Reduction of Steroid-Induced Intraocular Pressure Elevation in Sheep by Tissue Plasminogen Activator

PURPOSE. To investigate whether tissue plasminogen activator (tPA) can prevent and/or reverse steroid-induced IOP elevation in an ovine model. METHODS. Three animal groups were subjected to bilateral steroid-induced IOP elevation using thrice daily topical ocular prednisolone administration. In the first group (N ¼ 8), one eye each of two sheep was injected intravitreally with 100 lg, 200 lg, 500 lg, or 1 mg human recombinant tPA, while contralateral eyes received vehicle. In the second group (N ¼ 2), one eye was injected intravitreally with tPA (100 lg), while contralateral eyes received vehicle containing L-arginine. In the third group (N ¼ 4), each animal received intravitreal tPA in one eye concurrently with initiation of bilateral steroid administration. IOP was monitored for the duration of the experiment. Tissues from eyes of the third group were used to determine relative gene expression. RESULTS. In the first and second groups, IOP decreased by 9.7 (62.8) and 9.7 (61.6) mm Hg, respectively, 24 hours after tPA administration. In the third group, tPA-treated eyes did not develop IOP elevation with DIOP of 11.8 (61.3) mm Hg 8 days later. In all tPA-treated eyes, IOP remained low until the end of the study. mRNA levels in the trabecular meshwork were decreased for plasminogen activator tissue (PLAT), increased for matrix-metalloproteinase 1 (MMP-1), and stable for plasminogen activator inhibitor 1 (PAI-1), MMP-2, MMP-9, and MMP- 13 in tPA-treated eyes compared with contralateral controls. PAI-1 mRNA levels in ciliary processes also remained similar. CONCLUSIONS. Recombinant human tPA is effective in both preventing and reversing steroidinduced IOP elevation in sheep. Tissue plasminogen activator may be useful as a therapeutic agent in steroid-induced glaucoma.Fil: Gerometta, Rosana María del Rosario. University Of New York; Estados Unidos. Universidad Nacional del Nordeste. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kumar, Sandeep. State University Of New York; Estados UnidosFil: Shah, Shaily. State University Of New York; Estados UnidosFil: Alvarez, Larry. State University Of New York; Estados UnidosFil: Candia, Oscar. State University Of New York; Estados UnidosFil: Danias, John. State University Of New York; Estados Unido

Tissue Plasminogen Activator in Trabecular Meshwork Attenuates Steroid Induced Outflow Resistance in Mice

Tissue plasminogen activator, a serine protease encoded by the PLAT gene is present in the trabecular meshwork (TM) and other ocular tissues and has been reported to be downregulated by treatment with steroids in vitro. Steroids are known to cause changes in outflow facility of aqueous humor in many species. In the present study, we tested whether overexpression of PLAT can prevent and/or reverse the outflow facility of mouse eyes treated with steroids. Animals received bilateral injection with 20 µl of triamcinolone acetonide (TA) (40mg/ml) suspension subconjunctivally to induce outflow facility changes. Some animals received unilateral intracameral injection with 2 µl of adenoviral suspension [3-4x1012 virus genomes per milliliter (vg/ml)] carrying sheep PLAT cDNA (AdPLAT) either concurrently with TA injection or one week after TA injection, whereas others received bilateral intracameral injection with 2µl of adenoviral suspension (9x1012 vg/ml) carrying no transgene (AdNull) concurrently with TA injection. Animals were sacrificed one week after AdPLAT or AdNull treatment. Endogenous mRNA expression levels of mouse PAI-1 and MMP-2, -9 and -13 were also measured using qRT-PCR. Outflow facility one week after AdPLAT administration was increased by 60% and 63% respectively for animals that had not or had been pretreated with steroids. Overexpression of PLAT significantly upregulated expression of PAI-1, MMP-2, -9 and -13 compared to the levels found in TA only treated eyes. These findings suggest that overexpression of PLAT in TM of mouse eyes can both prevent and reverse the decrease in outflow facility caused by steroid treatment and is associated with upregulation of MMPs

Subsurface scientific exploration of extraterrestrial environments (MINAR 5): analogue science, technology and education in the Boulby Mine, UK

The deep subsurface of other planetary bodies is of special interest for robotic and human exploration. The subsurface provides access to planetary interior processes, thus yielding insights into planetary formation and evolution. On Mars, the subsurface might harbour the most habitable conditions. In the context of human exploration, the subsurface can provide refugia for habitation from extreme surface conditions. We describe the fifth Mine Analogue Research (MINAR 5) programme at 1 km depth in the Boulby Mine, UK in collaboration with Spaceward Bound NASA and the Kalam Centre, India, to test instruments and methods for the robotic and human exploration of deep environments on the Moon and Mars. The geological context in Permian evaporites provides an analogue to evaporitic materials on other planetary bodies such as Mars. A wide range of sample acquisition instruments (NASA drills, Small Planetary Impulse Tool (SPLIT) robotic hammer, universal sampling bags), analytical instruments (Raman spectroscopy, Close-Up Imager, Minion DNA sequencing technology, methane stable isotope analysis, biomolecule and metabolic life detection instruments) and environmental monitoring equipment (passive air particle sampler, particle detectors and environmental monitoring equipment) was deployed in an integrated campaign. Investigations included studying the geochemical signatures of chloride and sulphate evaporitic minerals, testing methods for life detection and planetary protection around human-tended operations, and investigations on the radiation environment of the deep subsurface. The MINAR analogue activity occurs in an active mine, showing how the development of space exploration technology can be used to contribute to addressing immediate Earth-based challenges. During the campaign, in collaboration with European Space Agency (ESA), MINAR was used for astronaut familiarization with future exploration tools and techniques. The campaign was used to develop primary and secondary school and primary to secondary transition curriculum materials on-site during the campaign which was focused on a classroom extra vehicular activity simulation

Proteomics insights into the fungal-mediated bioremediation of environmental contaminants

As anthropogenic activities continue to introduce various contaminants into the environment, the need for effective monitoring and bioremediation strategies is critical. Fungi, with their diverse enzymatic arsenal, offer promising solutions for the biotransformation of many pollutants. While conventional research reports on ligninolytic, oxidoreductive, and cytochrome P450 (CYP) enzymes, the vast potential of fungi, with approximately 10 345 protein sequences per species, remains largely untapped. This review describes recent advancements in fungal proteomics instruments as well as software and highlights their detoxification mechanisms and biochemical pathways. Additionally, it highlights lesser-known fungal enzymes with potential applications in environmental biotechnology. By reviewing the benefits and challenges associated with proteomics tools, we hope to summarize and promote the studies of fungi and fungal proteins relevant in the environment

Reduction of Steroid-Induced Intraocular Pressure Elevation in Sheep by Tissue Plasminogen Activator

PURPOSE. To investigate whether tissue plasminogen activator (tPA) can prevent and/or reverse steroid-induced IOP elevation in an ovine model. METHODS. Three animal groups were subjected to bilateral steroid-induced IOP elevation using thrice daily topical ocular prednisolone administration. In the first group (N ¼ 8), one eye each of two sheep was injected intravitreally with 100 lg, 200 lg, 500 lg, or 1 mg human recombinant tPA, while contralateral eyes received vehicle. In the second group (N ¼ 2), one eye was injected intravitreally with tPA (100 lg), while contralateral eyes received vehicle containing L-arginine. In the third group (N ¼ 4), each animal received intravitreal tPA in one eye concurrently with initiation of bilateral steroid administration. IOP was monitored for the duration of the experiment. Tissues from eyes of the third group were used to determine relative gene expression. RESULTS. In the first and second groups, IOP decreased by 9.7 (62.8) and 9.7 (61.6) mm Hg, respectively, 24 hours after tPA administration. In the third group, tPA-treated eyes did not develop IOP elevation with DIOP of 11.8 (61.3) mm Hg 8 days later. In all tPA-treated eyes, IOP remained low until the end of the study. mRNA levels in the trabecular meshwork were decreased for plasminogen activator tissue (PLAT), increased for matrix-metalloproteinase 1 (MMP-1), and stable for plasminogen activator inhibitor 1 (PAI-1), MMP-2, MMP-9, and MMP-13 in tPA-treated eyes compared with contralateral controls. PAI-1 mRNA levels in ciliary processes also remained similar. CONCLUSIONS. Recombinant human tPA is effective in both preventing and reversing steroidinduced IOP elevation in sheep. Tissue plasminogen activator may be useful as a therapeutic agent in steroid-induced glaucoma

Map of AdPLAT Construct.

<p>PLAT gene was amplified from cDNA synthesized from RNA extracted from sheep brain tissue. The expression cassette (<b>A</b>) was constructed by placing amplified and gel purified PLAT cDNA adjacent to a human histone B (H2B) tagged fluorescent reporter gene (mCherry) with internal ribosome entry site (IRES) in a suitable cloning vector. The expression cassette was then subcloned downstream of a CMV promoter at the multiple cloning site of the shuttle vector (<b>B</b>) and used to generate adenovirus (AdPLAT).</p

Outflow facility of eyes receiving AdPLAT one week after TA injection.

<p>Mean ± SD outflow facility in eyes receiving TA alone (TA, n=24), eyes injected with AdPLAT showing robust mCherry expression (TA+/+ AdPLAT, n=6), and eyes injected with AdPLAT showing minimal or no mCherry expression (TA+/-AdPLAT, n=11). Group means are significantly different (ANOVA, p<0.01). Asterisks indicate significant differences on post hoc analysis, **p<0.01.</p

Experimental Design.

<p>12 week-old C57BL/6 female mice received bilateral injections with 20 ul of triamcinolone acetonide (TA) suspension subconjunctivally. Animals were then divided into three treatment groups: 1) animals that also received unilateral intracameral injection with 2 ul of AdPLAT concurrently with TA injection while the contralateral eye remained uninjected, 2) animals that received bilateral intracameral injection with 2 ul of AdNull concurrently with TA injection, and 3) animals that received unilateral intracameral injection with 2 ul of AdPLAT one week after TA injection while the contralateral eye remained uninjected. Animals were sacrificed for outflow facility determination one week after AdPLAT or AdNull treatment.</p

mCherry expression in flat mounts of TM in AdPLAT-injected Eyes.

<p>Flatmount of anterior segments of mouse eyes from (<b>A</b>) animal injected with AdPLAT that showed robust mCherry expression (TA+/+ AdPLAT) (<b>B</b>) animal with minimal mCherry expression despite injection with AdPLAT (TA+/-AdPLAT), and (<b>C</b>) treatment-naïve mouse. (<b>D</b>) qRT-PCR quantification of transgenic PLAT expression in mouse eyes. Levels of PLAT are undetectable in all eyes except those showing robust mCherry expression (TA+/+ AdPLAT). CP = ciliary processes, TM = trabecular meshwork. Arrows indicate mCherry-positive cells.</p

Gene expression changes.

<p>Normalized fold change (mean ± SD) of (<b>A</b>) PAI-1 and (<b>B</b>) MMP-2 expression and normalized fold difference of (<b>C</b>) MMP-9 and (<b>D</b>) MMP-13 expression in TA eyes (n=10), TA+/+ AdPLAT eyes (n=9), and TA+/-AdPLAT eyes (n=10) from animals receiving AdPLAT one week after TA injection. Group means are significantly different (ANOVA PAI-1, p<0.01, MMP-2, p<0.001). Asterisks indicate differences on post hoc analysis, **p<0.01, ***p<0.001. No mRNA was detected for MMP-9 and MMP-13 in the TA or TA+/-AdPLAT groups.</p