Osmoregulation and the Anthozoan-Dinoflagellate Symbiosis

<p>This study investigated the responses of the temperate anemone Anthopleura aureoradiata, and the tropical coral Acropora aspera to osmotic stress and the role that free amino acids (FAAs) may play in the osmoregulatory mechanism of these anthozoan-dinoflagellate symbioses. Specimens were exposed to a range of hypo- and hyper-saline conditions for durations of 1, 12, 48 and 96 hours, whereupon respiration and photosynthetic rates were measured as physiological indicators of osmotic stress. High performance liquid chromatography was used to quantify 15 FAAs within the anthozoan host tissues to establish the response of FAA pools to osmotic stress and whether FAAs are used in an osmoregulatory capacity. Aposymbiotic specimens of A. aureoradiata were similarly tested to establish if the presence of symbiotic dinoflagellates alters the host’s capacity to respond to osmotic stress given that the symbionts are known to release FAAs into the host cytoplasm. In A. aureoradiata, significant changes in respiration were only observed with exposure to the extreme hypo-osmotic salinity of 12‰, with respiration decreasing by 67% after 1 hour of exposure. No significant changes in respiration were seen at 25, 43 or 50‰, despite a 52% decrease in respiration seen at the hyper-saline treatment of 50‰. The response of the coral A. aspera was markedly different, showing an increase in respiration in response to hypo-salinity (22 and 28‰). Interestingly, the most pronounced respiratory increase of up to 460% occurred in the less extreme hypo-saline treatment of 28‰. The response of photosynthesis also showed differences between the two species. In the symbiotic A. aureoradiata, photosynthesis declined by 61% after the 1 hour exposure to 12‰ and further decreased to 72% below control rates after 96 hours. While in A. aspera, photosynthesis showed no significant deviation from control levels at any of the treatment salinities. FAA pools in both A. aureoradiata and A. aspera showed significant responses to osmotic stress. In symbiotic A. aureoradiata, exposure to 12‰ caused total FAA pools to decline by 50% after 1 hour, after which a seemingly stable state was reached. A hyper-osmotic treatment of 50‰ resulted in a similar trend with a more than 50% decrease after 1 hour of exposure. In A. aspera, the response of the FAA pool was markedly different, with the concentration increasing by up to 200% with exposure to 22‰ and by more than 260% at 28‰. Interestingly, one on the main constituents of FAA pools in A. aureoradiata, Taurine (15% of FAA pools at 35‰), was not present in measurable quantities within A. aspera host tissue. In aposymbiotic individuals of A. aureoradiata exposed to extreme hypo- and hyper-saline treatments of 12 and 50‰ a significant impact on respiration was only observed at 12‰, with a 77% decrease in respiration after 96 hours. Changes in FAA pools of aposymbiotic A. aureoradiata were only seen after 12 hours exposure to 50‰ with a significant 26% decrease. However, the direct comparison between symbiotic and aposymbiotic A. aureoradiata did serve to highlight the contribution of symbiont-derived FAAs to the host pool of FAAs, with FAA pools in aposymbiotic anemones up to 41% lower than those found in symbiotic anemones. The results seen here were not suggestive of FAAs being regulated for the explicit use as compatible organic osmolytes. Rather, changes in FAA pools showed changes consistent with other stress responses. Moreover, the response of anthozoan-dinoflagellate symbioses to osmotic stress appears to be species specific, or at least taxa specific, as the responses of respiration, photosynthesis and FAA pools were very different between the temperate anemone A. aureoradiata and the tropical coral A. aspera. Nevertheless, differences in the respiratory response between symbiotic and apo-symbiotic anemones did indicate some influence of the dinoflagellate symbionts on the ability of the anthozoan host to mediate osmotic stress. It may therefore be that other symbiont-derived compounds are utilised as compatible organic osmolytes (COOs), with a primary candidate being glycerol. This warrants further investigation.</p>

Osmoregulation and the Anthozoan-Dinoflagellate Symbiosis

<p>This study investigated the responses of the temperate anemone Anthopleura aureoradiata, and the tropical coral Acropora aspera to osmotic stress and the role that free amino acids (FAAs) may play in the osmoregulatory mechanism of these anthozoan-dinoflagellate symbioses. Specimens were exposed to a range of hypo- and hyper-saline conditions for durations of 1, 12, 48 and 96 hours, whereupon respiration and photosynthetic rates were measured as physiological indicators of osmotic stress. High performance liquid chromatography was used to quantify 15 FAAs within the anthozoan host tissues to establish the response of FAA pools to osmotic stress and whether FAAs are used in an osmoregulatory capacity. Aposymbiotic specimens of A. aureoradiata were similarly tested to establish if the presence of symbiotic dinoflagellates alters the host’s capacity to respond to osmotic stress given that the symbionts are known to release FAAs into the host cytoplasm. In A. aureoradiata, significant changes in respiration were only observed with exposure to the extreme hypo-osmotic salinity of 12‰, with respiration decreasing by 67% after 1 hour of exposure. No significant changes in respiration were seen at 25, 43 or 50‰, despite a 52% decrease in respiration seen at the hyper-saline treatment of 50‰. The response of the coral A. aspera was markedly different, showing an increase in respiration in response to hypo-salinity (22 and 28‰). Interestingly, the most pronounced respiratory increase of up to 460% occurred in the less extreme hypo-saline treatment of 28‰. The response of photosynthesis also showed differences between the two species. In the symbiotic A. aureoradiata, photosynthesis declined by 61% after the 1 hour exposure to 12‰ and further decreased to 72% below control rates after 96 hours. While in A. aspera, photosynthesis showed no significant deviation from control levels at any of the treatment salinities. FAA pools in both A. aureoradiata and A. aspera showed significant responses to osmotic stress. In symbiotic A. aureoradiata, exposure to 12‰ caused total FAA pools to decline by 50% after 1 hour, after which a seemingly stable state was reached. A hyper-osmotic treatment of 50‰ resulted in a similar trend with a more than 50% decrease after 1 hour of exposure. In A. aspera, the response of the FAA pool was markedly different, with the concentration increasing by up to 200% with exposure to 22‰ and by more than 260% at 28‰. Interestingly, one on the main constituents of FAA pools in A. aureoradiata, Taurine (15% of FAA pools at 35‰), was not present in measurable quantities within A. aspera host tissue. In aposymbiotic individuals of A. aureoradiata exposed to extreme hypo- and hyper-saline treatments of 12 and 50‰ a significant impact on respiration was only observed at 12‰, with a 77% decrease in respiration after 96 hours. Changes in FAA pools of aposymbiotic A. aureoradiata were only seen after 12 hours exposure to 50‰ with a significant 26% decrease. However, the direct comparison between symbiotic and aposymbiotic A. aureoradiata did serve to highlight the contribution of symbiont-derived FAAs to the host pool of FAAs, with FAA pools in aposymbiotic anemones up to 41% lower than those found in symbiotic anemones. The results seen here were not suggestive of FAAs being regulated for the explicit use as compatible organic osmolytes. Rather, changes in FAA pools showed changes consistent with other stress responses. Moreover, the response of anthozoan-dinoflagellate symbioses to osmotic stress appears to be species specific, or at least taxa specific, as the responses of respiration, photosynthesis and FAA pools were very different between the temperate anemone A. aureoradiata and the tropical coral A. aspera. Nevertheless, differences in the respiratory response between symbiotic and apo-symbiotic anemones did indicate some influence of the dinoflagellate symbionts on the ability of the anthozoan host to mediate osmotic stress. It may therefore be that other symbiont-derived compounds are utilised as compatible organic osmolytes (COOs), with a primary candidate being glycerol. This warrants further investigation.</p>

The Pharmacokinetics, Pharmacodynamics, and Safety of Intravenous Pegcetacoplan Treatment in Healthy Subjects

Background: The complement cascade is part of innate immunity and is involved in multiple inflammatory processes and implicated in several diseases. Pegcetacoplan (PEG) is a pegylated, cyclic peptide that binds to complement protein C3 and is a broad inhibitor of the complement cascade. Subcutaneous (SC) dosing of PEG has demonstrated efficacy in the treatment of chronic conditions, such as paroxysmal nocturnal hemoglobinuria (PNH) and was recently approved by the FDA for the treatment of PNH in adults. Intravenous (IV) PEG administration may allow for more rapid and robust reduction of uncontrolled complement activation, especially in an acute setting, such as an acute hemolytic episode in PNH. Aims: To determine the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of IV PEG in acetate-buffered saline treatment in a Phase 1 single ascending dose study (ACTRN12616000700437) in healthy subjects. Methods: On Day 1, four cohorts with PEG doses (200mg, 600mg, 1500mg, 2300mg) received a single bolus of PEG IV (or matching placebo) administered over 30min. Blood samples for PK analyses of PEG concentration and PD analyses of alternative complement pathway hemolytic activity (AH50), total complement hemolytic activity (CH50), C3 and C3a levels were collected at 15, 30, and 60min, 4, 8, 12, and 24hrs, and Days 3, 4, 5, 6, 7, 8, 15, 22, 29, and 43. Subjects were monitored during a safety period from Day 2 to 8 by physical examination, ECG, hematology, serum chemistry, monitoring for injection site reaction and treatment emergent adverse events (TEAEs). Follow-up safety assessments were performed on Days 15, 22, 29, and 43. Results: Twenty subjects were enrolled and allocated 4:1 to PEG or placebo per cohort (PEG-200mg, n=4; PEG-600mg, n=4; PEG-1500mg, n=4; PEG-2300mg, n=4; pooled placebo, n=4). Following a single IV dose, peak concentration (C max) of PEG was observed at 1hr post-dose (infusion start) for most cohorts (mean serum concentration: PEG-200mg, 61μg/mL; PEG-600mg, 193μg/mL; PEG-2300mg, 708μg/mL) except PEG-1500mg (occurred at 4hrs, 542μg/mL). PEG concentration at the end of infusion was similar to the observed C max. PEG concentration declined in a mono-exponential manner, with a terminal elimination half-life ranging from 200 to 285 hrs (Figure). Total body clearance of PEG after IV administration was similar across cohorts. Early, immediate decreases in mean AH50 values were detected within 1hr in all PEG cohorts, with 1500 and 2300mg doses decreasing AH50 to undetectable levels (Figure). Decreases in mean AH50 values were maintained for at least 12, 72, 144 and 168hrs after single doses of 200, 600, 1500 and 2300mg PEG, respectively. All PEG groups had an initial rapid decrease within 1hr in mean C3a levels, with all dose groups having trough mean C3a levels within 24hrs of dosing. Dose related decreases in mean C3a were not observed, all doses recorded a max mean decrease of 47% to 57%. No changes seen with placebo for C3a. C3 and CH50 results will be forthcoming. Of the twenty subjects included in the study, 11 (55.0%) experienced a treatment-related adverse event (TEAE). The most common TEAEs in the PEG group were headache, (n=6, 37.5%); upper respiratory infections attributed to seasonal viral infection (n=2, 12.5%); diarrhea (n=2, 12.5%). No serious adverse events, deaths, or severe TEAEs occurred. One subject (5.0%) in the PEG-2300mg cohort experienced a moderate TEAE (infusion-related reaction, dizziness, clamminess, nausea) that led to study discontinuation. Conclusions: These results suggest that administration of IV PEG in a sodium acetate solution has a favorable safety profile and effectively increases PEG serum concentrations while decreasing complement activity within the first hour post-dose in healthy subjects. Although the safety and efficacy of SC PEG treatment has been demonstrated in patients with PNH, IV PEG administration could serve as a useful therapeutic option for patients with a need for rapid control of complement activity. While this formulation is different than the commercially available PEG (EMPAVELI), which is administered SC and is suspended in sorbitol, the results suggest that IV PEG is well tolerated and provides the grounds for future investigations of IV PEG administration. Figure 1 Figure 1. Disclosures Grossi: Apellis Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Yeh: Apellis Pharmaceuticals, Inc.: Current Employment, Current equity holder in publicly-traded company. Xu: Apellis Pharmaceuticals: Current Employment. Deschatelets: Apellis Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. OffLabel Disclosure: Pegcetacoplan, a subcutaneously administered C3-inhibitor that was recently approved by the US FDA for the treatment of PNH, controls IVH and prevents EVH. While subcutaneous pegcetacoplan is safe and effective, the aim of this study was to determine the safety, pharmacokinetics, and pharmacodynamics of IV pegcetacoplan in acetate-buffered saline treatment, different from current FDA approved formulation.

Combined Therapy With Avastin, a PAF Receptor Antagonist and a Lipid Mediator Inhibited Glioblastoma Tumor Growth

Glioblastoma multiforme (GBM) is an aggressive, highly proliferative, invasive brain tumor with a poor prognosis and low survival rate. The current standard of care for GBM is chemotherapy combined with radiation following surgical intervention, altogether with limited efficacy, since survival averages 18 months. Improvement in treatment outcomes for patients with GBM requires a multifaceted approach due to the dysregulation of numerous signaling pathways. Recently emerging therapies to precisely modulate tumor angiogenesis, inflammation, and oxidative stress are gaining attention as potential options to combat GBM. Using a mouse model of GBM, this study aims to investigate Avastin (suppressor of vascular endothelial growth factor and anti-angiogenetic treatment), LAU-0901 (a platelet-activating factor receptor antagonist that blocks pro-inflammatory signaling), Elovanoid; ELV, a novel pro-homeostatic lipid mediator that protects neural cell integrity and their combination as an alternative treatment for GBM. Female athymic nude mice were anesthetized with ketamine/xylazine, and luciferase-modified U87MG tumor cells were stereotactically injected into the right striatum. On post-implantation day 13, mice received one of the following: LAU-0901, ELV, Avastin, and all three compounds in combination. Bioluminescent imaging (BLI) was performed on days 13, 20, and 30 post-implantation. Mice were perfused for ex vivo MRI on day 30. Bioluminescent intracranial tumor growth percentage was reduced by treatments with LAU-0901 (43%), Avastin (77%), or ELV (86%), individually, by day 30 compared to saline treatment. In combination, LAU-0901/Avastin, ELV/LAU-0901, or ELV/Avastin had a synergistic effect in decreasing tumor growth by 72, 92, and 96%, respectively. Additionally, tumor reduction was confirmed by MRI on day 30, which shows a decrease in tumor volume by treatments with LAU-0901 (37%), Avastin (67%), or ELV (81.5%), individually, by day 30 compared to saline treatment. In combination, LAU-0901/Avastin, ELV/LAU-0901, or ELV/Avastin had a synergistic effect in decreasing tumor growth by 69, 78.7, and 88.6%, respectively. We concluded that LAU-0901 and ELV combined with Avastin exert a better inhibitive effect in GBM progression than monotherapy. To our knowledge, this is the first study that demonstrates the efficacy of these novel therapeutic regimens in a model of GBM and may provide the basis for future therapeutics in GBM patients.

Effects of Papaya Seed Oil (Carica papaya Linn.) on Food Consumption, Weight Gain and Hormones of Animals Treated With a High Calorie Diet

Objectives To evaluate the effects of papaya seed oil (Carica papaya Linn.) on food consumption, weight gain and hormonal of animals treated with a high calorie diet. Methods The project was approved by protocol no980/2018 (CEUA). Swiss, male, adult mice were used and divided into the experimental groups: control group (CT - Nuvital® diet - saline treatment), AIN-93M group (AIN-93M diet - saline treatment), HPL group (hypercaloric diet - saline treatment), HPL OS group (hypercaloric diet - soybean oil treatment), HPL AZ group (hypercaloric diet - olive oil treatment) and HPL OM group (hypercaloric diet - papaya seed oil treatment). The animals received treatment daily by gavage, 1 mL/kg, for 8 weeks. Body weight and food consumption were evaluated (Camry® analytical digital scale). At the end of the experiment, the animals were submitted to euthanasia and the blood was collected for quantification of leptin, insulin and resistin (commercial kit MADKMAG-71K®-Merck). The results were expressed as mean ± standard deviation, using Prisma 5.0 software (GraphPad Software, USA) (P ≤ 0.05).It was observed that the papaya seed oil reduced food consumption and body weight, as well as increased the concentration of leptin, maintaining insulin and resistin, thus being effective in combating the metabolic changes caused by the high fat diet. Results In the first month of the study, food consumption was lower in the HPL AZ and HPL OM groups than in the CT (P &lt; 0.005), and in the second month all groups that received HPL diet consumed less if compared to the CT, being that HPL OM have significantly lower consumption than groups AIN-93M, CT and HPL (P &lt; 0.05). At the beginning of the experiment, all animals were weighed and evenly distributed in the groups (P = 0.938). In the first and second weeks, HLP OM had a lower weight than HLP OS and HPL (P &lt; 0.05). In the weeks that followed, the weight gain of the HLP OM group was lower compared to the groups that received a high-fat diet, but without significant difference. In the evaluation of the hormone leptin, a higher value was found in the HPL OM group (P &lt; 0.001), with values of resistin and insulin similar to the control groups. Conclusions It was observed that the papaya seed oil reduced food consumption and body weight, as well as increased the concentration of leptin, maintaining insulin and resistin. Funding Sources CNPQ.

The outward Shaker channel OsK5.2 is beneficial to the plant salt tolerance through its role in K+ translocation and its control of leaf transpiration

High soil salinity constitutes a major environmental constraint to crop production worldwide, and the identification of genetic determinants of plant salt tolerance is awaited by breeders. While the leaf K+ to Na+ homeostasis is considered as key parameter of plant salt tolerance, the underlying mechanisms are not fully identified. Especially, the contribution of K+ channels to this homeostasis has been scarcely examined. Here, we show, using a reverse genetics approach, that the outwardly-rectifying K+ channel OsK5.2, involved in K+ translocation to the shoot and K+ release by guard cells for stomatal closure, is a strong determinant of rice salt tolerance. Upon saline treatment, OsK5.2 function in xylem sap K+ load was maintained, and even transiently increased, in roots. OsK5.2 selectively handled K+ in roots and was not involved in xylem sap Na+ load. In shoots, OsK5.2 expression was up-regulated from the onset of the saline treatment, enabling fast reduction of stomatal aperture, decreased transpirational water flow and therefore decreased trans-plant Na+ flux and reduced leaf Na+ accumulation. Thus, the OsK5.2 functions allowed shoot K+ nutrition while minimizing arrival of Na+, and appeared highly beneficial to the leaf K+ to Na+ homeostasis, the avoidance of salt toxicity and plant growth maintaining.

Pre-Treat Xenogenic Collagenous Blocks of Bone Substitutes with Saline Facilitate Their Manipulation and Guarantee High Bone Regeneration Rates, Qualitatively and Quantitatively

Deproteinized bovine bone mineral particles embedded in collagen (DBBM-C) are widely used for bone regenerations with excellent, albeit sometimes variable clinical outcomes. Clinicians usually prepare DBBM-C by mixing with blood. Replacing blood by saline represents an alternative. We investigated if saline treatment could improve DBBM-C i. handling in vitro and ii. biological performances in a rabbit calvarial model. In vitro, DBBM-C blocks soaked in saline or blood were submitted to compression tests. In vivo, four poly ether ether ketone (PEEK)cylinders were placed on 16 rabbit skulls, filled with DBBM-C soaked in blood or saline for 2–4–8–12 weeks before histomorphometry. DBBM-C blocks were fully hydrated after 30 s in saline when 120 s in blood could not hydrate blocks core. Stiffness gradually decreased 2.5-fold after blood soaking whereas a six-fold decrease was measured after 30 s in saline. In vivo, saline treatment allowed 50% more bone regeneration during the first month when compared to blood soaking. This difference was then no longer visible. New bone morphology and maturity were equivalent in both conditions. DBBM-C saline-soaking facilitated its handling and accelerated bone regeneration of highly qualitative tissues when compared to blood treatment. Saline pretreatment thus may increase the clinical predictability of bone augmentation procedures.