Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes.

Vascular bio-scaffolds produced from decellularized tissue offer a promising material for treatment of several types of cardiovascular diseases. These materials have the potential to maintain the functional properties of the extracellular matrix (ECM), and allow for growth and remodeling in vivo. The most commonly used methods for decellularization are based on chemicals and enzymes combinations, which often damage the ECM and cause cytotoxic effects in vivo. Mild methods involving pressurized CO2-ethanol (EtOH)-based fluids, in a supercritical or near supercritical state, have been studied for decellularization of cardiovascular tissue, but results are controversial. Moreover, data are lacking on the amount and type of lipids remaining in the tissue. Here we show that pressurized CO2-EtOH-H2O fluids (average molar composition, ΧCO2 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized CO2-limonene fluids (ΧCO2 0.88) and neat supercritical CO2 (scCO2) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized CO2-limonene followed by enzyme treatment, resulted in efficient DNA removal. The structure of elastic fibers was preserved after pressurized treatment, regardless solvent composition. In conclusion, pressurized CO2-ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO2-limonene fluids facilitate subsequent enzymatic removal of DNA.

Fossil insect eyes shed light on trilobite optics and the arthropod pigment screen.

Fossilized eyes permit inferences of the visual capacity of extinct arthropods1-3. However, structural and/or chemical modifications as a result of taphonomic and diagenetic processes can alter the original features, thereby necessitating comparisons with modern species. Here we report the detailed molecular composition and microanatomy of the eyes of 54-million-year-old crane-flies, which together provide a proxy for the interpretation of optical systems in some other ancient arthropods. These well-preserved visual organs comprise calcified corneal lenses that are separated by intervening spaces containing eumelanin pigment. We also show that eumelanin is present in the facet walls of living crane-flies, in which it forms the outermost ommatidial pigment shield in compound eyes incorporating a chitinous cornea. To our knowledge, this is the first record of melanic screening pigments in arthropods, and reveals a fossilization mode in insect eyes that involves a decay-resistant biochrome coupled with early diagenetic mineralization of the ommatidial lenses. The demonstrable secondary calcification of lens cuticle that was initially chitinous has implications for the proposed calcitic corneas of trilobites, which we posit are artefacts of preservation rather than a product of in vivo biomineralization4-7. Although trilobite eyes might have been partly mineralized for mechanical strength, a (more likely) organic composition would have enhanced function via gradient-index optics and increased control of lens shape.

Efficient methodology for the extraction and analysis of lipids from porcine pulmonary artery by supercritical fluid chromatography coupled to mass spectrometry.

Pulmonary artery grafts are needed as cardiovascular bioprosthetics. For successful tissue recellularization after transplantation, lipids have to be removed from the donor artery. Developing a selective process to remove lipids without damaging the extracellular matrix greatly depends on knowing the amount and type of lipid compounds in the specific tissue. Here we present an efficient methodology for the study of lipids present in porcine pulmonary arteries. The performance of six extraction methods to recover lipids from artery was evaluated. For this purpose, a supercritical fluid chromatography method coupled to quadrupole time-of-flight mass spectrometry detection (UHPSFC/QTOF-MS) was adapted. The method enabled separation of lipids of a wide range of polarity according to lipid class in less than 7 min. One dichloromethane-based extraction method was shown to be the most efficient one for the recovery of lipids from pulmonary artery. However, one MTBE-based extraction method was able to show the highest fatty acid extraction yields (to the expense of longer extraction times). Lipids were relative quantified according to class, and the major species within each class were identified. Triacylglycerols and glycerophospholipids were the most abundant classes, followed by sphingomyelins, monoacylglycerols and fatty acyls. The matrix effect exerted no interference on the analytical method, except for some few combinations of extraction method and lipid class. These results are of relevance for lipidomic studies from solid tissue, in particular for studies on pulmonary and cardiovascular diseases. Finally, our work sets the basis for the further development of a selective processes to remove lipids from pulmonary artery without damaging the tissue prior to transplantation.

Soft-tissue evidence for homeothermy and crypsis in a Jurassic ichthyosaur.

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius. Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.

Data on saponins, xylan and cellulose yield obtained from quinoa stalks after pressurized hot water extraction.

The data we present below are linked to our research paper "Integrated process for sequential extraction of saponins, xylan and cellulose from quinoa stalks (Chenopodium quinoa Willd.)" (Gil-Ramírez et al., 2018) [1]. The objective is to provide supplementary information in order to facilitate the comprehension of the central composite experimental design (rotatable 22) used in the integrated process of extractions. Two factors, temperature and time of extraction are considered in the design. The responses are the yield of saponin, xylan and cellulose. First, the desirable linear regression obtained by the observed vs. predicted yields plot for each variable response confirm the validation of the model (Fig. 1). Second, the data presented here through Standardized Pareto Charts (Fig. 2), provides information about the effect of the time and temperature, as well as their interactions, in the yield of saponins, xylan and cellulose obtained in an integrated sequential extraction.

Supercritical fluid extraction of lipids from linseed with on-line evaporative light scattering detection.

Supercritical fluid extraction (SFE) is a green alternative method of extraction for neutral lipids in seeds compared to conventional methods utilizing organic solvents. In this work, a novel method where SFE is hyphenated with an evaporative light scattering detector is presented. The method was subsequently applied to determine lipid content in crushed linseed. The new method enables rapid quantification of extracted lipids as well as be ability to continuously monitor the extraction rate in real-time, thus being able to determine the time point of completed extraction. Both the detector and the method was validated. The results show that any of several tested oils can be used to calibrate the detection method for the determination of lipids extraction from linseed. The overall method repeatability and intermediate precision was 2.6% relative standard deviations. The extracted amount was significantly less than that obtained using the standard method of Soxhlet with petroleum ether, 26.0±0.4% (95% CI, n=9) compared to 32.3±1.3% (95% CI, n=3) of extracted amounts. It was found that channeling effects were present, and by either performing sequential repeated extractions with decompression in-between or by using a relatively large vessel a more complete extraction could be obtained. Interestingly, a substantially higher extracted amount (approximately 50%) was obtained compared to both a single extraction by SFE and the Soxhlet method. Therefore, it is recommended that an additional extraction including a rapid decompression in-between should be included in the validation of a method using supercritical fluid extraction, in order to either rule out channeling effects or to acquire a full recovery.

The effect of superhydrophobic wetting state on corrosion protection--the AKD example.

Corrosion is of considerable concern whenever metal is used as construction material. In this study we address whether superhydrophobic coatings could be used as part of an environmentally friendly corrosion-protective system, and specific focus is put on how the wetting regime of a superhydrophobic coating affects corrosion inhibition. Superhydrophobic alkyl ketene dimer (AKD) wax coatings were produced, using different methods resulting in hierarchical structures, where the coatings exhibit the same surface chemistry but different wetting regimes. Contact angle measurements, ESEM, confocal Raman microscopy, open circuit potential and electrochemical impedance spectroscopy were used to evaluate the surfaces. Remarkably high impedance values of 10(10)Ω cm(2) (at 10(-2) Hz) were reached for the sample showing superhydrophobic lotus-like wetting. Simultaneous open circuit potential measurements suggest that the circuit is broken, most likely due to the formation of a thin air layer at the coating-water interface that inhibits ion transport from the electrolyte to the metal substrate. The remaining samples, showing superhydrophobic wetting in the rose state and hydrophobic Wenzel-like wetting, showed less promising corrosion-protective properties. Due to the absence of air films on these surfaces the coatings were penetrated by the electrolyte, which allowed the corrosion reaction to proceed.

Determination of carotenoids in microalgae using supercritical fluid extraction and chromatography.

A method was developed based on supercritical fluid chromatography for quantitative determination of carotenoids in extracts of Scenedesmus sp. By utilizing the low backpressure in supercritical fluid chromatography, a C18 column and a 2-ethyl pyridine column were coupled in series. It was concluded that even minor changes in temperature had a substantial effect on selectivity. A standard mixture of 8 carotenoids and microalgae extracts obtained through supercritical fluid extraction with and without 10% ethanol as a co-solvent were successfully separated. All of the carotenoids were separated within 10 min, while the total analysis time was 20 min. The method was validated and the carotenoids of microalgae extracts were quantified. Furthermore, the method should be seen as a more rapid and environmentally sustainable alternative to traditional high-performance liquid chromatography methods utilizing organic solvents.

Metabolomics of transgenic maize combining Fourier transform-ion cyclotron resonance-mass spectrometry, capillary electrophoresis-mass spectrometry and pressurized liquid extraction.

In this work, the potential of combining capillary electrophoresis-time-of-flight-mass spectrometry (CE-TOF-MS) and Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) for metabolomics of genetically modified organisms (GMOs) is demonstrated. Thus, six different varieties of maize, three of them transgenic (PR33P66 Bt, Tietar Bt and Aristis Bt) and their corresponding isogenic lines (PR33P66, Tietar and Aristis) grown under the same field conditions, were analyzed. Based on the ultrahigh resolution and remarkable mass accuracy provided by the 12-T FT-ICR-MS it was possible to directly analyze a good number of metabolites whose identity could be proposed based on their specific isotopic pattern. For identification of metabolite isomers, CE-TOF-MS was also used combining the information on nominal mass with electrophoretic mobility corroborating in that way the identity of several new biomarkers. Furthermore, PLE extractions were evaluated in order to establish selective extraction as an additional criterion to obtain useful information in maize metabolomics. Differences in the metabolite levels were found between the three transgenic maize varieties compared with their wild isogenic lines in some specific metabolic pathways. To our knowledge, this is the first time that an approach as the one presented in this work (pressurized liquid extraction+FT-ICR-MS+CE-TOF-MS) is shown for a metabolomic study.

Fast and easy coating for capillary electrophoresis based on a physically adsorbed cationic copolymer.

In this work, a new copolymer synthesized in our laboratory is used as physically adsorbed coating for capillary electrophoresis (CE). The copolymer is composed of ethylpyrrolidine methacrylate (EPyM) and methylmethacrylate (MMA). The capillary coating is easily obtained by simply flushing into the tubing an EPyM/MMA solution. It is demonstrated that the composition of the EPyM/MMA copolymer together with the selection of the background electrolyte (BGE) and pH allow tailoring the direction and magnitude of the electroosmotic flow (EOF) in CE. It is also shown that the EOF obtained for the EPyM/MMA-coated capillaries was reproducible in all cases independently on pH or polymer composition. Thus, RSD values lower than 1.9% (n=5) for the same capillary and day were obtained for the migration time, while the repeatability interdays (n=5) was observed to provide RSD values lower than 0.5%. The stability of the coating procedure was also tested between capillaries (n=3) obtaining RSD values lower than 0.6%. It is demonstrated with several examples that the use of EPyM/MMA coatings in CE can drastically reduce the analysis time and/or to improve the resolution of the separations. It is shown that EPyM/MMA-coated capillaries allow the separation of basic proteins by reducing their adsorption onto the capillary wall. Also, EPyM/MMA-coated capillaries provide a faster separation of samples containing simultaneously positive and negative analytes. Moreover, it is demonstrated that the use of EPyM/MMA-coated capillaries can incorporate an additional chromatographic-like interaction with nucleosides that highly improves the separation of this group of solutes.