LC-MS/MS Confirms That COX-1 Drives Vascular Prostacyclin Whilst Gene Expression Pattern Reveals Non-Vascular Sites of COX-2 Expression

There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF_1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF_1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.     

Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs

Due to structural flexibility, RNase sensitivity, and serum instability, RNA nanoparticles with concrete shapes for in vivo application remain challenging to construct. Here we report the construction of 14 RNA nanoparticles with solid shapes for targeting cancers specifically. These RNA nanoparticles were resistant to RNase degradation, stable in serum for >36 h, and stable in vivo after systemic injection. By applying RNA nanotechnology and exemplifying with these 14 RNA nanoparticles, we have established the technology and developed “toolkits” utilizing a variety of principles to construct RNA architectures with diverse shapes and angles. The structure elements of phi29 motor pRNA were utilized for fabrication of dimers, twins, trimers, triplets, tetramers, quadruplets, pentamers, hexamers, heptamers, and other higher-order oligomers, as well as branched diverse architectures via hand-in-hand, foot-to-foot, and arm-on-arm interactions. These novel RNA nanostructures harbor resourceful functionalities for numerous applications in nanotechnology and medicine. It was found that all incorporated functional modules, such as siRNA, ribozymes, aptamers, and other functionalities, folded correctly and functioned independently within the nanoparticles. The incorporation of all functionalities was achieved prior, but not subsequent, to the assembly of the RNA nanoparticles, thus ensuring the production of homogeneous therapeutic nanoparticles. More importantly, upon systemic injection, these RNA nanoparticles targeted cancer exclusively in vivo without accumulation in normal organs and tissues. These findings open a new territory for cancer targeting and treatment. The versatility and diversity in structure and function derived from one biological RNA molecule implies immense potential concealed within the RNA nanotechnology field.     

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and “pearls of wisdom”/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.     

Development of an Indicator to Monitor Mediterranean Wetlands

Wetlands are sensitive ecosystems that are increasingly subjected to threats from anthropogenic factors. In the last decades, coastal Mediterranean wetlands have been suffering considerable pressures from land use change, intensification of urban growth, increasing tourism infrastructure and intensification of agricultural practices. Remote sensing (RS) and Geographic Information Systems (GIS) techniques are efficient tools that can support monitoring Mediterranean coastal wetlands on large scales and over long periods of time. The study aims at developing a wetland indicator to support monitoring Mediterranean coastal wetlands using these techniques. The indicator makes use of multi-temporal Landsat images, land use reference layers, a 50m numerical model of the territory (NMT) and Corine Land Cover (CLC) for the identification and mapping of wetlands. The approach combines supervised image classification techniques making use of vegetation indices and decision tree analysis to identify the surface covered by wetlands at a given date. A validation process is put in place to compare outcomes with existing local wetland inventories to check the results reliability. The indicator´s results demonstrate an improvement in the level of precision of change detection methods achieved by traditional tools providing reliability up to 95% in main wetland areas. The results confirm that the use of RS techniques improves the precision of wetland detection compared to the use of CLC for wetland monitoring and stress the strong relation between the level of wetland detection and the nature of the wetland areas and the monitoring scale considered.     

Sawgrass (Cladium jamaicense) responses as early indicators of low-level phosphorus enrichment in the Florida Everglades

Anthropogenic phosphorus (P) inputs to the Florida Everglades have produced dramatic changes in the wetland vegetation of this otherwise oligotrophic system. While the proliferation of undesirable plant species in response to enrichment has been well documented, nutrient-related changes in the physiological and morphological attributes of existing vegetation, prior to any shifts in species composition or changes in the spatial extent of certain taxa, have yet to be adequately characterized. In this experiment, three sawgrass-dominated areas were enriched with P for 3 years at rates of 0.4 g P/m²/year (HP), 0.1 g P/m²/year (LP), or 0 g P/m²/year (controls) to assess potential impacts of P-enriched discharges from stormwater treatment areas into the Everglades. Elevated concentrations of TP in rhizomes and leaves and reduced ratios of leaf N:P were detected in HP plants within ~1 year at most sites. Live leaf densities, plant heights, and plant densities of the HP groups were generally higher than LP and control groups after 2 years, a pattern that was evident even after major fire events. Total aboveground biomass was significantly elevated in both HP and LP treatments at two of the three sites after 3 years. No change in species composition was detected during the study. Planned hydrologic restoration measures will increase P loads into parts of the Everglades that have not previously experienced anthropogenic P enrichment. Monitoring native vegetation such as sawgrass can be a sensitive and relatively robust means of detecting unintended P enrichment in these areas prior to shifts in vegetation community composition or changes in area cover of key species.     

LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.     

Apolipoprotein E genotyping in women with recurrent pregnancy loss: An in silico and experimental hybrid study

The role of apolipoprotein E gene polymorphisms in the pathogenesis of recurrent pregnancy loss remains controversial. Therefore, our objective was to investigate the association between recurrent pregnancy loss and apolipoprotein E gene polymorphisms among northwest Iranian women, and also to predict the impact of these nonsynonymous single nucleotide polymorphisms on structure and function of apolipoprotein E protein. The subjects of our current study consisted of 100 women that have had two or more consecutive idiopathic first trimester miscarriages, and one hundred healthy women from the same geographical areas were used as a control group. After DNA extraction, we used a polymerase chain reaction–restriction fragment length polymorphism to genotype of the apolipoprotein E gene. In addition, we predicted the possible effects of amino acid substitutions at codons 112 and/or 158 on the structure and function of apolipoprotein E protein using Polymorphism Phenotyping online software v2. Our results showed that the rate of apolipoprotein E ε4 carriers and the frequency of the ε4 allele in the case group were statistically and significantly higher than those in the control group (P < 0.05). Therefore, our data support the association of the Apo ε4 allele with RPL; however, in silico analysis predicted that the amino acid substitution at residue 112 (Apo ε4 allele) is a benign mutation. Accordingly, further studies are required to elucidate the mechanism(s) underlying the link between RPL pathogenesis and the Apo ε4 allele.     

Immunomodulatory effects of IL-12 released from poly-N-acetyl glucosamine gel matrix during schistosomiasis infection

We have reported recently that Interleukin-12 (IL-12) released from poly-N-acetyl glucosamine gel matrix (F2 gel/IL-12) is more effective than free IL-12 to enhance vaccination of mice with Schistosoma soluble worm antigen preparation. The aim of this study is to evaluate the effect of F2 gel/IL-12 on the inflammatory responses in mice undergoing schistosomiasis infection in absence of vaccination. To achieve this, mice undergoing Schistosoma mansoni infection or cured from this infection, after treatment with praziquantil (PZQ), were treated with subcutaneous injection of IL-12 for 3 consecutive days or once with F2 gel loaded with IL-12 (F2 gel/IL-12). The treatment was started on day 35 days after infection. For infection, mice were infected with 100 cercariae of S. mansoni using tail immersion method. We found that treatment with F2 gel/IL-12 induced significant decreases in the egg burden with a moderate reduction in the size of granuloma and decrease in the cellular granulomatous reaction in the lung as compared to infected mice treated with IL-12. These effects of F2 gel/IL-12 were more pronounced in infected mice previously treated with the anti-schistosomal drug PZQ. The total numbers of white blood cells in all treated mice showed similar profile. Treatment with IL-12 or F2 gel/IL-12, however, showed significant reduction in the number of mononuclear cells when compared with non-treated infected mice. In conclusion, this study showed the ability of IL-12 released from F2 gel to lower the inflammatory response to Schistosoma infection even in absence of vaccination.