanced omega-6/omega-3 ratio, and can potentially be used as a circulating biomarker for monitoring the health status and the efficacy of omega-3 intervention in humans.
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MicroRNA-214 Antagonism Protects against Renal Fibrosis.
J. Am. Soc. Nephrol.
PUBLISHED: 10-24-2013
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Renal tubulointerstitial fibrosis is the common end point of progressive renal disease. MicroRNA (miR)-214 and miR-21 are upregulated in models of renal injury, but the function of miR-214 in this setting and the effect of its manipulation remain unknown. We assessed the effect of inhibiting miR-214 in an animal model of renal fibrosis. In mice, genetic deletion of miR-214 significantly attenuated interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Treatment of wild-type mice with an anti-miR directed against miR-214 (anti-miR-214) before UUO resulted in similar antifibrotic effects, and in vivo biodistribution studies demonstrated that anti-miR-214 accumulated at the highest levels in the kidney. Notably, in vivo inhibition of canonical TGF-? signaling did not alter the regulation of endogenous miR-214 or miR-21. Whereas miR-21 antagonism blocked Smad 2/3 activation, miR-214 antagonism did not, suggesting that miR-214 induces antifibrotic effects independent of Smad 2/3. Furthermore, TGF-? blockade combined with miR-214 deletion afforded additional renal protection. These phenotypic effects of miR-214 depletion were mediated through broad regulation of the transcriptional response to injury, as evidenced by microarray analysis. In human kidney tissue, miR-214 was detected in cells of the glomerulus and tubules as well as in infiltrating immune cells in diseased tissue. These studies demonstrate that miR-214 functions to promote fibrosis in renal injury independent of TGF-? signaling in vivo and that antagonism of miR-214 may represent a novel antifibrotic treatment in the kidney.
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Prehospital hypertonic saline resuscitation attenuates the activation and promotes apoptosis of neutrophils in patients with severe traumatic brain injury.
Shock
PUBLISHED: 10-04-2013
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Activation of polymorphonuclear neutrophils (PMNs) is thought to contribute to traumatic brain injury (TBI). Since hypertonic fluids can inhibit PMN activation, we studied whether hypertonic fluid resuscitation can reduce excessive PMN activation in TBI patients.
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The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis.
Nat Commun
PUBLISHED: 08-10-2013
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MicroRNAs (miRNAs) regulate cardiovascular biology and disease, but the role of flow-sensitive microRNAs in atherosclerosis is still unclear. Here we identify miRNA-712 (miR-712) as a mechanosensitive miRNA upregulated by disturbed flow (d-flow) in endothelial cells, in vitro and in vivo. We also show that miR-712 is derived from an unexpected source, pre-ribosomal RNA, in an exoribonuclease-dependent but DiGeorge syndrome critical region 8 (DGCR8)-independent manner, suggesting that it is an atypical miRNA. Mechanistically, d-flow-induced miR-712 downregulates tissue inhibitor of metalloproteinase 3 (TIMP3) expression, which in turn activates the downstream matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) and stimulate pro-atherogenic responses, endothelial inflammation and permeability. Furthermore, silencing miR-712 by anti-miR-712 rescues TIMP3 expression and prevents atherosclerosis in murine models of atherosclerosis. Finally, we report that human miR-205 shares the same seed sequence as murine-specific miR-712 and also targets TIMP3 in a flow-dependent manner. Targeting these mechanosensitive athero-miRs may provide a new treatment paradigm in atherosclerosis.
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Influenza-associated neurological complications.
Neurocrit Care
PUBLISHED: 08-08-2013
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While mostly diagnosed in the pediatric population, neurological complications of pandemic influenza A infection may affect young and previously healthy adults, and may follow a fulminant, severe, and occasionally fatal course. We reviewed severe neurological complications secondary to influenza infection reported in the literature, in attempt to outline recurrent syndromes that may assist the clinician in making a timely diagnosis. Vigilance and awareness of these clinical entities are key in the neurologist and intensivists role in surveillance and early recognition of pandemic influenza, and in ensuring improved survival for affected patients.
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Canonical Transforming Growth Factor-? Signaling Regulates Disintegrin Metalloprotease Expression in Experimental Renal Fibrosis via miR-29.
Am. J. Pathol.
PUBLISHED: 07-29-2013
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Fibrosis pathophysiology is critically regulated by Smad 2- and Smad 3-mediated transforming growth factor-? (TGF-?) signaling. Disintegrin metalloproteases (Adam) can manipulate the signaling environment, however, the role and regulation of ADAMs in renal fibrosis remain unclear. TGF-? stimulation of renal cells results in a significant up-regulation of Adams 10, 17, 12, and 19. The selective Smad2/3 inhibitor SB 525334 reversed these TGF-?-induced changes. In vivo, using ureteral obstruction to model renal fibrosis, we observed increased Adams gene expression that was blocked by oral administration of SB 525334. Similar increases in Adam gene expression also occurred in preclinical models of hypertension-induced renal damage and glomerulonephritis. miRNAs are a recently discovered second level of regulation of gene expression. Analysis of 3 untranslated regions of Adam12 and Adam19 mRNAs showed multiple binding sites for miR-29a, miR-29b, and miR-29c. We show that miR-29 family expression is decreased after unilateral ureter obstruction and this significant decrease in miR-29 family expression was observed consistently in preclinical models of renal dysfunction and correlated with an increase in Adam12 and Adam19 expression. Exogenous overexpression of the miR-29 family blocked TGF-?-mediated up-regulation of Adam12 and Adam19 gene expression. This study shows that Adams are involved in renal fibrosis and are regulated by canonical TGF-? signaling and miR-29. Therefore, both Adams and the miR-29 family represent therapeutic targets for renal fibrosis.
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Bone marrow-derived endothelial progenitor cells protect postischemic axons after traumatic brain injury.
J. Cereb. Blood Flow Metab.
PUBLISHED: 07-27-2013
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White matter sparing after traumatic brain injury (TBI) is an important predictor of survival and outcome. Blood vessels and axons are intimately associated anatomically and developmentally. Neural input is required for appropriate vascular patterning, and vascular signaling is important for neuron development and axon growth. Owing to this codependence between endothelial cells and axons during development and the contribution of endothelial progenitor cells (EPCs) in ischemic injury, we hypothesized that EPCs are important in axonal survival after TBI. We examined the effects of allogenic-cultured EPCs on white matter protection and microvascular maintenance after midline fluid percussion injury in adult Sprague-Dawley rats. We used two in vitro models of injury, mechanical stretch and oxygen-glucose deprivation (OGD), to examine the effects of EPCs on the mechanical and ischemic components of brain trauma, respectively. Our results indicate that EPCs improve the white matter integrity and decrease capillary breakdown after injury. Cultured cortical neurons exposed to OGD had less axon degeneration when treated with EPC-conditioned media, whereas no effect was seen in axons injured by mechanical stretch. The results indicate that EPCs are important for the protection of the white matter after trauma and represent a potential avenue for therapy.Journal of Cerebral Blood Flow & Metabolism advance online publication, 4 December 2013; (2013) 0, 000-000. doi:10.1038/jcbfm.2013.216.
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Assessment of a novel, capsid-modified adenovirus with an improved vascular gene transfer profile.
J Cardiothorac Surg
PUBLISHED: 04-26-2013
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Cardiovascular disorders, including coronary artery bypass graft failure and in-stent restenosis remain significant opportunities for the advancement of novel therapeutics that target neointimal hyperplasia, a characteristic of both pathologies. Gene therapy may provide a successful approach to improve the clinical outcome of these conditions, but would benefit from the development of more efficient vectors for vascular gene delivery. The aim of this study was to assess whether a novel genetically engineered Adenovirus could be utilised to produce enhanced levels of vascular gene expression.
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MicroRNA regulation of endothelial homeostasis and commitment-implications for vascular regeneration strategies using stem cell therapies.
Free Radic. Biol. Med.
PUBLISHED: 04-22-2013
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Human embryonic (hESC) and induced pluripotent (hiPSC) stem cells have broad therapeutic potential in the treatment of a range of diseases, including those of the vascular system. Both hESCs and hiPSCs have the capacity for indefinite self-renewal, in addition to their ability to differentiate into any adult cell type. These cells could provide a potentially unlimited source of cells for transplantation and, therefore, provide novel treatments, e.g. in the production of endothelial cells for vascular regeneration. MicroRNAs are short, noncoding RNAs that act posttranscriptionally to control gene expression and thereby exert influence over a wide range of cellular processes, including maintenance of pluripotency and differentiation. Expression patterns of these small RNAs are tissue specific, and changes in microRNA levels have often been associated with disease states in humans, including vascular pathologies. Here, we review the roles of microRNAs in endothelial cell function and vascular disease, as well as their role in the differentiation of pluripotent stem cells to the vascular endothelial lineage. Furthermore, we discuss the therapeutic potential of stem cells and how knowledge and manipulation of microRNAs in stem cells may enhance their capacity for vascular regeneration.
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Practical considerations for the dosing and adjustment of continuous renal replacement therapy in the intensive care unit.
J Crit Care
PUBLISHED: 04-15-2013
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Familiarity with the initiation, dosing, adjustment, and termination of continuous renal replacement therapy (CRRT) is a core skill for contemporary intensivists. Guidelines for how to administer CRRT in the intensive care unit are not well documented. The purpose of this review is to discuss the modalities, terminology, and components of CRRT, with an emphasis on the practical aspects of dosing, adjustments, and termination. Management of electrolyte and acid-base derangements commonly encountered with acute renal failure is emphasized. Knowledge regarding the practical aspects of managing CRRT in the intensive care unit is a prerequisite for achieving desired physiological end points.
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Identification of novel small molecule inhibitors of adenovirus gene transfer using a high throughput screening approach.
J Control Release
PUBLISHED: 03-28-2013
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Due to many favourable attributes adenoviruses (Ads) are the most extensively used vectors for clinical gene therapy applications. However, following intravascular administration, the safety and efficacy of Ad vectors are hampered by the strong hepatic tropism and induction of a potent immune response. Such effects are determined by a range of complex interactions including those with neutralising antibodies, blood cells and factors, as well as binding to native cellular receptors (coxsackie adenovirus receptor (CAR), integrins). Once in the bloodstream, coagulation factor X (FX) has a pivotal role in determining Ad liver transduction and viral immune recognition. Due to difficulties in generating a vector devoid of multiple receptor binding motifs, we hypothesised that a small molecule inhibitor would be of value. Here, a pharmacological approach was implemented to block adenovirus transduction pathways. We developed a high throughput screening (HTS) platform to identify small molecule inhibitors of FX-mediated Ad5 gene transfer. Using an in vitro fluorescence and cell-based HTS, we evaluated 10,240 small molecules. Following sequential rounds of screening, three compounds, T5424837, T5550585 and T5660138 were identified that ablated FX-mediated Ad5 transduction with low micromolar potency. The candidate molecules possessed common structural features and formed part of the one pharmacophore model. Focused, mini-libraries were generated with structurally related molecules and in vitro screening revealed novel hits with similar or improved efficacy. The compounds did not interfere with Ad5:FX engagement but acted at a subsequent step by blocking efficient intracellular transport of the virus. In vivo, T5660138 and its closely related analogue T5660136 significantly reduced Ad5 liver transgene expression at 48 h post-intravenous administration of a high viral dose (1×10¹¹ vp/mouse). Therefore, this study identifies novel and potent small molecule inhibitors of the Ad5 transduction which may have applications in the Ad gene therapy setting.
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miRNA-21 is dysregulated in response to vein grafting in multiple models and genetic ablation in mice attenuates neointima formation.
Eur. Heart J.
PUBLISHED: 03-25-2013
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The long-term failure of autologous saphenous vein bypass grafts due to neointimal thickening is a major clinical burden. Identifying novel strategies to prevent neointimal thickening is important. Thus, this study aimed to identify microRNAs (miRNAs) that are dysregulated during neointimal formation and determine their pathophysiological relevance following miRNA manipulation.
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Vectored dispersal of Symbiodinium by larvae of a Caribbean gorgonian octocoral.
Mol. Ecol.
PUBLISHED: 03-17-2013
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The ability of coral reefs to recover from natural and anthropogenic disturbance is difficult to predict, in part due to uncertainty regarding the dispersal capabilities and connectivity of their reef inhabitants. We developed microsatellite markers for the broadcast spawning gorgonian octocoral Eunicea (Plexaura) flexuosa (four markers) and its dinoflagellate symbiont, Symbiodinium B1 (five markers), and used them to assess genetic connectivity, specificity and directionality of gene flow among sites in Florida, Panama, Saba and the Dominican Republic. Bayesian analyses found that most E. flexuosa from the Florida reef tract, Saba and the Dominican Republic were strongly differentiated from many E. flexuosa in Panama, with the exception of five colonies from Key West that clustered with colonies from Panama. In contrast, Symbiodinium B1 was more highly structured. At least seven populations were detected that showed patterns of isolation by distance. The symbionts in the five unusual Key West colonies also clustered with symbionts from Panama, suggesting these colonies are the result of long-distance dispersal. Migration rate tests indicated a weak signal of northward immigration from the Panama population into the lower Florida Keys. As E. flexuosa clonemates only rarely associated with the same Symbiodinium B1 genotype (and vice versa), these data suggest a dynamic host-symbiont relationship in which E. flexuosa is relatively well dispersed but likely acquires Symbiodinium B1 from highly structured natal areas prior to dispersal. Once vectored by host larvae, these symbionts may then spread through the local population, and/or host colonies may acquire different local symbiont genotypes over time.
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An analysis of the function and expression of D6 on lymphatic endothelial cells.
Blood
PUBLISHED: 03-11-2013
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The mechanisms by which CC chemokine receptor (CCR)7 ligands are selectively presented on lymphatic endothelium in the presence of inflammatory chemokines are poorly understood. The chemokine-scavenging receptor D6 is expressed on lymphatic endothelial cells (LEC) and contributes to selective presentation of CCR7 ligands by suppressing inflammatory chemokine binding to LEC surfaces. As well as preventing inappropriate inflammatory cell attachment to LECs, D6 is specifically involved in regulating the ability of LEC to discriminate between mature and immature dendritic cells (DCs). D6 overexpression reduces immature DC (iDC) adhesion to LECs, whereas D6 knockdown increases adhesion of iDCs that displace mature DCs. LEC D6 expression is regulated by growth factors, cytokines, and tumor microenvironments. In particular, interleukin-6 and interferon-? are potent inducers, indicating a preferential role for D6 in inflamed contexts. Expression of the viral interleukin-6 homolog from Kaposi sarcoma-associated herpesvirus is also sufficient to induce significant D6 upregulation both in vitro and in vivo, and Kaposi sarcoma and primary effusion lymphoma cells demonstrate high levels of D6 expression. We therefore propose that D6, which is upregulated in both inflammatory and tumor contexts, is an essential regulator of inflammatory leukocyte interactions with LECs and is required for immature/mature DC discrimination by LECs.
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DNA barcoding reveals the coral "laboratory-rat", Stylophora pistillata encompasses multiple identities.
Sci Rep
PUBLISHED: 03-05-2013
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Stylophora pistillata is a widely used coral "lab-rat" species with highly variable morphology and a broad biogeographic range (Red Sea to western central Pacific). Here we show, by analysing Cytochorme Oxidase I sequences, from 241 samples across this range, that this taxon in fact comprises four deeply divergent clades corresponding to the Pacific-Western Australia, Chagos-Madagascar-South Africa, Gulf of Aden-Zanzibar-Madagascar, and Red Sea-Persian/Arabian Gulf-Kenya. On the basis of the fossil record of Stylophora, these four clades diverged from one another 51.5-29.6?Mya, i.e., long before the closure of the Tethyan connection between the tropical Indo-West Pacific and Atlantic in the early Miocene (16-24?Mya) and should be recognised as four distinct species. These findings have implications for comparative ecological and/or physiological studies carried out using Stylophora pistillata as a model species, and highlight the fact that phenotypic plasticity, thought to be common in scleractinian corals, can mask significant genetic variation.
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Critical research needs for identifying future changes in Gulf coral reef ecosystems.
Mar. Pollut. Bull.
PUBLISHED: 02-24-2013
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Expert opinion was assessed to identify current knowledge gaps in determining future changes in Arabian/Persian Gulf (thereafter Gulf) coral reefs. Thirty-one participants submitted 71 research questions that were peer-assessed in terms of scientific importance (i.e., filled a knowledge gap and was a research priority) and efficiency in resource use (i.e., was highly feasible and ecologically broad). Ten research questions, in six major research areas, were highly important for both understanding Gulf coral reef ecosystems and also an efficient use of limited research resources. These questions mirrored global evaluations of the importance of understanding and evaluating biodiversity, determining the potential impacts of climate change, the role of anthropogenic impacts in structuring coral reef communities, and economically evaluating coral reef communities. These questions provide guidance for future research on coral reef ecosystems within the Gulf, and enhance the potential for assessment and management of future changes in this globally significant region.
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Treatment with a highly selective ?? antagonist causes dose-dependent impairment of cerebral perfusion after hemodilution in rats.
Anesth. Analg.
PUBLISHED: 02-11-2013
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Acute ?-blockade has been associated with a dose-dependent increase in adverse outcomes, including stroke and mortality. Acute blood loss contributes to the incidence of these adverse events. In an attempt to link the risks of acute blood loss and ?-blockade, animal studies have demonstrated that acute ?-blockade impairs cerebral perfusion after hemodilution. We expanded on these findings by testing the hypothesis that acute ?-blockade with a highly ?(1)-specific antagonist (nebivolol) causes dose-dependent cerebral hypoxia during hemodilution.
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Profiling of transcriptional and epigenetic changes during directed endothelial differentiation of human embryonic stem cells identifies FOXA2 as a marker of early mesoderm commitment.
Stem Cell Res Ther
PUBLISHED: 02-08-2013
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INTRODUCTION: Differentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage. METHODS: We performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes. RESULTS: We identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC-EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation. CONCLUSION: This enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease.
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MicroRNAs in pulmonary arterial remodeling.
Cell. Mol. Life Sci.
PUBLISHED: 02-05-2013
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Pulmonary arterial remodeling is a presently irreversible pathologic hallmark of pulmonary arterial hypertension (PAH). This complex disease involves pathogenic dysregulation of all cell types within the small pulmonary arteries contributing to vascular remodeling leading to intimal lesions, resulting in elevated pulmonary vascular resistance and right heart dysfunction. Mutations within the bone morphogenetic protein receptor 2 gene, leading to dysregulated proliferation of pulmonary artery smooth muscle cells, have been identified as being responsible for heritable PAH. Indeed, the disease is characterized by excessive cellular proliferation and resistance to apoptosis of smooth muscle and endothelial cells. Significant gene dysregulation at the transcriptional and signaling level has been identified. MicroRNAs are small non-coding RNA molecules that negatively regulate gene expression and have the ability to target numerous genes, therefore potentially controlling a host of gene regulatory and signaling pathways. The major role of miRNAs in pulmonary arterial remodeling is still relatively unknown although research data is emerging apace. Modulation of miRNAs represents a possible therapeutic target for altering the remodeling phenotype in the pulmonary vasculature. This review will focus on the role of miRNAs in regulating smooth muscle and endothelial cell phenotypes and their influence on pulmonary remodeling in the setting of PAH.
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Clinical outcome prediction in aneurysmal subarachnoid hemorrhage using Bayesian neural networks with fuzzy logic inferences.
Comput Math Methods Med
PUBLISHED: 01-24-2013
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The novel clinical prediction approach of Bayesian neural networks with fuzzy logic inferences is created and applied to derive prognostic decision rules in cerebral aneurysmal subarachnoid hemorrhage (aSAH).
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An AT-hook domain in MeCP2 determines the clinical course of Rett syndrome and related disorders.
Cell
PUBLISHED: 01-22-2013
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Mutations in the X-linked MECP2 cause Rett syndrome, a devastating neurological disorder typified by a period of apparently normal development followed by loss of cognitive and psychomotor skills. Data from rare male patients suggest symptom onset and severity can be influenced by the location of the mutation, with amino acids 270 and 273 marking the difference between neonatal encephalopathy and death, on the one hand, and survival with deficits on the other. We therefore generated two mouse models expressing either MeCP2-R270X or MeCP2-G273X. The mice developed phenotypes at strikingly different rates and showed differential ATRX nuclear localization within the nervous system, over time, coinciding with phenotypic progression. We discovered that MeCP2 contains three AT-hook-like domains over a stretch of 250 amino acids, like HMGA DNA-bending proteins; one conserved AT-hook is disrupted in MeCP2-R270X, lending further support to the notion that one of MeCP2s key functions is to alter chromatin structure.
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A fast one-step extraction and UPLC-MS/MS analysis for E2/D 2 series prostaglandins and isoprostanes.
Lipids
PUBLISHED: 01-18-2013
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Prostaglandins (PG) and isoprostanes (iso-PG) may be derived through cyclooxygenase or free radical pathways and are important signaling molecules that are also robust biomarkers of oxidative stress. Their quantification is important for understanding many biological processes where PG, iso-PG, or oxidative stress are involved. One of the common methods for PG and iso-PG quantifications is LC-MS/MS that allows a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the currently used LC-MS/MS methods require a multi-step extraction and a long (within an hour) LC separation to achieve simultaneous separation and analysis of the major iso-PG. The developed and validated for brain tissue analysis one-step extraction protocol and UPLC-MS/MS method significantly increases the recovery of the PG extraction up to 95 %, and allows for a much faster (within 4 min) major iso-PGE2 and -PGD2 separation with 5 times narrower chromatographic peaks as compared to previously used methods. In addition, it decreases the time and cost of analysis due to the one-step extraction approach performed in disposable centrifuge tubes. All together, this significantly increases the sensitivity, and the time and cost efficiency of the PG and iso-PG analysis.
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Lower leg neuromuscular changes following fibular reposition taping in individuals with chronic ankle instability.
Man Ther
PUBLISHED: 01-07-2013
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Soleus and peroneus longus arthrogenic muscle inhibition has been reported in patients with chronic ankle instability. Fibular reposition taping (FRT) has been shown to improve outcomes however, the influence of FRT on neuromuscular function is unknown. The purpose was to determine the effects of FRT on soleus and peroneus longus h-reflex amplitude in patients with chronic ankle instability. This was a randomized, single blind, sham-controlled crossover laboratory experiment. Twelve young adults with chronic ankle instability (age: 21.5 ± 1.6, height: 173.8 ± 10.4, mass: 72.8 ± 16.3) participated in two testing sessions. We measured peak h-reflex and M-wave of the soleus and peroneus longus and v-wave of the soleus only. Measurements were recorded before and after the application of FRT or a sham tape intervention. Sessions were separated by a week and counterbalanced, h-reflex and v-wave were normalized to M-wave at each time point. Significant increase in h/M ratio was observed in the soleus following FRT compared to baseline, but not after the sham intervention. No difference in peroneus longus h/M ratio or soleus v/M ratio was observed in any session. FRT may be an effective modality for increasing soleus h-reflex for patients with chronic ankle instability.
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Fast growth may impair regeneration capacity in the branching coral Acropora muricata.
PLoS ONE
PUBLISHED: 01-01-2013
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Regeneration of artificially induced lesions was monitored in nubbins of the branching coral Acropora muricata at two reef-flat sites representing contrasting environments at Réunion Island (21°07S, 55°32E). Growth of these injured nubbins was examined in parallel, and compared to controls. Biochemical compositions of the holobiont and the zooxanthellae density were determined at the onset of the experiment, and the photosynthetic efficiency (Fv/Fm ) of zooxanthellae was monitored during the experiment. Acropora muricata rapidly regenerated small lesions, but regeneration rates significantly differed between sites. At the sheltered site characterized by high temperatures, temperature variations, and irradiance levels, regeneration took 192 days on average. At the exposed site, characterized by steadier temperatures and lower irradiation, nubbins demonstrated fast lesion repair (81 days), slower growth, lower zooxanthellae density, chlorophyll a concentration and lipid content than at the former site. A trade-off between growth and regeneration rates was evident here. High growth rates seem to impair regeneration capacity. We show that environmental conditions conducive to high zooxanthellae densities in corals are related to fast skeletal growth but also to reduced lesion regeneration rates. We hypothesize that a lowered regenerative capacity may be related to limited availability of energetic and cellular resources, consequences of coral holobionts operating at high levels of photosynthesis and associated growth.
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Application of blood-based biomarkers in human mild traumatic brain injury.
Front Neurol
PUBLISHED: 01-01-2013
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Traumatic Brain Injury (TBI) is a global health concern. The majority of TBIs are mild, yet our ability to diagnose and treat mild traumatic brain injury (mTBI) is lacking. This deficiency results from a variety of issues including the difficulty in interpreting ambiguous clinically presented symptoms, and ineffective imaging techniques. Thus, researchers have begun to explore cellular and molecular based approaches to improve both diagnosis and prognosis. This has been met with a variety of challenges, including difficulty in relating biological markers to current clinical symptoms, and overcoming our lack of fundamental understanding of the pathophysiology of mTBI. However, recent adoption of high throughput technologies and a change in focus from the identification of single to multiple markers has given just optimism to mTBI research. The purpose of this review is to highlight a number of current experimental peripheral blood biomarkers of mTBI, as well as comment on the issues surrounding their clinical application and utility.
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The value of serum biomarkers in prediction models of outcome after mild traumatic brain injury.
J Trauma
PUBLISHED: 11-11-2011
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To determine, using a civilian model of mild traumatic brain injury (TBI), the added value of biomarker sampling upon prognostication of outcome at 1 week and 6 weeks postinjury.
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Controlled blast exposure during forced explosive entry training and mild traumatic brain injury.
J Trauma
PUBLISHED: 11-11-2011
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There is a paucity of data regarding the pathophysiology and short- and long-term neurologic consequences of primary blast injury in humans. The purpose of this investigation was to test the feasibility of implementing a research protocol in the context of a forced explosive entry training course.
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MicroRNA and vascular remodelling in acute vascular injury and pulmonary vascular remodelling.
Cardiovasc. Res.
PUBLISHED: 11-07-2011
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Vascular remodelling is an integral pathological process central to a number of cardiovascular diseases. The complex interplay between distinct cell populations in the vessel wall following vascular injury leads to inflammation, cellular dysfunction, pro-growth signals in the smooth muscle cell (SMC) compartment, and the acquisition of a synthetic phenotype. Although the signals for vascular remodelling are diverse in different pathological contexts, SMC proliferation and migration are consistently observed. It is therefore critical to elucidate key mechanisms central to these processes. MicroRNAs (miRNAs) are small non-coding sequences of RNA that have the capacity to regulate many genes, pathways, and complex biological networks within cells, acting either alone or in concert with one another. In diseases such as cancer and cardiac disease, the role of miRNA in disease pathogenesis has been documented in detail. In contrast, despite a great deal of interest in miRNA, relatively few studies have directly assessed the role of miRNA in vascular remodelling. The potential for modulation of miRNA to achieve therapeutic benefits in this setting is attractive. Here, we focus on the role of miRNA in vascular inflammation and remodelling associated with acute vascular injury (vein graft disease, angioplasty restenosis, and in-stent restenosis) as well as in vascular remodelling associated with the development of pulmonary arterial hypertension.
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Sustained reduction of vein graft neointima formation by ex vivo TIMP-3 gene therapy.
Circulation
PUBLISHED: 09-14-2011
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Coronary artery vein graft failure, resulting from thrombosis, intimal thickening, and atherosclerosis, is a significant clinical problem, with approximately 50% of vein grafts failing within 10 years. Intimal thickening is caused by migration of vascular smooth muscle cells from the media to the intima, where they proliferate. Interventions using gene transfer to inhibit vascular smooth muscle cells proliferation and migration are attractive because ex vivo access to the graft is possible. The involvement of matrix-degrading metalloproteinases in intimal thickening is well established, and we previously showed that adenoviral-delivered overexpression of an endogenous inhibitor, the tissue inhibitor of metalloproteinases-3 (TIMP-3), significantly retarded intimal thickening in short-term autologous porcine arteriovenous interposition grafts (28 days). However, it is essential to determine whether this approach will provide longer-term benefits.
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Inhibition of in-stent stenosis by oral administration of bindarit in porcine coronary arteries.
Arterioscler. Thromb. Vasc. Biol.
PUBLISHED: 08-18-2011
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We have previously demonstrated that bindarit, a selective inhibitor of monocyte chemotactic proteins (MCPs), is effective in reducing neointimal formation in rodent models of vascular injury by reducing smooth muscle cell proliferation and migration and neointimal macrophage content, effects associated with the inhibition of MCP-1/CCL2 production. The aim of the current study was to evaluate the efficacy of bindarit on in-stent stenosis in the preclinical porcine coronary stent model.
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A cluster of basic amino acids in the factor X serine protease mediates surface attachment of adenovirus/FX complexes.
J. Virol.
PUBLISHED: 08-17-2011
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Hepatocyte transduction following intravenous administration of adenovirus 5 (Ad5) is mediated by interaction between coagulation factor X (FX) and the hexon. The FX serine protease (SP) domain tethers the Ad5/FX complex to hepatocytes through binding heparan sulfate proteoglycans (HSPGs). Here, we identify the critical HSPG-interacting residues of FX. We generated an FX mutant by modifying seven residues in the SP domain. Surface plasmon resonance demonstrated that mutations did not affect binding to Ad5. FX-mediated, HSPG-associated cell binding and transduction were abolished. A cluster of basic amino acids in the SP domain therefore mediates surface interaction of the Ad/FX complex.
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Patients and family members views on how clinicians enact and how they should enact incident disclosure: the "100 patient stories" qualitative study.
BMJ
PUBLISHED: 07-27-2011
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To investigate patients and family members perceptions and experiences of disclosure of healthcare incidents and to derive principles of effective disclosure.
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A model of low-level primary blast brain trauma results in cytoskeletal proteolysis and chronic functional impairment in the absence of lung barotrauma.
J. Neurotrauma
PUBLISHED: 07-26-2011
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Shock-wave exposure from improvised explosive devices (IEDs) has been implicated as a possible contributing factor to neurological impairment reported in combat veterans. However, evidence-based substantiation of this implication, particularly for low-level exposure in the absence of external signs of trauma, remain elusive. Accordingly, we constructed an open-ended shock tube producing a short-duration, low-amplitude shockwave. Low-level (11.5?kPa static overpressure) complex shock-wave exposure in rats resulted in no histological evidence of lung injury. By contrast, delayed cytoskeletal proteolysis of ?II-spectrin was detected in the cortex and hippocampus by 12?h post-injury. Cell death was minimal and localized predominantly in the corpus callosum and periventricular regions. These regions, with presumably different density interfaces, exhibit biological responses to shockwaves consistent with interface turbulence described by Richtmyer-Meshkov instability. Evoked compound action potential (CAP) recordings from the corpus callosum showed a significant increase in the duration of CAP responses at 14 and 30 days post-injury, and a gradual depression in the unmyelinated fiber amplitude. Shielding the head attenuated ?II-spectrin cytoskeletal breakdown, thus directly implicating low-level shock-wave exposure as a cause of brain injury in the rat. Despite anatomical and scaling differences in rats compared to humans, the results suggest the potential for undiagnosed traumatic brain pathologies occurring in combat veterans following shock-wave exposure.
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MicroRNAs regulating cell pluripotency and vascular differentiation.
Vascul. Pharmacol.
PUBLISHED: 06-03-2011
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Human embryonic stem cells (hESC) offer broad potential for regenerative medicine owing to their capacity for self renewal, exponential scale up and differentiation into any cell type in the adult body. hESC have been proposed as a potentially unlimited source for the generation of transplantable, healthy, functional vascular cells for repair of ischemic tissues. To optimally harness this potential necessitates precise control over biological processes that govern maintenance, pluripotency and cell differentiation including signalling cascades, gene expression profiles and epigenetic modification. Such control may be elicited by microRNAs, which are powerful negative regulators of gene expression. Here, we review the role for miRNAs in both the maintenance of pluripotency and differentiation of cells to a cardiovascular lineage including endothelial cells, vascular smooth muscle cells and cardiomyocytes and put this into context for regenerative medicine in the cardiovascular system.
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Profiling the indole alkaloids in yohimbe bark with ultra-performance liquid chromatography coupled with ion mobility quadrupole time-of-flight mass spectrometry.
Rapid Commun. Mass Spectrom.
PUBLISHED: 04-27-2011
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An ultra-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (UPLC/IM-QTOF-MS) method was developed for profiling the indole alkaloids in yohimbe bark. Many indole alkaloids with the yohimbine or ajmalicine core structure, plus methylated, oxidized and reduced species, were characterized. Common fragments and mass differences are described. It was shown that the use of IMS could provide another molecular descriptor, i.e. molecular shape by rotationally averaged collision cross-section; this is of great value for identification of constituents when reference materials are usually not available. Using the combination of high resolution (~40000) accurate mass measurement with time-aligned parallel (TAP) fragmentation, MS(E) (where E represents collision energy), ion mobility mass spectrometry (IMS) and UPLC chromatography, a total 55 indole alkaloids were characterized and a few new indole alkaloids are reported for the first time.
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Effect of implantation site and injury condition on host response to human-derived fascia lata ECM in a rat model.
J. Orthop. Res.
PUBLISHED: 04-15-2011
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The host response and remodeling of ECM scaffolds are believed to be critical determinants of success or failure in repair or reconstructive procedures. Host response has been investigated in subcutaneous or abdominal wall implantation models. The extent to which evaluation of the host response to ECM intended for tendon or ligament repair should be performed in an orthotopic site is not known. This study compared the host response to human-derived fascia lata ECM among various implantation sites in the rat model. Results showed that a xenograft in the rat shoulder does not exhibit a different host response at 7 days from xenograft in the body wall, suggesting that either site may be appropriate to study the early host response to biologic grafts as well as the effect of various treatments aimed to modify the early host response. By 28 days, a xenograft in the rat shoulder does elicit a unique host response from that seen in the body wall. Therefore, it may be more appropriate to use an orthotopic shoulder model for investigating the long-term host response and remodeling of biologic grafts to be used for rotator cuff repair.
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miR-21 and miR-214 are consistently modulated during renal injury in rodent models.
Am. J. Pathol.
PUBLISHED: 04-01-2011
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Transforming growth factor (TGF)-? is one of the main fibrogenic cytokines that drives the pathophysiology of progressive renal scarring. MicroRNAs (miRNAs) are endogenous non-coding RNAs that post-transcriptionally regulate gene expression. We examined the role of TGF-?-induced expression of miR-21, miRNAs in cell culture models and miRNA expression in relevant models of renal disease. In vitro, TGF-? changed expression of miR-21, miR-214, and miR-145 in rat mesangial cells (CRL-2753) and miR-214, miR-21, miR-30c, miR-200b, and miR-200c during induction of epithelial-mesenchymal transition in rat tubular epithelial cells (NRK52E). miR-214 expression was robustly modulated in both cell types, whereas in tubular epithelial cells miR-21 was increased and miR-200b and miR-200c were decreased by 58% and 48%, respectively, in response to TGF-?. TGF-? receptor-1 was found to be a target of miR-200b/c and was down-regulated after overexpression of miR-200c. To assess the differential expression of these miRNAs in vivo, we used the anti-Thy1.1 mesangial glomerulonephritis model and the unilateral ureteral obstruction model in which TGF-? plays a role and also a genetic model of hypertension, the stroke-prone spontaneously hypertensive rat with and without salt loading. The expressions of miR-214 and miR-21 were significantly increased in all in vivo models, showing a possible miRNA signature of renal damage despite differing causes.
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Teacher-student relationship climate and school outcomes: implications for educational policy initiatives.
J Youth Adolesc
PUBLISHED: 03-07-2011
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In recent discussions regarding concerns about the academic achievement of US students, educational policy makers have suggested the implementation of certain teacher policies. To address the limited empirical research on the putative educational impact of such policies, this study used multilevel structural equation models to investigate the longitudinal associations between teacher evaluation and reward policies, and student mathematics achievement and dropout with a national sample of students (n = 7,779) attending one of 431 public high schools. The student sample included an equal number of boys and girls averaging 16 years of age, and included a White (53%) majority. This study examined whether associations between teacher policies and student achievement were mediated by the teacher-student relationship climate. Results of this study were threefold. First, teacher evaluation policies that allowed students to evaluate their teachers were associated with more positive student reports of the classroom teaching climate. Second, schools with teacher reward policies that included assigning higher performing teachers with higher performing students had a negative association with student perceptions of the teaching climate. Lastly, schools with better student perceptions of the teaching climate were associated with lower student dropout rates by students senior year. These findings are discussed in light of their educational policy implications.
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Serotonin transporter, sex, and hypoxia: microarray analysis in the pulmonary arteries of mice identifies genes with relevance to human PAH.
Physiol. Genomics
PUBLISHED: 02-08-2011
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Pulmonary arterial hypertension (PAH) is up to threefold more prevalent in women than men. Female mice overexpressing the serotonin transporter (SERT; SERT+ mice) exhibit PAH and exaggerated hypoxia-induced PAH, whereas male SERT+ mice remain unaffected. To further investigate these sex differences, microarray analysis was performed in the pulmonary arteries of normoxic and chronically hypoxic female and male SERT+ mice. Quantitative RT-PCR analysis was employed for validation of the microarray data. In relevant groups, immunoblotting was performed for genes of interest (CEBP?, CYP1B1, and FOS). To translate clinical relevance to our findings, CEBP?, CYP1B1, and FOS mRNA and protein expression was assessed in pulmonary artery smooth muscle cells (PASMCs) derived from idiopathic PAH (IPAH) patients and controls. In female SERT+ mice, multiple pathways with relevance to PAH were altered. This was also observed in chronically hypoxic female SERT+ mice. We selected 10 genes of interest for qRT-PCR analysis (FOS, CEBP?, CYP1B1, MYL3, HAMP2, LTF, PLN, NPPA, UCP1, and C1S), and 100% concordance was reported. Protein expression of three selected genes, CEBP?, CYP1B1, FOS, was also upregulated in female SERT+ mice. Serotonin and 17?-estradiol increased CEBP?, CYP1B1, and FOS protein expression in PASMCs. In addition, CEBP?, CYP1B1, and FOS mRNA and protein expression was also increased in PASMCs derived from IPAH patients. Here, we have identified a number of genes that may predispose female SERT+ mice to PAH, and these findings may also be relevant to human PAH.
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Development and characterization of a mouse floxed Bmp2 osteoblast cell line that retains osteoblast genotype and phenotype.
Cell Tissue Res.
PUBLISHED: 01-27-2011
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Bone morphogenetic protein 2 (Bmp2) is essential for osteoblast differentiation and osteogenesis. Generation of floxed Bmp2 osteoblast cell lines is a valuable tool for studying the effects of Bmp2 on osteoblast differentiation and its signaling pathways during skeletal metabolism. Due to relatively limited sources of primary osteoblasts, we have developed cell lines that serve as good surrogate models for the study of osteoblast cell differentiation and bone mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 osteoblast cell lines. Primary mouse floxed Bmp2 osteoblasts were transfected with pSV3-neo and clonally selected. These transfected cells were verified by PCR and immunohistochemistry. To determine the genotype and phenotype of the immortalized cells, cell morphology, proliferation, differentiation and mineralization were analyzed. Also, expression of osteoblast-related gene markers including Runx2, Osx, ATF4, Dlx3, bone sialoprotein, dentin matrix protein 1, osteonectin, osteocalcin and osteopontin were examined by quantitative RT-PCR and immunohistochemistry. These results showed that immortalized floxed Bmp2 osteoblasts had a higher proliferation rate but preserved their genotypic and phenotypic characteristics similar to the primary cells. Thus, we, for the first time, describe the development of immortalized mouse floxed Bmp2 osteoblast cell lines and present a useful model to study osteoblast biology mediated by BMP2 and its downstream signaling transduction pathways.
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Deregulation of microRNA-503 contributes to diabetes mellitus-induced impairment of endothelial function and reparative angiogenesis after limb ischemia.
Circulation
PUBLISHED: 01-10-2011
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Diabetes mellitus impairs endothelial cell (EC) function and postischemic reparative neovascularization by molecular mechanisms that are not fully understood. microRNAs negatively regulate the expression of target genes mainly by interaction in their 3 untranslated region.
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Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors.
Mol. Ther.
PUBLISHED: 10-26-2010
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Retroviral vectors remain the most efficient and widely applied system for induction of pluripotency. However, mutagenic effects have been documented in both laboratory and clinical gene therapy studies, principally as a result of dysregulated host gene expression in the proximity of defined integration sites. Here, we report that cells with characteristics of pluripotent stem cells can be produced from normal human fibroblasts in the absence of reprogramming transcription factors (TFs) during lentiviral (LV) vector-mediated gene transfer. This occurred via induced alterations in host gene and microRNA (miRNA) expression and detrimental changes in karyotype. These findings demonstrate that vector-induced genotoxicity may alone play a role in somatic cell reprogramming derivation and urges caution when using integrating vectors in this setting. Clearer understanding of this process may additionally reveal novel insights into reprogramming pathways.
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Out-of-hospital hypertonic resuscitation following severe traumatic brain injury: a randomized controlled trial.
JAMA
PUBLISHED: 10-07-2010
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Hypertonic fluids restore cerebral perfusion with reduced cerebral edema and modulate inflammatory response to reduce subsequent neuronal injury and thus have potential benefit in resuscitation of patients with traumatic brain injury (TBI).
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Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells.
Biochem. J.
PUBLISHED: 10-07-2010
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hESCs (human embryonic stem cells) have enormous potential for use in pharmaceutical development and therapeutics; however, to realize this potential, there is a requirement for simple and reproducible cell culture methods that provide adequate numbers of cells of suitable quality. We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with EHNA [erythro-9-(2-hydroxy-3-nonyl)adenine], an established inhibitor of ADA (adenosine deaminase) and cyclic nucleotide PDE2 (phosphodiesterase 2). hESCs maintained in feeder-free conditions with EHNA for more than ten passages showed no reduction in hESC-associated markers including NANOG, POU5F1 (POU domain class 5 transcription factor 1, also known as Oct-4) and SSEA4 (stage-specific embryonic antigen 4) compared with cells maintained in feeder-free conditions containing bFGF (basic fibroblast growth factor). Spontaneous differentiation was reversibly suppressed by the addition of EHNA, but, upon removing EHNA, hESC populations underwent efficient spontaneous, multi-lineage and directed differentiation. EHNA also acts as a strong blocker of directed neuronal differentiation. Chemically distinct inhibitors of ADA and PDE2 lacked the capacity of EHNA to suppress hESC differentiation, suggesting that the effect is not driven by inhibition of either ADA or PDE2. Preliminary structure-activity relationship analysis found the differentiation-blocking properties of EHNA to reside in a pharmacophore comprising a close adenine mimetic with an extended hydrophobic substituent in the 8- or 9-position. We conclude that EHNA and simple 9-alkyladenines can block directed neuronal and spontaneous differentiation in the absence of exogenous cytokine addition, and may provide a useful replacement for bFGF in large-scale or cGMP-compliant processes.
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Pluripotent stem cell differentiation into vascular cells: a novel technology with promises for vascular re(generation).
Pharmacol. Ther.
PUBLISHED: 10-04-2010
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Several types of stem and progenitor cells are currently under investigation for their potential to accomplish vascular regeneration. This review focuses on embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We will discuss the technologies allowing for their derivation, culture expansion and maintenance in a pluripotent status. Moreover, both ESCs and iPSCs can be differentiated in endothelial cells (ECs) and mural cell, including vascular smooth muscle cells (VSMCs). Here, we will describe the involvements of growth factors (vascular endothelial growth factors-VEGFs-, platet-derived growth factors-PDGFs-), Wnt and Notch signal pathways, reactive oxygen species (ROS), histone deacetylases (HDACs), and microRNAs (miRNAs) in vascular cell differentiation from pluripotent stem cells. We will additionally describe the therapeutic potential of stem cells for vascular medicine.
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Vascular-targeting antioxidant therapy in a model of hypertension and stroke.
J. Cardiovasc. Pharmacol.
PUBLISHED: 09-15-2010
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Oxidative stress is implicated in the pathogenesis of hypertension and stroke. Superoxide is produced by NAD(P)H oxidase in the vasculature and reduces nitric oxide bioavailability, which leads to increased blood pressure. The objective of this study was to determine whether targeting an antioxidant peptide to the vasculature would increase the antioxidant effect and reduce systolic blood pressure (SBP) in a model of genetic hypertension, the stroke-prone spontaneously hypertensive rat. Vascular-targeting peptides CRPPR and CSGMARTKC were identified by phage display in mice. These peptides retain their selectivity across species and target the aorta (CRPPR) and cardiac vasculature (CSGMARTKC) in the stroke-prone spontaneously hypertensive rat. These vascular-targeting peptides were linked to the antioxidant peptide gp91ds, which selectively inhibits assembly of NAD(P)H oxidase, thereby reducing superoxide production. SBP was determined for 1 week before treatment followed by 3 weeks of study duration before euthanasia. SBP in the control animals increased from 178.1 ± 4.1 mmHg to 201.6 ± 9.0 mmHg. The SBP of the animals treated with gp91ds alone, HIV-tat-gp91ds, and CSGMARTKC-gp91ds increased from 177.8 ± 3.5 mmHg, 179.8 ± 4.7 mmHg, and 177.9 ± 5.2 mmHg, respectively, to 201.6 ± 10.8 mmHg, 200.3 ± 11.7 mmHg and 205.7 ± 10.9 mmHg, respectively. This increase in SBP was significantly attenuated in animals receiving CRPPR-gp91ds (maximum SBP 187.5 mmHg ± 5.2, *P , 0.001 versus other treatment groups and control group). Additionally, animals treated with CRPPR-gp91ds, CSGMARTKC-gp91ds, and gp91ds alone showed significantly improved nitric oxide bioavailability determined by large vessel myography. Therefore, targeting an antioxidant to the aortic vasculature in vivo using peptides can significantly improve nitric oxide bioavailability and attenuate the time-dependent and progressive increase in SBP in the stroke-prone spontaneously hypertensive rat. This study has demonstrated the importance and potential benefit of targeting a biologically active peptide in the context of a preclinical model of endothelial dysfunction and hypertension.
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