For gas chromatography - mass spectrometry (GC-MS) analyses performed in situ, pH and salts (e.g., chlorides, sulfates) may enhance or inhibit the detection of targeted molecules of interest for ...astrobiology (e.g. amino acids, fatty acids, nucleobases). Obviously, salts influence the ionic strength of the solutions, the pH value, and the salting effect. But the presence of salts may also produce complexes or mask ions in the sample (masking effect on hydroxide ion, ammonia, etc.). For future space missions, wet chemistry will be conducted before GC-MS analyses to detect the full organic content of a sample. The defined organic targets for space GC-MS instrument requirements are generally strongly polar or refractory organic compounds, such as amino acids playing a role in the protein production and metabolism regulations for life on Earth, nucleobases essential for DNA and RNA formation and mutation, and fatty acids that composed most of the eukaryote and prokaryote membranes on Earth and resist to environmental stress long enough to still be observed on Mars or ocean worlds in geological well-preserved records. The wet-chemistry chemical treatment consists of reacting an organic reagent with the sample to extract and volatilize polar or refractory organic molecules (i.e. dimethylformamide dimethyl acetal (DMF-DMA) in this study). DMF-DMA derivatizes functional groups with labile H in organics, without modifying their chiral conformation. The influence of pH and salt concentration of extraterrestrial materials on the DMF-DMA derivatization remains understudied. In this research, we studied the influence of different salts and pHs on the derivatization of organic molecules of astrobiological interest with DMF-DMA, such as amino acids, carboxylic acids, and nucleobases. Results show that salts and pH influence the derivatization yield, and that their effect depend on the nature of the organics and the salts studied. Second, monovalent salts lead to a higher or similar organic recovery compared to divalent salts regardless of pH below 8. However, a pH above 8 inhibits the DMF-DMA derivatization influencing the carboxylic acid function to become an anionic group without labile H. Overall, considering the negative effect of the salts on the detection of organic molecules, future space missions may have to consider a desalting step prior to derivatization and GC-MS analyses.
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•DMF-DMA derivatization is a fast one-pot reaction helpful to break the matrix, desorb, derivatize, and volatilize analytes.•DMF-DMA derivatizes polar and refractory organic molecules as well as enantiomeric forms separated with a chiral column.•GC-MS with a DMF-DMA extraction identifies hundreds of organic compounds (from pmol to mmol of molecule) in a bulk sample.•DMF-DMA is suited for space applications (spaceflight and return sample missions) and (biogeo)chemistry laboratory work.
PurposeFibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to ...negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro.MethodsCorneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed.ResultsExplants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14-16 for explants with fibrin glue.ConclusionsFibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy.
The Sample Analysis at Mars (SAM) suite instrument on board NASA's Curiosity rover has characterized the inorganic and organic chemical composition of seven samples from the Glen Torridon (GT) ...clay‐bearing unit. A variety of organic molecules were detected with SAM using pyrolysis (up to ∼850°C) and wet chemistry experiments coupled with evolved gas analysis (EGA) and gas chromatography‐mass spectrometry. SAM EGA and GCMS analyses revealed a greater diversity and abundance of sulfur‐bearing aliphatic and aromatic organic compounds in the sediments of this Gale crater unit than earlier in the mission. We also report the detection of nitrogen‐containing, oxygen‐containing, and chlorine‐containing molecules, as well as polycyclic aromatic hydrocarbons found in GT, although the sources of some of these organics may be related to the presence of chemical reagents in the SAM instrument background. However, sulfur‐bearing organics released at high temperature (≥600°C) are likely derived from Martian sources (e.g., igneous, hydrothermal, atmospheric, or biological) or exogenous sources and consistent with the presence of recalcitrant organic materials in the sample. The SAM measurements of the GT clay‐bearing unit expand the inventory of organic matter present in Gale crater and is also consistent with the hypothesis that clay minerals played an important role in the preservation of ancient refractory organic matter on Mars. These findings deepen our understanding of the past habitability and biological potential of Gale crater.
Plain Language Summary
Organic molecules are essential to all life as we know it. Clay minerals are known on Earth for their high organic preservation potential and can be key indicators of past habitable environments. On Mars, the Glen Torridon (GT) region in Gale crater was first identified from orbit as a priority target for the Mars Science Laboratory mission due to its abundant clay minerals. To evaluate the organic preservation potential of this region, seven rock samples were collected and characterized using the Sample Analysis at Mars (SAM) instrument onboard the Curiosity rover. The SAM investigation indicated the presence of various organic compounds, including the first observation on Mars of some sulfur‐containing and ring‐structured organics and the highest abundance of sulfur organics observed to date. Our investigation of the sources of these organics revealed that while some of the sulfur‐bearing organics are likely Martian, a portion may also be related to the presence of chemical reagents carried in SAM, making attribution to a definitive source challenging. Nevertheless, these new SAM results confirm that ancient organic matter is preserved in the clay mineral bearing sediments of GT. Its origin—either meteoritic, abiotic or biotic—has yet to be established.
Key Points
Curiosity explored the Glen Torridon region of Gale crater, which has a smectite‐rich mineralogy with high organic preservation potential
The greatest diversity and abundance of sulfur‐bearing organics to date were detected in the solid samples by the SAM instrument
S‐bearing organics extracted ≥600°C and some aromatic compounds likely come from martian refractory organic matter
Aromatic organic salts such as benzoates or phthalates may be widespread degradation products of organic molecules at the surface of Mars. The low volatility of these aromatic carboxylic salts could ...have compromised their detection through thermal extraction in situ analyses such as those performed by the Viking landers. However, over the years, analytical chemistry laboratories on board current and future Martian surface missions, such as the Sample Analysis at Mars (SAM) instrument suite on board the Curiosity rover and the Mars Organic Molecule Analyzer (MOMA) instrument of the Rosalind Franklin ExoMars rover, respectively, have evolved. These instruments have improved in efficiency to detect refractory and polar organic compounds, which could influence the detection of aromatic organic salts. To evaluate the capability of detecting aromatic organic salts on Mars with in situ instruments, we performed laboratory experiments under Viking, SAM, and MOMA-like Gas Chromatography-Mass Spectrometry (GC–MS) conditions with two carboxylic acid/salt couples: phthalic acid/calcium phthalate and benzoic acid/calcium benzoate. We studied the behavior and signatures of both molecular forms when using pyrolysis and derivatization experiments and the implications of these results in the search for organic molecules on Mars. This study showed that the Viking experiments could not have detected the presence of aromatic carboxylic salts in Martian samples because its maximum pyrolysis temperature was too low (500 °C). However, we showed that calcium benzoate and calcium phthalate, despite their refractory nature, could be identified indirectly through the detection of thermal and derivatized degradation products, both with SAM and MOMA. No conclusive proof of the presence of these aromatic organic salt species have been found in the SAM in situ data but given the right instrumental set-up they could be detected if present. The conclusions of this work raise essential questions on the detectability of refractory molecules, the analytical efficiency of flight instruments, and the interpretation of in situ data.
•Calcium phthalate and benzoate could be indirectly detected on Mars with SAM and MOMA.•Calcium carboxylic salts produce characteristic pyrolysis byproducts.•Calcium salts can be derivatized with MTBSTFA, DMFDMA and TMAH.•Tandem analyses are necessary to identify the presence of aromatic carboxylic salts.
A high-resolution bacterial tracking technique, ferrographic capture, was used to enumerate fluorescent-stained bacterial cells that were injected into groundwater during a field experiment. The goal ...of the experiment was to investigate whether detachment of previously injected stained resident cells attached to aquifer sediment was enhanced in the presence of the newly injected mobile cells. This injection was an improvement on past experiments in that the attached (resident) cells were stained, allowing their concentrations to be enumerated directly by ferrographic capture (upon detachment). Contrary to expectations based on previous experiments, enhanced detachment of stained resident cells did not occur upon the arrival of injected cells. Consistent with previous experiments, however, was the observation of ephemeral increases in unstained cell concentrations coincident with the arrival of the stained injected cells. The ephemeral pulses of unstained cells were previously speculated to represent enhanced detachment of unstained indigenous cells in response to hydrodynamic collision with injected cells. The lack of enhanced detachment of stained resident cells in the present experiments indicates that increased concentrations of unstained cells may have occurred by mechanisms other than hydrodynamic collision. Visually observed variations in stain intensity indicated that increased unstained cell concentrations may have resulted from cell division at the low-concentration fringe of the injected plume.
X-ray diffraction analysis has been performed on a series of 1-palmitoyl-2-dibromostearoyl-phosphatidylcholines (BRPCs) with bromine atoms at the 6, 7-, the 11, 12-, or the 15, 16-positions on the ...sn-2 acyl chains. The diffraction patterns indicate that, when hydrated, each of these lipids forms liquid-crystalline bilayers at 20 degrees C. For each lipid, electron density profiles and continuous Fourier transforms were calculated by the use of swelling experiments. In the electron profiles, high-density peaks, due to the bromine atoms, are observed. The separation between these bromine peaks in the profile decreases as the bromine atoms are moved toward the terminal methyl of the acyl chain. For the 6, 7- and 11, 12-bromolipids, experimental Fourier transforms can be approximated by the sum of the transform of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and the transform of two symmetrically placed peaks of electron density (the bromines). For the case of the 15, 16-bromolipids, a better fit is obtained for the transforms of a model bilayer where the thickness of the methylene chain region of the bilayer is 3 A greater than that of POPC. Our analysis indicates the following: for each of these bromolipids, the bromines are well localized in the bilayer; the distance of the bromines from the head-group-hydrocarbon boundary are 3.5, 8.0, and 14 A, for 6, 7-, 11, 12-, and 15, 16-BRPC, respectively; the bilayer thickness and perturbation to bilayer hydrocarbon chain packing caused by the bromine atoms depend on the position of the bromines on the hydrocarbon chain.
Purpose Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation can significantly ...influence the bio‐ chemical and physical properties of AM, affecting clinical efficacy. This study investigates a novel drying technique and the biochemical and structural effects on AM and following pre‐treatment of AM with novel lyoprotectants.
Methods AM was cryopreserved (CPAM) or dried (DAM) with and without pre‐treatment with lyoprotectants. Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunofluorescence and immunoassays. The biocompatibility of AM preparations co‐cultured with primary corneal epithelial cell (CEC) or keratocyte monolayers were assessed using standard cellular health assays.
Results Drying devitalised AM epithelium, but less so than cryopreservation, and cellular damage was reduced in DAM pre‐treated with lyoprotectant. DAM alone, and pre‐treated with lyoprotectant showed greater factor retention efficiencies and bioavailability compared to CPAM. In addition cellular health assays showed that DAM alone and pre‐treated with lyoprotectant are compatible and superior substrates compared to CPAM for CEC expansion, with increased proliferation and reduced cytotoxicity profiles. This was supported by improved wound healing in CEC co‐cultured with DAM and pre‐treated DAM, compared to CPAM.
Conclusion Our modified preservation process and our resultant optimised DAM has enhanced structural properties and biochemical stability and is a superior substrate to conventional CPAM.
To confirm that human corneal rims left over from DALK/DSEK/PK surgeries could be useful sources for ex vivo endothelial cell expansion. Human corneal rims remaining from DALK/DSEK/PK surgeries were ...utilized (1:1 sex ratio, age 63+20 years, endothelial cell density >2,500 cells/mm2). The time from death to use varied between 3 days and 1.5 months. Endothelial cells isolated using a two‐step, peel‐and‐digest method, whereby the Descemet’s membrane and endothelial cells were peeled off under a dissecting microscope, followed by digestion in collagenase. The isolated cells were suspended in TrypLE prior to plating onto FNC‐coated tissue culture plates. The cells were then cultured in Ham’s F12:M199 (1:1) media supplemented with, ascorbic acid, transferrin, sodium selenite and bFGF. Characterisation of the cultured cells was performed by RT‐qPCR and immunofluorescence staining accordingly. The number of isolated endothelial cells was repeatedly low (< 20,000 cells). However, improved techniques allowed to reduce stromal cell contamination. It was observed that endothelial cell proliferation was improved when the culture surface area was reduced. Furthermore, typical endothelial cobble stone morphology was observed when the cell density was high. Cell morphology and growth showed notable difference related to donor age and preservation time. ZO‐1, Na/K‐ATPase and PITX2 were used to confirm the endothelial phenotype. Preserved human corneal rims can be utilized for ex vivo expansion of corneal endothelial cells but further optimization is needed.
The purpose of the current study was to explore the relationship between critical consciousness and multicultural awareness among undergraduate students at Auburn University. Due to the constantly ...changing makeup of the United States population and the American Counseling Association’s Code of Ethics requiring counselors to be multiculturally competent with diverse populations, counselor educators are under more pressure than ever to promote multicultural competence in counselors-in-training (Cohn & Caumont, 2016; ACA, 2014; Hill, Vereen, McNeal, and Stotesbury, 2013; Zalaquett, Foley, Tillotson, Dinsmore, & Hof, 2008; Decker, Manis, & Paylo, 2015). This study found both political awareness and engagement in advocacy activities to be strong predictors of increased multicultural awareness among the participants. These predictors provide evidence linking critical consciousness and multicultural awareness, which can potentially be used by counselor educators to increase multicultural competence in counselors-in-training. Additionally, a discussion regarding the further discussion of the implications for counselor educators is provided.
Purpose Keratocytes of the corneal stroma are quiescent, dendritic cells, which maintain the extracellular matrix. It is believed that there is a subpopulation of keratocytes that act as multipotent ...progenitor cells. These cells repopulate the stroma after damage and may play a role in the regeneration of other layers of the cornea, such as the epithelium. Optimisation of culture of these cells is integral for developing potential cell therapies.
Methods Primary human corneal stromal stem cells (hCSC) were extracted from the limbal region of the cornea. A range of culture media were tested for the effect on stem cell phenotype and differentiation potential. The effect of extended passaging and three‐dimensional culture of the cells as spheroids was assessed by RT‐qPCR and immunocytochemistry.
Results The phenotype of hCSC is strongly affected by extraction, passage and the culture medium. The cells form spheroids and upregulate markers, such as CD34. Under the correct conditions, hCSC show potential as a multipotent stem cell, expressing the indicative markers and differentiating in vitro down osteogenic, chondrogenic and adipogenic lineages. The CD34+ population of hCSC demonstrate a higher stem cell potential and differentiate to corneal epithelial cells, with a rounded morphology and high expression of key markers such as cytokeratin 3, HES1 and desmoglein 3.
Conclusion Corneal stromal stem cells demonstrate a multipotent stem cell potential, independent of the role they are traditionally associated with in the cornea. In vitro, this potential depends on the culture environment and on isolation of certain subpopulations. In future, these cells show potential in the regeneration of the ocular surface in cases of disease or trauma.
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