Acute Tolerance Research Articles

A Novel Ferroptosis Inhibitor UAMC-3203, a Potential Treatment for Corneal Epithelial Wound.

Corneal wound, associated with pain, impaired vision, and even blindness, is the most common ocular injury. In this study, we investigated the effect of a novel ferroptosis inhibitor, UAMC-3203 (10 nM-50 µM), in corneal epithelial wound healing in vitro in human corneal epithelial (HCE) cells and ex vivo using alkali-induced corneal wounded mice eye model. We evaluated in vivo acute tolerability of the compound by visual inspection, optical coherence tomography (OCT), and stereomicroscope imaging in rats after its application (100 µM drug solution in phosphate buffer pH 7.4) twice a day for 5 days. In addition, we studied the partitioning of UAMC-3203 in corneal epithelium and corneal stroma using excised porcine cornea. Our study demonstrated that UAMC-3203 had a positive corneal epithelial wound healing effect at the optimal concentration of 10 nM (IC50 value for ferroptosis) in vitro and at 10 µM in the ex vivo study. UAMC-3203 solution (100 µM) was well tolerated after topical administration with no signs of toxicity and inflammation in rats. Ex-vivo distribution study revealed significantly higher concentration (~12-38-fold) and partition coefficient (Kp) (~52 times) in corneal epithelium than corneal stroma. The UAMC-3203 solution (100 µM) was stable for up to 30 days at 4 °C, 37 °C, and room temperature. Overall, UAMC-3203 provides a new prospect for safe and effective therapy for corneal wounds.

Temperatures leading to heat escape responses in Antarctic marine ectotherms match acute thermal limits.

Thermal tolerance windows are key indicators of the range of temperatures tolerated by animals and therefore, a measure of resilience to climate change. In the ocean, where ectotherms are immersed, body temperatures are tightly coupled to environmental temperature and species have few options for thermoregulation. However, mobile species do have the ability to orientate towards optimal temperatures and move away from sub-optimal or dangerous temperatures. Escape responses are one such locomotory behavior, which typically manifests as a series of violent flicking movements that move individuals out of dangerous environments. We tested 11 species of Antarctic marine ectotherms, from one of the most stable shallow water marine environments, with an annual temperature range of -2°C to +2°C, that are vulnerable to small degrees of warming. Three species, the clam Laternula elliptica, the sea cucumber Cucumaria georgiana, and the brittlestar Ophionotus victoriae, showed no, or virtually no, escape response to temperature. Escape responses from a further eight species had a median response temperature of 11.2 (interquartile range, 10°C-15.7°C), which is well above current environmental temperatures but close to the range for acute lethal limits of Antarctic marine ectotherms (CTmax range, 17.2°C-26.6°C). This highlights that both acute tolerance limits and escape responses, fall outside current environmental temperatures, but also those predicted for 100s of years in the Southern Ocean. In a warmer Southern Ocean Antarctic fauna may not have the capacity to use temperature to select optimal thermal conditions, which leaves adaptation as a primary mechanism for their persistence.

Magnitude–duration relationships of physiological sensitivity and environmental exposure improve climate change vulnerability assessments

Integrating thermal physiology with environmental temperature is essential to understanding distributions of species and vulnerability to climate change. Warming tolerance – the difference between an organism's maximum thermal tolerance (Tmax) and maximum habitat temperature (Thab) – is frequently used to integrate organismal sensitivity and environmental exposure. Traditionally, applications of warming tolerance define Tmax and Thab as invariable magnitudes, yet tolerance magnitude depends on exposure duration, and diel temperature cycles expose organisms to a range of temperature magnitudes and durations. How traditional (i.e. acute) estimates of warming tolerance compare to estimates from prolonged exposures remains poorly understood. In this study, magnitude–duration curves for tolerances of one cold‐water, two cool‐water and one warm‐water species of freshwater fish were compiled from the literature and compared to magnitude–duration exposures from 66 streams across the eastern United States. Warming tolerances were estimated for exposure durations spanning 0.01–24 h. Current acute (0.01 h) warming tolerances ranged from median 6.30°C for the cold‐water species to 9.68°C for the warm‐water species. The lowest warming tolerances corresponded to prolonged exposures lasting median 3.85–5.30 h among species and were 2.51–4.38°C lower than acute estimates. Although acute estimates remained positive in historically occupied and unoccupied streams (6.30 versus 2.33°C), estimates based on prolonged exposure were positive at occupied streams of the cold‐water species but transitioned to negative in unoccupied streams (2.19 versus −1.12°C). Acute warming tolerances for the cold‐water species also remained positive under future climate (6.29–4.23°C) but approached zero at prolonged durations (2.19–0.09°C) and transitioned to negative for 47.2% of streams. Results demonstrate that acute measures of Tmax and Thab overestimate warming tolerances and therefore underestimate climate change vulnerability. Integrating magnitude–duration relationships into warming tolerance estimates can elucidate physiological mechanisms underlying species distributions and can improve accuracy of climate change vulnerability assessments.

Genome-wide association study and genomic prediction of tolerance to acute hypoxia in rainbow trout

Hypoxia is one of the major threats to the aquaculture sector resulting in substantial economic losses to the fish farmers. Thus, tolerance to hypoxia is of high economic interest to be genetically improved by breeding programs. Rainbow trout (Oncorhynchus mykiss) is one of the most cultured salmonid species worldwide, with well-developed breeding programs. Still, studies of genetic potential to improve hypoxia tolerance in this species are rare. In the present study, 1320 individuals of rainbow trout were used for a genome-wide association study of acute hypoxia tolerance based on imputed high-density genotypes to explore the genetic architecture and related candidate genes affecting hypoxia response. Three significant (Omy31_1, Omy31_2, Omy20) and two putative (Omy15, Omy28) quantitative trait loci (QTLs) were detected, but each of them only explained between 0.2% and 0.8% of the genetic variance of acute hypoxia tolerance. However, heritability was estimated at a moderate value of 0.24–0.28, that suggests a solid potential to improve hypoxia tolerance in the studied rainbow trout population by genetic selection. Moreover, it was shown that genomic prediction for hypoxia tolerance would lead to a relative increase of ∼11% for genomic selection (GS) accuracy compared to the pedigree-based selection, considering a reference population of 1000 individuals. Finally, fifteen genes (ids, fmr1, arx, lonrf3, commd5, map4k4, smu1, b4galt1, re1, abca1, noa1, igfbp7, noxo1, bcl2a, mylk3) were proposed as potential functional candidates involved in hypoxia tolerance. Taking all proposed candidate genes (6 out of 15 genes) and high linkage disequilibrium (r2) values within the main QTL (Omy31_1), we may hypothesize that the complex response to acute hypoxia in rainbow trout, i.e., the interplay between behavioural, morphological, and physiological responses, is primarily encoded by a supergene. However, further functional validation of their effects may help to specify the biological mechanisms triggering a response to acute hypoxia in rainbow trout.

Analgesic tolerance and cross-tolerance to the bifunctional opioid/neuropeptide FF receptors agonist EN-9 and μ-opioid receptor ligands at the supraspinal level in mice

The chimeric peptide EN-9 was reported as a κ-opioid/neuropeptide FF receptors bifunctional agonist that modulated chronic pain with no tolerance. Many lines of evidence have shown that the effect of the κ-opioid receptor is mediated by not only its specific activation but also downstream events participation, especially interaction with the μ-opioid receptor pathway in antinociception and tolerance on most occasions. The present study investigated the acute and chronic cross-tolerance of EN-9 with μ-opioid receptor agonist EM-2, DAMGO, and morphine after intracerebroventricularly (i.c.v) injection in the mouse tail-flick test. In the acute tolerance test, EN-9 showed symmetrical acute cross-tolerance to DAMGO but no cross-tolerance to EM2. In the chronic tolerance test, EN-9 had no tolerance after eight days of repeated administration. However, EN-9 illustrated complete cross-tolerance to morphine and symmetrical cross-tolerance to EM2. In addition, inhibition of NPFF receptor could induce the tolerance development of EN-9. These findings indicated that supraspinal EN-9-induced antinociception contains additional components, which are mediated by the downstream μ-opioid receptor pathway both in acute and chronic treatment, whereas the subtypes of μ-opioid receptor or NPFF system pathway involved in antinociceptive effects induced by EN-9 are complex. Identifying the receptor mechanism could help design preferable bifunctional opioid compounds.

Cardiovascular oxygen transport and peripheral oxygen extraction capacity contribute to acute heat tolerance in European seabass

This study evaluated whether different parameters describing cardiovascular function, energy metabolism, oxygen transport and oxidative stress were related to the critical thermal maximum (CTMAX) of European seabass (Dicentrarchus labrax) and if there were differential changes in these parameters during and after heat shock in animals with different CTMAX in order to characterize which physiological features make seabass vulnerable to heat waves. Seabass (n = 621) were tested for CTMAX and the physiological parameters were measured in individuals with good or poor temperature tolerance before and after a heat shock (change in temperature from 15 °C to 28 °C in 1.5 h). Fish with good thermal tolerance had larger ventricles with higher maximal heart rate during the heat shock than individuals with poor tolerance. Furthermore, they initially had a high ventricular Ca2+-ATPase activity, which was reduced to a similar level as in fish with poor tolerance following heat shock. The activity of heart lactate dehydrogenase increased in fish with high tolerance, when they were exposed to heat shock, while the aerobic enzyme activity did not differ between groups. The tolerant individuals had smaller red muscle fibers with higher myoglobin content than the poorly tolerant ones. The poorly tolerant individuals had higher hematocrit, which increased with heat shock in both groups. The poorly tolerant individuals had also higher activity of enzymes related to oxidative stress especially after heat shock. In general, CTMAX was not depending on merely one physiological factor but several organ and cellular parameters were related to the CTMAX of seabass and when working in combination they might protect the highly tolerant seabass from future heat waves.

The effects of marine heatwaves on acute heat tolerance in corals.

Scleractinian coral populations are increasingly exposed to conditions above their upper thermal limits due to marine heatwaves, contributing to global declines of coral reef ecosystem health. However, historic mass bleaching events indicate there is considerable inter- and intra-specific variation in thermal tolerance whereby species, individual coral colonies and populations show differential susceptibility to exposure to elevated temperatures. Despite this, we lack a clear understanding of how heat tolerance varies across large contemporary and historical environmental gradients, or the selective pressures that underpin this variation. Here we conducted standardised acute heat stress experiments to identify variation in heat tolerance among species and isolated reefs spanning a large environmental gradient across the Coral Sea Marine Park. We quantified the photochemical yield (Fv /Fm ) of coral samples in three coral species, Acropora cf humilis, Pocillopora meandrina, and Pocillopora verrucosa, following exposure to four temperature treatments (local ambient temperatures, and + 3°C, +6°C and + 9°C above local maximum monthly mean). We quantified the temperature at which Fv /Fm decreased by 50% (termed ED50) and used derived values to directly compare acute heat tolerance across reefs and species. The ED50 for Acropora was 0.4-0.7°C lower than either Pocillopora species, with a 0.3°C difference between the two Pocillopora species. We also recorded 0.9°C to 1.9°C phenotypic variation in heat tolerance among reefs within species, indicating spatial heterogeneity in heat tolerance across broad environmental gradients. Acute heat tolerance had a strong positive relationship to mild heatwave exposure over the past 35 years (since 1986) but was negatively related to recent severe heatwaves (2016-2020). Phenotypic variation associated with mild thermal history in local environments provides supportive evidence that marine heatwaves are selecting for tolerant individuals and populations; however, this adaptive potential may be compromised by the exposure to recent severe heatwaves.

Integrated transcriptomic and metabolic analysis response in gills, hepatopancreas, and muscle metabolism in oriental river prawn Macrobrachium nipponense in response to acute high salinity stress

Salinity is an ecological factor that affects the physiological metabolism, survival, and distribution of crustaceans. Although some crustaceans can tolerate an extensive range of salinity, drastic fluctuations can induce damage and even cause death. The oriental river prawn, Macrobrachium nipponense, is a major commercial aquaculture species in China, Japan, and Southeast Asian countries and can survive in a salinity range of 7–20. To explore the metabolic responses and molecular mechanisms of salinity tolerance in M. nipponense, a liquid chromatography–mass spectrometry-based metabolomic analysis and high-throughput RNA sequencing were combined to evaluate the metabolic effects and primary regulatory pathways in gills, hepatopancreas, and muscle of M. nipponense in response to acute high salinity stress in a time-dependent manner. Differentially expressed genes (DGEs) were identified, and total of 632, 836, and 1246 DEGs with a cutoff of significant two-fold change were differentially expressed in hepatopancreas, gills, and muscle tissues, respectively. The DEGs of hepatopancreas and gill tissues were mainly enriched in PPAR signaling pathway, longevity regulating pathway, protein digestion and absorption, and the DEGs of muscle tissue in arginine biosynthesis, adrenergic signaling in cardiomyocytes, cardiac muscle contraction, and cGMP-PKG signaling pathway. The transcriptomic response suggested that M. nipponense exposed to acute high salinity stress may regulate mechanisms related to ion exchange, metabolism, and immune responses to adapt to the environmental alteration. Through LC-MS analysis, 1432 metabolites (589 negative and 843 positive metabolites) were identified. Metabolomic analysis revealed that multiple amino acids and fatty acids were affected. Integrated transcriptome and metabolome analyses indicated that 16 pathways were identified in three tissues of M. nipponense. In addition, 24 metabolites and 34 related DEGs were recorded. Hence, salinity exposure affected metabolic processes in M. nipponense and a higher expression of aminophospholipid genes and enhanced fatty acids may be related to strengthening tolerance to acute high salinity. Overall, these results provide valuable insights into the mechanisms underlying acute high salinity stress responses and tolerance in M. nipponense.

Reduced acute functional tolerance and enhanced preference for ethanol in Caenorhabditis elegans exposed to lead during development: Potential role of alcohol dehydrogenase

Despite its relative simplicity, the invertebrate Caenorhabditis elegans (C. elegans) has become a powerful tool to evaluate toxicity. Lead (Pb) persistence in the environment and its distinctive characteristic as a neurodevelopmental toxicant determine the potential effects of this metal against challenging events later in life. Additionally, among other psychoactive substances, low to moderate ethanol (EtOH) doses have been pointed out to induce behaviors such as acute functional tolerance (AFT) and drug-induced chemotaxis. In the present study, we aimed to study the impact of early-life Pb exposure on EtOH-induced motivational and stimulant effects in C. elegans by assessing the preference for EtOH and the participation of alcohol dehydrogenase (ADH, sorbitol dehydrogenase -SODH in worms) in the AFT response. Thus, N2 (wild type) and RB2114 (sod-1 −/−) strains developmentally exposed to 24 μM Pb were evaluated in their AFT to 200 mM EtOH alone and in combination with acetaldehyde (ACD). We ascribed the enhanced EtOH-induced AFT observed in the N2 Pb-exposed animals to a reduced ADH functionality as evaluated by both, ADH activity determination and the allyl alcohol test, which altogether suggest excess EtOH accumulation rather than low ACD formation in these animals. Moreover, the Pb-induced preference for EtOH indicates enhanced motivational effects of this drug as a consequence of early-life exposure to Pb, results that resemble our previous reports in rodents and provide a close association between EtOH stimulant and motivational effects in these animals.

The interplay between prior selection, mild intermittent exposure, and acute severe exposure in phenotypic and transcriptional response to hypoxia.

Hypoxia has profound and diverse effects on aerobic organisms, disrupting oxidative phosphorylation and activating several protective pathways. Predictions have been made that exposure to mild intermittent hypoxia may be protective against more severe exposure and may extend lifespan. Here we report the lifespan effects of chronic, mild, intermittent hypoxia, and short‐term survival in acute severe hypoxia in four clones of Daphnia magna originating from either permanent or intermittent habitats. We test the hypothesis that acclimation to chronic mild intermittent hypoxia can extend lifespan through activation of antioxidant and stress‐tolerance pathways and increase survival in acute severe hypoxia through activation of oxygen transport and storage proteins and adjustment to carbohydrate metabolism. Unexpectedly, we show that chronic hypoxia extended the lifespan in the two clones originating from intermittent habitats but had the opposite effect in the two clones from permanent habitats, which also showed lower tolerance to acute hypoxia. Exposure to chronic hypoxia did not protect against acute hypoxia; to the contrary, Daphnia from the chronic hypoxia treatment had lower acute hypoxia tolerance than normoxic controls. Few transcripts changed their abundance in response to the chronic hypoxia treatment in any of the clones. After 12 h of acute hypoxia treatment, the transcriptional response was more pronounced, with numerous protein‐coding genes with functionality in oxygen transport, mitochondrial and respiratory metabolism, and gluconeogenesis, showing upregulation. While clones from intermittent habitats showed somewhat stronger differential expression in response to acute hypoxia than those from permanent habitats, contrary to predictions, there were no significant hypoxia‐by‐habitat of origin or chronic‐by‐acute treatment interactions. GO enrichment analysis revealed a possible hypoxia tolerance role by accelerating the molting cycle and regulating neuron survival through upregulation of cuticular proteins and neurotrophins, respectively.

Apremilast-induced increases in acute ethanol intoxication and decreases in ethanol drinking in mice involve PKA phosphorylation of GABAA β3 subunits

We previously showed that apremilast, an FDA-approved PDE4 inhibitor, selectively alters behavioral responses to ethanol and certain GABAergic drugs in a PKA-dependent manner in C57BL6/J mice. Here, we investigated if PKA phosphorylation of β3 GABAA receptor subunits is involved in apremilast regulation of ethanol, propofol, or diazepam responses. Apremilast prolonged rotarod ataxia and loss of the righting reflex by ethanol and propofol in wild-type mice, but not in β3-S408A/S409A knock-in mice. In contrast, apremilast hastened recovery from the ataxic and sedative effects of diazepam in both genotypes. These findings suggest that apremilast modulation of ethanol and propofol behaviors in wild-type mice is mediated by β3 subunit phosphorylation, whereas its actions on diazepam responses involve a different mechanism. The PKA inhibitor H-89 prevented apremilast modulation of ethanol-induced ataxia. Apremilast sensitized wild-type males to ethanol-induced ataxia and decreased acute functional tolerance (AFT) in females but had no effect in β3-S408A/S409A mice of either sex. These results could not be attributed to genotype differences in blood ethanol clearance. There were also no baseline genotype differences in ethanol consumption and preference in two different voluntary drinking procedures. However, the ability of apremilast to reduce ethanol consumption was diminished in β3-S408A/S409A mice. Our results provide strong evidence that PKA-dependent phosphorylation of β3 GABAA receptor subunits is an important mechanism by which apremilast increases acute sensitivity to alcohol, decreases AFT, and decreases ethanol drinking.

The impact of macronutrient composition on metabolic regulation: An Islet-Centric view.

The influence of dietary carbohydrates and fats on weight gain is inconclusively understood. We studied the acute impact of these nutrients on the overall metabolic state utilizing the insulin:glucagon ratio (IGR). Following in vitro glucose and palmitate treatment, insulin and glucagon secretion from islets isolated from C57Bl/6J mice was measured. Our human in vivo study included 21 normoglycaemia (mean age 51.9 ± 16.5 years, BMI 23.9 ± 3.5kg/m2 , and HbA1c 36.9 ± 3.3 mmol/mol) and 20 type 2 diabetes (T2D) diagnosed individuals (duration 12 ± 7 years, mean age 63.6 ± 4.5 years, BMI 29.1 ± 2.4kg/m2 , and HbA1c 52.3 ± 9.5 mmol/mol). Individuals consumed a carbohydrate-rich or fat-rich meal (600 kcal) in a cross-over design. Plasma insulin and glucagon levels were measured at -30, -5, and 0min, and every 30 min until 240 min after meal ingestion. The IGR measured from mouse islets was determined solely by glucose levels. The palmitate-stimulated hormone secretion was largely glucose independent in the analysed mouse islets. The acute meal tolerance test demonstrated that insulin and glucagon secretion is dependent on glycaemic status and meal composition, whereas the IGR was dependent upon meal composition. The relative reduction in IGR elicited by the fat-rich meal was more pronounced in obese individuals. This effect was blunted in T2D individuals with elevated HbA1c levels. The metabolic state in normoglycaemic individuals and T2D-diagnosed individuals is regulated by glucose. We demonstrate that consumption of a low carbohydrate diet, eliciting a catabolic state, may be beneficial for weight loss, particularly in obese individuals.

Acute CO2 tolerance in fishes is associated with air breathing but not the Root effect, red cell βNHE, or habitat

High CO2 (hypercapnia) can impose significant physiological challenges associated with acid-base regulation in fishes, impairing whole animal performance and survival. Unlike other environmental conditions such as temperature and O2, the acute CO2 tolerance thresholds of fishes are not understood. While some fish species are highly tolerant, the extent of acute CO2 tolerance and the associated physiological and ecological traits remain largely unknown. To investigate this, we used a recently developed ramping assay, termed the Carbon Dioxide maximum (CDmax), that increases CO2 exposure until loss of equilibrium (LOE) is observed. We investigated if there was a relationship between CO2 tolerance and the Root effect, β-adrenergic sodium proton exchanger (βNHE), air-breathing, and fish habitat in 17 species. We hypothesized that CO2 tolerance would be higher in fishes that lack both a Root effect and βNHE, breathe air, and reside in tropical habitats. Our results showed that CDmax ranged from 2.7 to 26.7 kPa, while LOE was never reached in four species at the maximum PCO2 we could measure (26.7 kPa); CO2 tolerance was only associated with air-breathing, but not the presence of a Root effect or a red blood cell (RBC) βNHE, or fish habitat. This study demonstrates that the diverse group of fishes investigated here are incredibly tolerant of CO2 and that although this tolerance is associated with air-breathing, further investigations are required to understand the basis for CO2 tolerance.

Early Outcome, Cosmetic Result and Tolerability of an IOERT-Boost Prior to Adjuvant Whole-Breast Irradiation.

Simple SummaryOur study with 139 breast cancer patients treated with intraoperative radiotherapy reports favorable data on the cosmetic outcome as well as the acute and early long-term side effects. Our oncologic control rates are comparable to the previous literature.Background/Aims: Due to its favorable dose distribution and targeting of the region at highest risk of recurrence due to direct visualization of tumor bed, intraoperative electron radiation therapy (IOERT) is used as part of a breast-conserving treatment approach. The aim of this study was to analyze tumor control and survival, as well as the toxicity profile, and cosmetic outcomes in patients irradiated with an IOERT boost for breast cancer. Materials and Methods: 139 Patients treated at our institution between January 2010 and January 2015 with a single boost dose of 10 Gy to the tumor bed during breast-conserving surgery followed by whole-breast irradiation were retrospectively analyzed. Results: 139 patients were included in this analysis. The median age was 54 years (range 28–83 years). The preferred surgical strategy was segmental resection with sentinel lymphonodectomy (66.5%) or axillary dissection (23.1%). Regarding adjuvant radiotherapy, the vast majority received 5 × 1.8 Gy to 50.4 Gy. At a median follow-up of 33.6 months, recurrence-free and overall survival were 95.5% and 94.9%, respectively. No patient developed an in-field recurrence. Seven patients (5.0%) died during the follow-up period, including two patients due to disease recurrence (non-in-field). High-grade (CTCAE > 2) perioperative adverse events attributable to IOERT included wound healing disorder (N = 1) and hematoma (N = 1). High-grade late adverse events (LENT-SOMA grade III) were reported only in one patient with fat necrosis. Low-grade late adverse events (LENT-SOMA grade I-II) included pain (18.0%), edema (10.5%), fibrosis (21%), telangiectasia (4.5%) and pigmentation change (23.0%). The mean breast retraction assessment score was 1.66 (0–6). Both patients and specialists rated the cosmetic result “excellent/good” in 84.8% and 87.9%, respectively. Conclusion: Our study reports favorable data on the cosmetic outcome as well as the acute and early long-term tolerability for patients treated with an IOERT boost. Our oncologic control rates are comparable to the previous literature. However, prospective investigations on the role of IOERT in comparison to other boost procedures would be desirable.

Effect of low dissolved oxygen on the viability of juvenile Margaritifera margaritifera: Hypoxia tolerance ex situ

Abstract The decline of endangered freshwater pearl mussel (FPM, Margaritifera margaritifera) has been attributed to juvenile mortality caused by low concentrations of dissolved oxygen in the stream substrate resulting from fine sediments (siltation) that impede water exchange in the interstitial microhabitat of juveniles. If low oxygen concentration causes recruitment failure of FPMs, knowledge on the oxygen tolerance of juvenile FPMs is essential for the conservation of the species, as it will justify conservation efforts improving water exchange in the bottom gravel. However, the tolerance of low oxygen of FPM juveniles has not been directly studied. Juvenile FPMs (9–11 months old) were exposed in individual chambers equipped with optical oxygen measurement spots to different levels of dissolved oxygen at 19 °C and their viability was monitored for 10 days to assess the acute oxygen tolerance of juvenile FPMs. Oxygen concentration ranged between 8.8 and 6.2 mg L−1 in the high oxygen treatment (control), 5.0–0.4 mg L−1 in the medium treatment, and 1.3–0.04 mg L−1 in the low oxygen treatment (near‐anoxic conditions). Viability of juvenile FPMs depended on the concentration of available dissolved oxygen, such that all juveniles exposed to near‐anoxic conditions were classified as non‐viable, whereas all mussels exposed to high and medium concentrations were viable at the end of the 10 day experiment. Juveniles differed in their ability to tolerate near‐anoxic conditions, so that some individuals survived only 1 day and others survived up to 9 days. This study provides the first direct experimental evidence on the oxygen sensitivity of FPM juveniles and suggests that >10‐day events of very low dissolved oxygen at summer temperatures are fatal to juvenile FPMs, supporting the view that actions preventing low oxygen episodes in the substrate are essential for recruitment, and conservation, of FPMs.

A prospective, multicenter, clinical Study to evaluate the Safety, Pharmacokinetics, and Efficacy of Bleed Outcomes, with HemoRel-A® in severe Hemophilia A Patients.

To evaluate efficacy for an on-demand treatment of acute bleeding events, pharmacokinetics, safety, and tolerability of HemoRel-A® in severe hemophilia A. A total of 44 male subjects with severe hemophilia A with an annualized bleed rate of 12 while on-demand treatment with factor VIII (FVIII) were enrolled in the study and received HemoRel-A® for bleed treatment. The efficacy of HemoRel-A® was evaluated based on a four-point scale (excellent, good, moderate, or none). Six-point pharmacokinetic (PK) assessment was performed following a single dose of 50 IU/kg in 12 subjects after a 7-day wash-out period. Safety evaluations were performed at each visit and inhibitor testing was performed in all patients at screening and end of study. Forty-four male subjects received at least a single dose of the study medication and were included in the intent-to-treat (ITT) analysis and safety outcome. In 23 (7.52%) out of the 306 bleeding events, HemoRel-A® efficacy was rated as excellent, in 272 (88.89 %) bleeds it was rated as good, and in 11 (3.68%) bleeding events it was rated as moderate. No failure of efficacy was noted in any of the bleeding events. Thus overall out of 306 bleeding events, 295 (96.41%) showed excellent or good efficacy. Pharmacokinetic assessment based on plasma FVIII activity measured by the chromogenic assay in 12 patients showed comparative results similar to FVIII preparations. A total of 12 adverse events (AEs) were reported in this study. There was no inhibitor development in this previously treated patients (PTP) cohort. HemoRel-A® was established to be efficacious and safe in the treatment of acute bleeding events in subjects with severe hemophilia A. CTRI/2018/05/013790. Registration date: 9th May 2018.

Lipid-induced glucose intolerance is driven by impaired glucose kinetics and insulin metabolism in healthy individuals

AimsHypertriglyceridemia is associated with an increased risk of type 2 diabetes. We aimed to comprehensively examine the effects of hypertriglyceridemia on major glucose homeostatic mechanisms involved in diabetes progression. MethodsIn this randomized, cross-over, single-blinded study, two dual-labeled, 3-hour oral glucose tolerance tests were performed during 5-hour intravenous infusions of either 20 % Intralipid or saline in 12 healthy subjects (age 27.9 ± 2.6 years, 11 men, BMI 22.6 ± 1.4 kg/m2) to evaluate lipid-induced changes in insulin metabolism and glucose kinetics. Insulin sensitivity, β cell secretory function, and insulin clearance were assessed by modeling glucose, insulin and C-peptide data. Intestinal glucose absorption, endogenous glucose production, and glucose clearance were assessed from glucose tracers. The effect of triglycerides on β-cell secretory function was examined in perifusion experiments in murine pseudoislets and human pancreatic islets. ResultsMild acute hypertriglyceridemia impaired oral glucose tolerance (mean glucose: +0.9 [0.3, 1.5] mmol/L, p = 0.008) and whole-body insulin sensitivity (Matsuda index: −1.67 [−0.50, −2.84], p = 0.009). Post-glucose hyperinsulinemia (mean insulin: +99 [17, 182] pmol/L, p = 0.009) resulted from reduced insulin clearance (−0.16 [−0.32, −0.01] L min−1 m−2, p = 0.04) and enhanced hyperglycemia-induced total insulin secretion (+11.9 [1.1, 22.8] nmol/m2, p = 0.02), which occurred despite a decline in model-derived β cell glucose sensitivity (−41[−74, −7] pmol min−1 m−2 mmol−1 L, p = 0.04). The analysis of tracer-derived glucose metabolic fluxes during lipid infusion revealed lower glucose clearance (−96 [−152, −41] mL/kgFFM, p = 0.005), increased 2-hour oral glucose absorption (+380 [42, 718] μmol/kgFFM, p = 0.04) and suppressed endogenous glucose production (−448 [−573, −123] μmol/kgFFM, p = 0.005). High-physiologic triglyceride levels increased acute basal insulin secretion in murine pseudoislets (+11 [3, 19] pg/aliquot, p = 0.02) and human pancreatic islets (+286 [59, 512] pg/islet, p = 0.02). ConclusionOur findings support a critical role for hypertriglyceridemia in the pathogenesis of type 2 diabetes in otherwise healthy individuals and dissect the glucose homeostatic mechanisms involved, encompassing insulin sensitivity, β cell function and oral glucose absorption.

Abstract 3938: Discovery of a novel small molecule inhibitor of the YAP1/TAZ-TEAD transcriptional complex

Abstract Hippo pathway alterations in human cancers often result in dephosphorylation of yes-associated protein (YAP1) and its paralog TAZ (WWTR1), allowing the formation of an active complex with transcriptional enhanced associate domain transcription factors (TEADs). This complex formation results in the activation of pro-survival and pro-proliferative transcriptional programs in cancer cells. Many tumor types harbor alterations in the Hippo pathway, including mesothelioma, where a high percentage of tumors are driven by YAP1/TEAD activity. Although traditionally difficult to drug with small molecules, identification of autopalmitoylation sites in the hydrophobic palmitate pocket of TEADs necessary for YAP1 interaction has enabled modern drug discovery platforms to generate compounds that allosterically inhibit YAP1/TAZ-TEAD complex formation and transcriptional activity. We report the discovery and characterization of the novel YAP1/TAZ-TEAD inhibitor MRK-A from an aryl ether chemical series demonstrating potent and specific inhibition of YAP1/TAZ-TEAD activity. In biochemical thermal shift assays, MRK-A caused a concentration-dependent melting temperature shift of 8-12.5 and 0.6-1.5 degrees for TEAD1 and TEAD2, respectively, indicating direct binding to TEAD protein. In cellular assays, MRK-A demonstrated inhibition of a TEAD-based reporter assay, with little to no activity in multiple orthogonal off-target reporter assays such as WNT, NF-KB, TGFB and PPARG (8.4 nM vs. >10000 nM), which is consistent with the exquisite selectivity profile of this molecule (>1000x selectivity against 350+ measured kinases and other common off-targets). In the NF2-deficient mesothelioma cell line H226, MRK-A suppressed the transcription of endogenous YAP/TAZ-TEAD target genes CYR61, ERBB3, ANKRD1 and CTGF (50-75% inhibition at 100 nM), but not LATS1, a non-TEAD regulated Hippo pathway gene. In co-immunoprecipitation assays, MRK-A disrupted the interaction of YAP1 and TEAD in H226 cells at concentrations consistent with inhibition of target genes. In addition, MRK-A potently blocked the clonogenic growth and viability of H226 cells in a dose-dependent manner (maximal response at 1 µM compound >90% growth inhibition), while sparing the Hippo wild-type mesothelioma cell line H28. Furthermore, structurally similar control compounds, MRK-B and MRK-C, without the ability to block TEAD-mediated transcription (TEAD reporter MCF7 assay IC50 > 10000 nM), did not impact the clonogenic growth of H226 cells. In vivo, MRK-A did not show acute tolerability signals in mice and demonstrated pharmacokinetics suitable for daily oral dosing in efficacy studies. In summary, we report the structure and characterization of MRK-A demonstrating potent and specific inhibition of YAP1/TAZ-TEAD mediated transcriptional responses, with potential implications for treating malignancies driven by altered Hippo signaling. Citation Format: Casey J. Moure, Christopher Sondey, Mangeng Cheng, My Mansueto, Rafael Fernandez, Sebastian E. Schneider, Julia V. Ramirez, Brian Long, Erin DiMauro, Brandon Vara, Charles Yeung, Abe Achab, Jongwon Lim, Ronald Kim, Cayetana Zarate, Jonathan Bennett, Rachel Palte, Robert Foti, Vladimir Simov, Evan Barry. Discovery of a novel small molecule inhibitor of the YAP1/TAZ-TEAD transcriptional complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3938.

A novel osmoprotective liposomal formulation from synthetic phospholipids to reduce in vitro hyperosmolar stress in dry eye treatments

Dry eye disease (DED) is a worldwide, multifactorial disease mainly caused by a deficit in tear production or increased tear evaporation with an increase in tear osmolarity and inflammation. This causes discomfort and there is a therapeutic need to restore the homeostasis of the ocular surface. The aim of the present work was to develop a biodegradable and biocompatible liposomal formulation from the synthetic phospholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) that is able to reduce the effects of hypertonic stress by helping to restore the lipid layer of the tear film. Liposomes were made using the lipid film hydration method with synthetic phospholipids (10 mg/mL) with and without 0.2% HPMC. They were characterised in terms of size, osmolarity, pH, surface tension, and viscosity. Additionally, the in vitro toxicity of the formulation at 1 and 4 h in human corneal epithelial cells (hTERT-HCECs) and human conjunctival cells (IM-HConEpiC) was determined. Furthermore, osmoprotective activity was tested in a corneal model of hyperosmolar stress. In vivo acute tolerance testing was also carried out in albino New Zealand rabbits by topical application of the ophthalmic formulations every 30 min for 6 h. All the assayed formulations showed suitable physicochemical characteristics for ocular surface administration. The liposomal formulations were well-tolerated in cell cultures and showed osmoprotective activity in a hyperosmolar model. No alterations or discomfort were reported when they were topically administered in rabbits. According to the results, the osmoprotective liposomal formulations developed in this work are promising candidates for the treatment of DED.

1403-P: DA-1726, a Balanced GLP1R/GCGR Dual Agonist, Effectively Controls Both Body Weight and Blood Glucose

DA-1726 is a novel balanced long-acting oxyntomodulin (OXM) analogue, which is under preclinical development for the treatment of obesity. In this study, the pharmacological effects of DA-1726 were evaluated. DA-1726 exhibited a balanced dual action on GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in CHO-K1 cells transiently overexpressing each human receptor. In high-fat diet-induced obese mice, 3-week treatment of DA-1726 showed more body weight loss (BWL) and increasing energy expenditure than a pair-fed group. This means that the BWL effect of DA-1726 is due to decreased food intake and increased energy metabolism through GLP1R and GCGR action. In comparison with a GLP1R agonist, DA-1726 showed superior BWL, despite eating more food compared to semaglutide (SEMA) after 4-week treatment in obese mice. At the end of the study, DA-1726 significantly increased the expression of thermogenic genes (Ucp-1 and Ppargc1a) in white adipose tissue (WAT) , and increased WAT browning was histologically confirmed. In addition, DA-1726 inhibited adipocyte differentiation in vitro. Taken together, it suggests that the GCGR action of DA-1726 contributes to reduced adiposity by enhancing fat burning and inhibiting adipogenesis. OXM analogue biased to GCGR fails to control blood glucose despite GLP1R agonism. As a balanced analogue, DA-1726 effectively reduced postprandial glucose excursion in acute oral glucose tolerance test in normal mice. Notably, DA-1726 showed similar glycemic control and excellent BWL to SEMA in hyperglycemic and obese FATZO mice after 4-week treatment. Simultaneously, DA-1726 enhanced insulin sensitivity by significantly reducing fasting plasma insulin and glucose levels. Meanwhile, DA-1726 showed no hypoglycemia risk in overnight fasted normal mice unlike SEMA after a single injection, regardless of dose escalation. Our findings suggest that DA-1726 is a balanced GLP1R/GCGR dual agonist that effectively reduces body weight and blood glucose levels. Disclosure T.Kim: None. I.Jung: None. K.Kim: None. B.Lee: None. M.Kim: None. Y.Chae: None.