Saturating Dose Research Articles

Combination therapy with GalNAc-siRNA drugs targeting both PCSK9 and APOC3 resulted in enhanced lipid lowering in mice

Abstract Introduction Aggressive lowering of both atherogenic low-density lipoprotein cholesterol (LDL-C) and triglyceride-rich lipoproteins (TRLs) could reduce residual atherosclerotic cardiovascular disease (ASCVD) risk. Drugs targeting PCSK9 or APOC3 have shown good lipid-lowering effects in clinic. Here, we have developed GalNAc-conjugated siRNAs, RBD7022 targeting PCSK9 and RBD5044 targeting APOC3, as novel therapeutic agents to lower LDL-C and triglyceride (TG) plasma levels respectively. Each drug shows potent and durable effect in silencing its own target. However, it is unknown whether the combined therapy with siRNA drugs targeting both PCSK9 and APOC3 will generate enhanced effects in lipid lowering. Purpose To investigate the effects of fixed-dose combination of RBD7022 and RBD5044 on LDL-C, TG and total cholesterol (TC). Methods RBD7022 and RBD5044 completely match the human PCSK9 and APOC3 transcripts respectively. RBD7022 has 2 nucleotide mismatches with mouse pcsk9, but still shows silencing activity in mice, while RBD5044 has no activity due to 5 mismatches with mouse apoc3. Therefore, humanized APOC3 transgenic mice were used in this study, which had serious hypertriglyceridemia with relatively high cholesterol and TG levels. They were randomized into 8 groups with treatment of RBD7022 (1 mg/kg or 3 mg/kg), RBD5044 (3 mg/kg or 6 mg/kg), or 3 different dose combinations of RBD7022 and RBD5044, as well as PBS control. All animals were administrated once on Day 1. Plasma was collected to quantify lipid biomarkers. Results RBD7022 showed a robust effect on LDL-C reduction, with inhibition rates of 45% in 3 mg/kg and 43% in 6 mg/kg, indicating 3 mg/kg was a saturated dose for RBD7022 in LDL-C lowering. RBD5044 showed a dose-dependent effect on LDL-C reduction, with inhibition rate of 52% at 1 mg/kg and 59% at 3 mg/kg. Interestingly, significantly enhanced LDL-C reduction was observed in the combination groups, with 67% reduction in LDL-C at RBD5044 1 mg/kg plus RBD7022 3 mg/kg, 73% reduction in LDL-C at RBD5044 3 mg/kg plus RBD7022 3 mg/kg and 78% reduction in LDL-C at RBD5044 3 mg/kg plus RBD7022 6 mg/kg, showing dose-dependent effects (Figure 1A). Enhanced TC reduction was also found in combination at RBD5044 3 mg/kg plus RBD7022 6 mg/kg compared with RBD5044 or RBD7022 alone. RBD5044 showed a dose dependent effect on TG reduction with inhibition rates of 56% at 1 mg/kg and 83% at 3 mg/kg. RBD7022 treatment alone did not show significant effect on TG reduction. No added effects of RBD7022 on TG levels was found in the combination with RBD5044, indicating the TG lowering was induced by RBD5044. Conclusions The combination of RBD7022 targeting PCSK9 and RBD5044 targeting APOC3 simultaneously reduce LDL-C and TC levels. The combination of RBD7022 and RBD5044 could be a better therapeutic option to further reduce LDL-C, particularly for mixed hyperlipidemia patients and other high-risk individuals with both high LDL-C and high TG levels.Figure 1

Study of thermoluminescence characteristics of quartz for high radiation doses (>1kGy): Implications for extending the luminescence dating range

Quartz is an omnipresent abundant natural mineral, used for luminescence dating. Lately, quartz optically stimulated luminescence (OSL) technique is widely used to estimate the equivalent doses (De) for dating geological events (up to 250 Gy, limited by saturation). Some works report thermoluminescence (TL) saturation around ∼ (10–40) kGy. Still dose estimates for such high radiation dose (HRD) range are not achieved. Significant research exists about luminescence response for low dose ranges (<250 Gy) but limited studies are done for HRDs (>1 kGy). This work characterizes the luminescence response of quartz for HRDs (1–21 kGy) to improve existing understanding of luminescence mechanism. Results show that the characteristics of the trap (<200 °C) differ significantly at HRDs than low doses. TL in multi-spectral detection (UV–Visible) band suggest an increase in 340–380 °C peak intensity up to 11 kGy dose. The measurements of saturation dose suggest that it depends on the trapping centres but is independent of recombination centres for the samples used for study. The traps are found bleachable by sunlight, reducing TL signal to residual levels in 1 h. Further, the bleachability is found to be anti-correlated with luminescence emission wavelength. At HRDs luminescence sensitivity is influenced by dose given in previous cycle which is difficult to correct by routine normalization procedures. The work also explores the various normalization methods to find appropriate method for HRD estimation and recommends the use of mass normalization as other normalization methods do not correct the sensitivity changes at HRDs adequately.

Abstract 38: Loop Diuretic Resistance in a Preclinical Model of Cardiorenal Syndrome 2: Role of Enhanced Distal Convoluted Tubule Sodium Transport

Nearly 60% of heart failure (HF) patients develop a progressive decline in kidney function. Decompensated HF also causes edema, usually treated with loop diuretics, like furosemide, that inhibit the Na-K-2Cl cotransporter NKCC2 in the thick ascending limb (TAL). However, nearly 50% of HF patients do not respond adequately to loop diuretics and become resistant. The mechanisms causing loop diuretic resistance are unclear but may involve TAL-dependent and independent mechanisms. Few have used the mouse as a model of cardiorenal syndrome due to its resistance to renal damage. We hypothesize that mice develop loop diuretic resistance and lower GFR after chronic HF induced by myocardial infarction (MI). MI was induced in C57Bl6J mice through ligation of the left anterior descending coronary artery and compared to a sham surgery group. We assessed heart function by echocardiography and GFR (FITC inulin) in conscious mice. The response to diuretics was studied in metabolic cages. MI mice had a 50% reduction in ejection fraction (EF) one month after MI (MI: 29.94 ± 1.94, Sham: 67.02 ± 1.14%, n= 8, p&lt;0.05). EF was still lower 3 and 6 months after MI. The MI group showed decreased GFR, 6 months after MI (MI: 0.50 ± 0.06, Sham: 0.75±0.05 µl/min, n= 5, p&lt;0.05). Urinary albumin was similar in both groups (n=8). In metabolic cages, a saturating dose of the NKCC2 inhibitor bumetanide (20 mg/Kg), induced diuresis and natriuresis in sham mice (Delta UV: 1.96 ± 0.27 ml/4h, Delta UNa: 117.2 ± 11.02µmols/4h, n= 6) but had an attenuated response in MI mice (Delta UV: 0.88 ± 0.23 ml/4h, Delta UNa: 65.00 ± 4.53 µmols/4h, n= 4, p&lt;0.05 vs sham). To study the distal convoluted tubule (DCT) we measured the response to hydrochlorothiazide (HCTZ). HCTZ (100 mg/Kg) induced a natriuretic response in sham mice (Delta UNa: 75.24±11.18µmols/4h, n= 10) and a higher response in the MI group (Delta UNa: 129.54±11.59µmols/4h, n= 12, p&lt;0.05 vs sham). RNAseq in isolated mTALs from MI and sham mice identified 85 up- and 160 down-regulated genes (n=5, p&lt;0.025). Up-regulated genes involved those in the sphingosine pathway, protein trafficking, PKA signaling (PDE4B, PRKD2) and cell growth/tubule differentiation (FREM2, WFDC1). mRNA expression of TAL NaCl transporters was not affected. We conclude that HF decreases GFR, induces loop diuretic resistance and affects mTAL signaling in mice. Loop diuretic resistance may involve enhanced DCT NaCl reabsorption.

Isothermal thermoluminescence dating of speleothem growth – A case study from Bleßberg cave 2, Germany

Speleothems are a key archive of past climatic and environmental changes. 230Th/U dating is the most commonly used method to determine speleothem ages. However, incorporation of non-radiogenic thorium may hamper 230Th/U dating, and samples older than 600 ka also remain out-of-reach. Calcite exhibits a thermoluminescence (TL) signal at 280 °C with a high characteristic saturation dose, and provides significant potential to date carbonate samples over several million years. Hitherto, the application of TL dating for calcite has mainly been hindered by two factors: 1) a spurious TL signal occurring in the high temperature range, and 2) non-uniform dose rate due to U-series disequilibrium. Here we test an isothermal TL (ITL) dating method on a speleothem sample from Bleßberg cave 2, Germany. We show that the ITL signal measured at 240 °C can completely remove the 280 °C TL peak with a negligible TL contribution from the higher temperature range, thus reducing the influence from the spurious signal. The time-dependent dose rate variation can be simulated using the initial radioactivity of 238U, 234U, 230Th and their decay constants. We use the 230Th/U dating method to provide precise and accurate radiometric ages documenting that the speleothem grew between 425.5 ± 5.4 and 320.5 ± 9.7 ka. The ITL ages (421 ± 23 to 311 ± 23 ka) of four subsamples from the speleothem are consistent with the 230Th/U ages at isochronous sampling positions, showing the general reliability of the ITL dating method. ITL dating provides a pathway to construct chronologies for palaeoclimate reconstructions for speleothems beyond the range of the 230Th/U-method and for samples that are unsuitable for U-series dating methods.

High-temperature atomic layer deposition of silicon oxide films using Tris(dimethylamino)silane and ozone

Atomic layer deposition (ALD) benefits from high process temperatures when the substrate is not temperature-sensitive because the films deposited at higher temperatures exhibit better electrical properties. In this study, we investigated the ALD process of silicon oxide films by alternating injections of tris(dimethylamino)silane (tris-DMAS) and O3/O2 at temperatures ranging from 400 to 700 °C. The saturation dose of tris-DMAS was 1.5 × 106 L at 400 °C. Self-limiting growth with a growth rate of approximately 0.9 Å/cycle was observed up to 600 °C, allowing excellent step coverage. However, at 700 °C, the growth rate increased significantly to 4.6 Å/cycle, carbon and nitrogen impurities were detected by secondary ion mass spectrometry, and step coverage was poor, supporting the thermal decomposition of tris-DMAS at this temperature. Therefore, the optimum temperature for ALD SiO2 using tris-DMAS is 600 °C for application as an insulating film on high aspect ratio patterns. At this temperature, the leakage current density was 0.58 nA/cm2, and the oxide-trapped charge density was 4.0 × 1010 cm−2.

Novel elastomeric spiropyran-doped poly(dimethylsiloxane) optical waveguide for UV sensing

Novel poly(dimethylsiloxane) (PDMS) doped with two different spiropyran derivatives (SP) were investigated as potential candidates for the preparation of elastomeric waveguides with UV-dependent optical properties. First, free-standing films were prepared and evaluated with respect to their photochromic response to UV irradiation. Kinetics, reversibility as well as photofatigue and refractive index of the SP-doped PDMS samples were assessed. Second, SP-doped PDMS waveguides were fabricated and tested as UV sensors by monitoring changes in the transmitted optical power of a visible laser (633 nm). UV sensing was successfully demonstrated by doping PDMS using one spiropyran derivative whose propagation loss was measured as 1.04 dB/cm at 633 nm, and sensitivity estimated at 115% change in transmitted optical power per unit change in UV dose. The decay and recovery time constants were measured at 42 and 107 s, respectively, with an average UV saturation dose of 0.4 J/cm2. The prepared waveguides exhibited a reversible and consistent response even under bending. The sensor parameters can be tailored by varying the waveguide length up to 21 cm, and are affected by white light and temperatures up to 70 ℃. This work is relevant to elastomeric optics, smart optical materials, and polymer optical waveguide sensors.Graphical

Limitations of Patient-Controlled Epidural Analgesia Following Abdominoplasty.

Effective postoperative pain management is essential for patient satisfaction and an uneventful postoperative course, particularly in body contouring procedures. Systemic analgesic regimens can be supported by regional procedures, such as the transverse abdominis plane (TAP) block, but these have a limited duration of action. In contrast, thoracic epidural analgesia offers the possibility of a longer-lasting, individualized regional anesthesia administered by a patient-controlled analgesia pump. The aim of this study was to investigate the effects of a patient-controlled epidural analgesia to better classify the clinical value of this procedure in abdominoplasties. This work reviewed the digital medical charts of patients who underwent selective abdominoplasty without combined surgical procedures between September 2018 and August 2022. Evaluated data comprise the postoperative analgesia regimen, including on-demand medication, mobilization time, inpatient length of stay, and clinical outcome. The patients were grouped by the presence of a thoracic epidural catheter. This catheter was placed before anesthetic induction and a saturation dose was preoperatively applied. Postoperative PCEA patients received a basal rate and could independently administer boluses. Basal rate was individually adjusted during daily additional pain visits. The study cohort included 112 patients. Significant differences in the demand for supportive nonepidural opiate medication were shown between the patient-controlled epidural analgesia (PCEA) group (n = 57) and the non-PCEA group (n = 55), depending on the time after surgery. PCEA patients demanded less medication during the early postoperative days (POD 0: PCEA 0.13 (±0.99) mg vs non-PCEA 2.59 (±4.55) mg, P = 0.001; POD 1: PCEA 0.79 mg (±3.06) vs non-PCEA 2.73 (±3.98) mg, P = 0.005), but they required more during the later postoperative phase (POD 3: PCEA 2.76 (±5.60) mg vs non-PCEA 0.61 (±2.01) mg, P = 0.008; POD 4: PCEA 1.64 (±3.82) mg vs non-PCEA 0.07 (±2.01) mg, P = 0.003). In addition, PCEA patients achieved full mobilization later (PCEA 2.67 (±0.82) days vs non-PCEA 1.78 (±1.09) days, P = 0.001) and were discharged later (PCEA 4.84 (±1.23) days vs non-PCEA 4.31 (±1.37) days, P = 0.005). Because the postoperative benefits of PCEA are limited to potent analgesia immediately after abdominoplasty, less cumbersome, time-limited regional anesthesia procedures (such as TAP block) appear not only adequate but also more effective.

Associations between urine glyphosate levels and metabolic health risks: insights from a large cross-sectional population-based study

BackgroundThe prevalence of metabolic syndrome (MetS) in American adults increased from 37.6% in the 2011–12 period to 41.8% in 2017–2018. Environmental exposure, particularly to common compounds such as glyphosate, has drawn increasing attention as a potential risk factor.MethodsWe employed three cycles of data (2013–2018) from the National Health and Nutrition Examination Survey (NHANES) in a cross-sectional study to examine potential associations between urine glyphosate measurements and MetS incidence. We first created a MetS score using exploratory factor analysis (EFA) of the International Diabetes Federation (IDF) criteria for MetS, with data drawn from the 2013–2018 NHANES cycles, and validated this score independently on an additional associated metric, the albumin-to-creatinine (ACR) ratio. The score was validated via a machine learning approach in predicting the ACR score via binary classification and then used in multivariable regression to test the association between quartile-categorized glyphosate exposure and the MetS score.ResultsIn adjusted multivariable regressions, regressions between quartile-categorized glyphosate exposure and MetS score showed a significant inverted U-shaped or saturating dose‒response profile, often with the largest effect for exposures in quartile 3. Exploration of potential effect modification by sex, race, and age category revealed significant differences by race and age, with older people (aged > 65 years) and non-Hispanic African American participants showing larger effect sizes for all exposure quartiles.ConclusionsWe found that urinary glyphosate concentration is significantly associated with a statistical score designed to predict MetS status and that dose–response coefficient is nonlinear, with advanced age and non-Hispanic African American, Mexican American and other Hispanic participants exhibiting greater effect sizes.

Navigating methotrexate toxicity: Examining the therapeutic roles of folinic acid and glucarpidase.

Methotrexate (MTX) toxicity varies depending on factors such as dosing frequency (acute or repeated), dosage (low or high) and the administration route (oral, parenteral or intrathecal). Renal impairment can trigger or exacerbate MTX toxicity. Acute oral low-dose MTX (LDMTX) overdoses seldom lead to toxicity due to the saturable maximal bioavailable dose, but toxicity risks increase with repeated low doses (>3 days), high-dose MTX (HDMTX) or intrathecal poisoning. Folinic acid shares MTX transporters in the gut and cells and bypasses the MTX-induced dihydrofolate reductase inhibition. The required folinic acid dosage differs for low-dose and high-dose MTX toxicities. Acute LDMTX poisoning rarely requires folinic acid, while chronic LDMTX poisoning needs low-dose folinic acid until cellular function is restored. In HDMTX toxicities, early intravenous folinic acid administration is recommended, with dose and duration being guided by MTX concentrations and clinical improvement. In intrathecal MTX poisoning, folinic acid should be administered intravenously. Glucarpidase, a recombinant bacterial enzyme, has a high affinity for MTX and folate analogues in the intravascular or intrathecal systems. It decreases serum MTX concentrations by 90%-95% within 15 min. Its primary indication is for intrathecal MTX poisoning. It is rarely indicated in HDMTX toxicity unless patients have renal injury. However, there is no literature evidence supporting its use in HDMTX poisoning. Its use is limited by its significant cost and lack of availability. Haemodialysis can be potentially useful for MTX removal in cases where glucarpidase is not available. Additionally, fluid hydration, renal support and urine alkalinization are important adjunctive therapies for managing MTX toxicities.

Not too old to rock: ESR and OSL dating reveal Quaternary activity of the Periadriatic Fault in the Alps

The Periadriatic Fault system (PAF) ranks among the largest post-collisional structures of the European Alps. Recent geodetic data suggest that a fraction of the Adria–Europe convergence is still being accommodated in the Eastern Alps. However, the historical seismicity records along the easternmost segment of the PAF are ambiguous, and instrumental records indicate that seismotectonic deformation is mostly concentrated in the adjacent Southern Alps and Dinarides. Both electron spin resonance (ESR) and optically stimulated luminescence (OSL) dating methods can be used to date coseismic slip (with a combined range covering a few decades to a couple of million years) in slowly deforming fault zones, such as the PAF. Since the saturation doses of the quartz ESR signals are larger than quartz and feldspar OSL, ESR enables establishing a maximum age of the last resetting event of the system, while OSL allows constraining their minimum age when the signal is in saturation. We collected fault gouge samples from three localities along the easternmost segment of the PAF. For ESR, we measured the signals from the Al center in quartz comparing the results from the single aliquot additive dose (SAAD) and single aliquot regenerative (SAR) dose protocols. For OSL, we recorded the infrared stimulated luminescence signal at 50°C (IR50) and post-infrared infrared-stimulated luminescence signal at 225 °C (pIRIR225). Our dating results indicate that the studied segment of the PAF system accommodated seismotectonic deformation during the Quaternary, with a maximum age for the last resetting event of the system ranging from 1075 ± 48 to 552 ± 26 ka (ESR SAR) and minimum ages in the range from 196 ± 12 to 281 ± 16 ka (saturation of pIRIR225). The obtained ages suggest that the studied segment of the PAF could be considered at least as a potentially active fault.Graphical abstract

Sex-Specific Mitochondrial Bioenergetics Differences in Heart and Kidney Cortex

Rationale: Mitochondrial bioenergetics of different tissues have been studied widely, but their sex-specific differences have not been systematically studied. The goal of this study was therefore to investigate the sex-specific differences in mitochondrial respiration (oxygen consumption rate; OCR) and membrane potential (Δψ) in the heart and kidney cortex and to explore underlying mechanisms responsible for any observed differences. Methods: Mitochondria were isolated from adult male and female Sprague-Dawley rat heart and kidney cortex and were used to study their bioenergetics responses. Three different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), and succinate in the presence of complex I inhibitor rotenone (SR) were used. This was followed by two different ADP addition protocols to determine how different substrates influence mitochondrial OCR and Δψ to ADP perturbations during oxidative phosphorylation (OxPhos) in male and female heart and kidney cortex. In one, a single saturated dose of ADP, while in the other, sequentially increasing doses of ADP were added to mitochondria in the presence of different substrates. Mitochondrial OCR was measured using a dual chamber Oroboros Oxygraph-2k Instrument and Δψ was measured using a spectrofluorometer and rhodamine-123 dye. From these measurements, OCR and Δψ alterations under different respiratory states and as functions of added ADP concentration were determined for different substrates and for both male and female rats. Results: Male and female mitochondria exhibited distinct respiratory patterns in the kidney cortex and heart with different substrates. In the kidney cortex, male mitochondria showed significantly higher OCR than female mitochondria when fueled with PM or SR. However, no significant sex differences in the OCR were observed when GM were used as substrates. In contrast, heart mitochondria exhibited negligible sex differences in the OCR for PM and SR, but significant differences emerged with GM, where female mitochondria displayed higher OCR than their male counterparts. Interestingly, sex-specific variations of Δψ in ADP-stimulated respiration were not apparent for either tissue or substrate when a single saturated dose of ADP was used. Instead, a trend similar to the OCR emerged when ADP was administered in gradually increasing concentrations. Conclusion: Heart and kidney cortex mitochondrial Δψ for males and females did not differ conclusively with the two ADP addition protocols. However, there were significant differences in the OCR of mitochondria from the heart and kidney cortex of males and females, depending on the respiratory substrate utilized. Males were found to have enhanced OCR in the kidney cortex, whereas females exhibited higher OCR in the heart. These observed sex-specific differences in the OCR could be attributed to differential regulation of mitochondrial metabolic pathways by respiratory substrates and sex hormones. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Altered Hepatic Mitochondrial Bioenergetics and Oxidant Emission with Ischemia and Reperfusion

Rationale: Ischemia-reperfusion injury (IRI) is inevitable in liver transplantation (LT) which can be mitigated by normothermic machine perfusion (NMP) before LT. The current NMP liver viability criteria for LT mainly rely on bile production and perfusate lactate levels, factors that can also be affected by external variables such as NMP conditions and perfusate additives. Incorporating mitochondrial bioenergetic data could improve the predictive value of current NMP viability criteria for LT. Thus, the goal of this study was to evaluate mitochondrial oxygen consumption rate (OCR), membrane potential (Δψ), and H2O2 oxidant emission in control livers and in livers with IRI and to explore underlying mechanisms responsible for any observed differences. Methods: Healthy control livers and livers exposed to 60 minutes warm ischemia time (WIT) through in situ clamping of the portal vein and hepatic artery followed by 60 minutes reperfusion (IRI) were harvested from adult male Sprague-Dawley rats. Mitochondria from control and IRI livers were isolated using established protocols and assessed for their bioenergetics responses. Three different substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), and succinate were used, followed by addition(s) of ADP and the uncoupler FCCP. Two different ADP addition protocols were designed to determine how different substrates influence mitochondrial OCR, Δψ, and H2O2 emission during oxidative phosphorylation (OxPhos) between control and IRI conditions. In one protocol, a single saturated dose of ADP addition, and in the other, sequentially increasing doses of ADP additions were made following substrate addition. The OCR and Δψ were measured simultaneously using a dual chamber Oroboros Oxygraph-2k Instrument coupled to a fluorometer using the TMRM dye. The H2O2 emission was measured spectrofluorometrically using the amplex red and horseradish peroxidase assay. Results: The measured data show that the kinetics and effciency of OxPhos defining mitochondrial OCR and Δψ responses in the liver are negatively affected by IRI, characterized by enhanced H2O2 emission, in a substrate-dependent manner. Interestingly, the respiratory rates are higher when GM and succinate are utilized as substrates, whereas the use of PM shows comparatively lower respiration in both conditions. Control mitochondria exhibited higher respiratory rates than IRI mitochondria irrespective of the substrate utilized. The ADP-induced state 3 OCR in IRI mitochondria was 50% lower than that in control mitochondria resulting in doubling the duration of state 3 OCR. Similar differences were observed in Δψ for both ADP addition protocols. Compromised mitochondrial bioenergetics during IRI was observed using both single and sequentially increasing doses of ADP and was concomitant with a higher rate of H2O2 emission. Conclusion: This study provided novel quantitative data demonstrating the substrate specific changes in mitochondrial bioenergetics in hepatic IRI, which can be used to improve the NMP viability criteria for LT. The obtained bioenergetics and H2O2 emission data indicate that, during IRI, mitochondrial function is highly affected which may critically impact energy dependent metabolism and lactate/pyruvate ratio. NIH R01-HL151587. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loop Diuretic Resistance in a Mouse Model of Cardiorenal Syndrome

Nearly 30% of patients with heart failure (HF) develop Cardiorenal syndrome type 2 (CRS2), characterized by renal injury and lower glomerular filtration rate (GFR) secondary to lower cardiac function. Patients with uncompensated heart failure develop edema and are usually treated with a loop diuretic, such as furosemide, that acts in thick ascending limb (TAL) to inhibit NKCC2. However, over 50% of HF patients develop resistance to loop diuretics. The molecular mechanisms underlying the development of diuretic resistance are not clear and may involve decreased NKCC2-mediated NaCl transport, enhanced distal tubule NaCl reabsorption, or decreased accessibility of the diuretic drug to the nephron lumen. Few studies have used the mouse as a model of cardiorenal syndrome after myocardial infarction (MI). We hypothesize that C57Bl6J mice would develop kidney damage several months after myocardial infarction (MI) and resistance to loop diuretics. Heart failure was induced in C57Bl6J mice through ligation of the left anterior descending coronary artery (LAD) and classified as MI group and compared to a sham surgery group (without ligation) as controls. Both groups were assessed for heart function by echocardiography and renal filtration function by measuring GFR (FITC inulin) in conscious mice. The response to diuretics was studied in metabolic cages, 7-8 months after MI. MI mice had a 50% reduction in ejection fraction (EF) 4 weeks after MI compared to sham group (MI: 29.94 ± 1.94, Sham: 67.02 ± 1.14, n= 8 each group, p&lt;0.05). Decreased EF continued to be lower 6 and 12 months after MI. Six months after surgery, the MI group showed decreased GFR compared to Sham (MI: 0.50 ± 0.06, Sham: 0.75±0.05 μl/min, n= 5 MI, n=7 sham, p&lt;0.05). Despite lower GFR, 24h-urinary albumin excretion was similar in both groups (n=8 each group). In metabolic cage studies, a saturating dose of the NKCC2 inhibitor bumetanide (20 mg/Kg), induced diuresis and natriuresis in sham mice (Delta UV: 1.96 ± 0.27 ml/4h, Delta UNa: 117.2 ± 11.02μmols/4h, UCl: 175.65 ± 13.01 μmols/4h, n= 6) but had a smaller effect in MI mice which exhibited an attenuated diuretic response (Delta UV: 0.88 ± 0.23 ml/4h, Delta UNa: 65.00 ± 4.53 μmols/4h, UCl: 102.89 ± 14.08 μmols/4h, n= 4, p&lt;0.05 vs sham), indicative of loop diuretic resistance. To study if bumetanide resistance is secondary to enhanced distal convoluted tubule (DCT) NaCl reabsorption, we measured the diuretic response to the NCC inhibitor hydrochlorothiazide (HCTZ). HCTZ (100 mg/Kg) induced diuresis and natriuresis in sham mice (Delta UNa: 75.24±11.18μmols/4h, UCl: 39.44±10.15 μmols/4h, n= 10) and the diuretic response was slightly higher in the MI group (Delta UNa: 129.54±11.59μmols/4h, UCl: 94.61±11.56 μmols/4h, n= 12, p&lt;0.05 vs sham). We conclude that chronic heart failure in C57Bl6J mice, decreases GFR and induces loop diuretic resistance. Loop diuretic resistance after MI may be secondary to enhanced DCT NaCl transport in mice. This model could be useful to study the mechanisms causing decreased renal function and loop diuretic resistance after MI and pursuing new therapeutic avenues. No funding was received by the authors for conducting this study. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Correlations between quartz OSL dose-response curve and TL glow curve characteristics, and their implications for equivalent dose estimation

Optically stimulated luminescence (OSL) dating of quartz using the single-aliquot regenerative-dose (SAR) procedure requires construction of dose-response curves (DRCs). The shapes of DRCs and their characteristic saturation doses (D0 values) are well-known to be highly variable between samples and among individual grains from the same sample, but the factors that control DRC characteristics are imperfectly understood. Here we report correlations between OSL DRC shapes and thermoluminescence (TL) glow curve characteristics for a sample of quartz from northern Australia. Individual grains were measured using the SAR procedure and one of two preheat temperatures (160 °C and 260 °C), and grouped according to similarities in their DRC shapes. Grains from each DRC group were then physically transferred onto a separate disc for TL measurement as a multi-grain aliquot. A strong correlation was observed between DRC group and laboratory-irradiated TL glow curve shapes, along with some differences between the two preheats. Irrespective of the selected preheat, earlier saturating DRC groups are associated with the most intense 110 °C TL signal, and the least intense 325 °C TL signal, which corresponds to the main OSL trap. Later saturating DRC groups generally show the opposite trend. There are also trends within other regions of the glow curve and DRC group, such as prominent TL responses at 140–160 °C, 220–240 °C and 390–410 °C. We assessed the effect on the accuracy of equivalent dose (De) estimation using an associated radiocarbon age as an independent cross-check of the OSL ages for the DRC groups. De over- and under-estimates were obtained for some DRC groups. Most notably, grains preheated to 160 °C displayed a pattern of De values which decreased with an increase in D0. We relate this to variable concentrations of charge at the ∼230 °C TL trap prior to regenerative dose OSL measurements. OSL ages for most DRC groups preheated to 260 °C are close to the radiocarbon age, as are those of later saturating DRC groups preheated to 160 °C; the latter DRC groups have the smallest TL signals at ∼230 °C relative to the TL peak at 325 °C. The results of this study suggest that correlations between single-grain De and D0 values can occur due to underlying differences in the TL characteristics of grains with different DRC shapes. In the absence of independent age control and prior to measuring multi-grain aliquots of quartz, we recommend that OSL dating practitioners screen their samples for any single-grain patterns of De as a function of DRC shape, to determine the optimal SAR measurement conditions and data-analysis procedures for De estimation.

Effect of alpha irradiation on the saturation characteristics of fine-grained quartz OSL

A negative relationship has been reported for the characteristic saturation dose (D0) of the quartz optically stimulated luminescence (OSL) signal and the quartz grain size, of which the mechanism is still not understood. Here, the natural coarse-grained quartz samples (63–90 μm, 90–125 μm) are artificially ground into fine grains and the effect of alpha irradiation on the D0 values of the ground fine grains is investigated. The ground fine grains (4–11, 11–45, 45–63 μm) have similar D0 values as the coarse grains. Laboratory alpha irradiation is administered on the artificial 4–11 μm quartz fraction. The D0 shows an increasing trend with the amount of alpha irradiation; meanwhile, the proportion of the fast OSL component decreases and the proportions of the medium and slow components increase with alpha irradiation. The higher D0 after alpha irradiation may be a result of the higher proportion of electron traps related to the medium component. However, the increasing trend of D0 stops when the alpha dose reaches a certain amount, and the degree of D0 increase induced by alpha irradiation cannot fully account for the much higher D0 values of the fine quartz grains in nature.

The new concept of MIEX® resin dose categories. swelling effect, reactor steady-state balance, and nom removal process control

The use of powdered adsorbents for water purification has many advantages and one major drawback – lack of regeneration due to difficulty in separating powdered particles. This weakness is attempted to be broken by powdered magnetic adsorbents, in particular magnetic ion exchange resins, used to remove natural organic matter (NOM) from water. In this water treatment process, NOM removal is controlled by the adsorbent content in the reactor (adsorbent dose) and the degree of its saturation. The control over the dose and saturation is done by mutual relations between the regenerated resin stream directed to the reactor and the saturated resin stream received from the reactor. An obstacle in balancing these streams is a variable volume of the adsorbent resulting from its varied swelling, depending on the features of the solution and saturation of the adsorbent. For this reason, it was proposed to distinguish new resin dose and content categories adequate to these changes, the use of which allows full control of both streams. Thus, the reactor feed stream was associated with relative fresh resin content (RRC) and relative fresh resin dose (RRD), which indicate the volume occupied by the regenerated adsorbent in the solution of water during purification. However, the stream received from the reactor was associated with saturated resin content (SRC) and saturated resin dose (SRD), which indicate the volume occupied by saturated adsorbent in the solution of water under treatment. In turn, these two categories of contents/doses are related to the swelling degree (hSR). Another role was assigned to the third dose category, which is absolute fresh resin dose (ARD), referring to the volume occupied by the regenerated adsorbent in the solution of demineralized water. Thanks to two key features with reference properties (demineralized water, regenerated adsorbent), ARD allows one to transfer laboratory results to practice and to compare the results of various research. The resin loss factor described by the ηLS indicator was also included in this structure.

Improving Intracellular Delivery of an Antibody-Drug Conjugate Targeting Carcinoembryonic Antigen Increases Efficacy at Clinically Relevant Doses In Vivo.

Solid tumor antibody-drug conjugates (ADC) have experienced more clinical success in the last 5 years than the previous 18-year span since the first ADC approval in 2000. While recent advances in protein engineering, linker design, and payload variations have played a role in this success, high expression and readily internalized targets have also been crucial to solid tumor therapy. However, these factors are also paradoxically connected to poor tissue penetration and lower efficacy. Previous work shows that potent ADCs can benefit from slower internalization under subsaturating doses to improve tissue penetration and increase tumor response. In contrast, faster internalization is predicted to increase efficacy under higher, tumor saturating doses. In this work, the intracellular delivery of SN-38 conjugated to an anti-carcinoembryonic antigen (anti-CEA) antibody (Ab) is increased by coadministering a noncompeting (cross-linking) anti-CEA Ab to improve efficacy in a colorectal carcinoma animal model. The SN-38 payload enables broad tumor saturation with clinically-tolerable doses, and under these saturating conditions, using a second CEA receptor cross-linking Ab yields faster internalization, which increases tumor killing efficacy. Our spheroid results show indirect bystander killing can also occur, but the more efficient direct cell killing from targeted intracellular payload release drives a greater tumor response. These results provide a strategy to increase therapeutic effectiveness with improved intracellular delivery under tumor saturating doses with the potential to expand the ADC target repertoire.

The effective threshold dose of etanercept in patients with methotrexate-resistant rheumatoid arthritis.

The therapy of rheumatoid arthritis (RA) was advanced by biological agents, yet costly. This study aims to identify the effective threshold dose of etanercept (ENT) and cost-effectiveness in methotrexate (MTX)-resistant RA in real world. Eligible patients had an inadequate response (DAS28-ESR > 3.2) to initial MTX monotherapy, and subsequently received etanercept. The effective cut-off value of cumulative dose was identified to maintain remission response (DAS28-ESR < 2.6) at month 24 by using restricted cubic splines. Remission rate, low disease activity (LDA) rate, glucocorticoid exposure, safety, and cost-effectiveness were compared between the saturated and non-saturated dose groups divided by the cut-off dose. Seventy-eight (14.2%) of 549 enrolled patients were eligible, and 72 patients completed follow-up. The 2-year cumulative cut-off dose that maintained remission response at 24months was 1975mg. And the recommended threshold dosing strategy of etanercept was twice weekly (BIW) for the first 6months, every week (QW) for the next 6months, and every 2weeks (Q2W) and every month (QM) for the second year. Greater net changes in DAS28-ESR score were observed in the ENT saturated dose group than in the non-saturated dose group (average change 0.569, 95%CI 0.236-0.901, p = 0.001). The proportion of patients achieving remission (27.8% vs 72.2%, p < 0.001) and LDA (58.3% vs 83.3%, p = 0.020) in the non-saturated group was both significantly lower than that in the saturated group at 24months. The incremental cost-effectiveness ratio of the saturated group referred to the non-saturated group was 5791.2 $/QALY. In refractory RA patients, the effective cumulative cut-off dose of etanercept for sustained remission at 24months was calculated as 1975mg, and receiving saturated dose was more effective and cost-effective than with non-saturated dose. Key Points • The effective cumulative cut-off dose of etanercept for sustained remission at 24months in RA patients is calculated as 1975mg. • Receiving saturated dose of etanercept is more effective and cost-effective than with non-saturated dose in refractory RA patients.

A phase 1 study of AGEN2373, a novel CD137 agonist antibody designed to avoid hepatoxicity, in patients with advanced solid tumors.

2524 Background: We present results from the completely enrolled monotherapy arm of the first-in-human dose escalation study of AGEN2373, a novel CD137 agonist antibody engineered to maximize efficacy by circumventing the dose-limiting hepatotoxicity reported for prior CD137 agonists (NCT04121676). AGEN2373 binds to a unique epitope of CD137 on effector T and NK cells in the tumor and tumor-draining lymph nodes, promoting co-engagement of activating Fcγ receptors. CD137 (4-1BB) is a tumor necrosis factor receptor superfamily protein and co-stimulatory receptor that promotes anti-tumor activity of adaptive and innate immune cells, making it an attractive target for immunotherapy. Methods: 46 patients (pts) received AGEN2373 IV every 2 weeks (Q2W), Q3W, or Q4W at doses between 0.03 and 10 mg/kg in a 3+3 design. Key endpoints included safety, tolerability, PK, preliminary efficacy, and PD markers. Imaging was Q8W. Results: Pts were enrolled between Oct 2019 and May 2022 with a median follow-up of 7.2 months. The median age was 64 (range 33-82). The most common tumor types were colorectal cancer, 13 pts (28%) and sarcoma, 8 (17%). Median prior therapies were 4 (range 1-14) including 48% with prior immunotherapy. There were no dose-limiting toxicities (DLTs) and no treatment-related hepatitis. 37% of pts had treatment-related AEs (TRAEs). TRAEs &gt;10% were limited to fatigue (17%) and nausea (15%). There were no G3, 4 or 5 TRAEs. Pharmacokinetic parameters including half-life, clearance, and volume of distribution were consistent with a human IgG1 Fc backbone. Induction of soluble CD137 was dose dependent with 2 mg/kg as the lowest saturating dose and minimum predicted efficacious dose. In 19 pts treated at 2 mg/kg or higher who had at least one post-baseline scan, the overall response rate (ORR) was 11% (n=2); 37% had SD (n=7) resulting in a disease control rate (DCR) (CR, PR or SD) of 47%. Notable responses include: a pt with vulvar SCC who had progressed rapidly on pembrolizumab and had a confirmed partial response (cPR) while remaining on AGEN2373 x 2 mg/kg q2w for ~40 wks, and a pt with ampullary carcinoma with 4 prior regimens had a cPR on AGEN2373 x 6 mg/kg q3w with complete resolution of the pancreatic lesion. A pt with CRPC had a 38% tumor reduction in liver target lesions (confirmed) on AGEN2373 x 10mg/kg q3w but was non-evaluable due to palliative radiation to bone metastases. Conclusions: AGEN2373 showed objective responses as monotherapy in heavily pretreated pts with solid tumors and was well-tolerated with no evidence of hepatotoxicity. This distinguishes AGEN2373 from previous CD137-targeted agents that reported dose-limiting hepatotoxicity. Combination therapy with botensilimab, a novel Fc-enhanced CTLA-4 antagonist, is being studied in pts with PD(L)-1 pretreated melanoma. Clinical trial information: NCT04121676 .

Kisspeptin Modulates Mitochondrial Metabolic State and Potentiates Glucose Stimulated Insulin Secretion in INS 832/3 Cells and Pseudo-Islets

Glucagon Like Peptide-1 (GLP-1) and Kisspeptin (KP) receptors are co-expressed on pancreatic β-cells, and activation of both receptors potentiates glucose-stimulated insulin secretion (GSIS). Due to their roles in enhancing GSIS, GLP-1 and KP represent targets for controlling blood glucose in patients with diabetes. Unlike GLP-1, KP has a low affinity for receptor activation (EC50 GLP-1 ~20 nM vs KP ~1 μM) making it difficult to use as a therapeutic agent. GLP-1 has been shown to potentiate insulin secretion via cAMP dependent pathways. Conversely, previous studies in isolated rat islets of Langerhans indicated that KP potentiation of GSIS was not dependent on cAMP and may be related to changes in beta cell metabolism (Schwetz TA, et al., Differential Stimulation of Insulin Secretion by GLP-1 and Kisspeptin-10. PLoS ONE 9: e113020, 2014). In the current studies, we evaluated KP signaling in comparison to GLP-1, in order to determine if there are unique components to target in the KP signaling pathway. The effects of GLP-1 and a 10 amino acid analog of KP (KP10) on insulin secretion and cell signaling were compared in insulinoma INS 832/3 cells. As previously described, KP had no effect on insulin secretion in the absence of glucose. In the presence of stimulatory glucose (15.5 mM), KP10 (10 μM) doubled insulin secretion (GSIS without KP10= 471.3 +/- 83.8, GSIS with KP10 = 865.0 +/- 23.8 pgm/min; +/- SEM) with an EC50 of ~5-10 μM. At saturating doses, KP10 (100 μm) and GLP-1 (100 nM) had similar effects on GSIS. Importantly, in combination, they had an additive effect (GLP-1 = 612.5 +/- 25.0, GLP-1 + KP-10 = 739.8 +/- 70.0 pgm/min) consistent with activation of independent downstream signaling pathways. GLP-1 increased cAMP production (165.0 +/- 17.4% over control), whereas KP-10 had no effect. Neither GLP-1 nor KP-10 altered intracellular Ca2+ levels at basal or activating glucose concentrations. Conversely, the addition of KP-10 enhanced glucose-stimulated oxygen consumption (% change over baseline O_2 consumption= 221.2 +/- 44.2%) from INS 832/3 pseudo-islets (Hart et al. Insulinoma-derived pseudo-islets for diabetes research. Am. J. Physio. 321, C247-C256, 2021) which was correlated with a sustained increase in mitochondrial NADH fluorescence. Our findings indicate that KP modulation of mitochondrial metabolic state is related to its ability to potentiate GSIS. Moreover, the KP induced potentiation is synergistic with that promoted by GLP-1 indicating the potential for targeting the KP activated metabolic pathway to further enhance GLP-1 therapeutic effectiveness. Funded by the Juvenile Diabetes Research Foundation (2-SRA-2018-685-S-B), and Procyon Technologies LLC This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.