Efflux Rate Research Articles

The Effects of Fever on Macrophage Metabolism and Function in Young and Aged Cells

Abstract Fever is an adaptive immune response to infections. With aging comes a rise in background inflammation that does not protect against infection. Here, we investigate the metabolism and function of macrophages at fever temperatures in a mouse cell line, in young and aged peritoneal resident macrophages, and bone marrow derived macrophages. Fever temperatures generally enhance macrophage functions although the effects are complex and dynamic. We show in the RAW264.7 murine cell line, that a raise in the baseline body temperature of 37°C to a fever temperature of 39°C increased the speed in which macrophages shift from mitochondrial oxidative phosphorylation to glycolysis. Driving this metabolic reprogramming, is the production of nitric oxide. There is also an increase in global protein synthesis and an increase in the mRNA for iNOS, the enzyme that produces NO and inflammatory cytokine production. The metabolic shift was measured via the oxygen consumption and the proton efflux rate over time. Cells were stimulated with lipopolysaccharide and/or interferon-gamma (IFNg). Our data also shows that tissue resident PRMs from aged mice exhibit a much stronger response to fever than those of young mice, while bone marrow derived microphages (BMDM) appeared to show a similar response to that of RAW264.7 cell line. However, with the addition of a cyclooxygenase2 inhibitor to block the production of prostaglandin E2, BMDMs of aged mice show an exaggerated increase in their response to LPS+IFNg activation. This work uncovers differences in fever responses between young and aged individuals that may explain the overall outcomes of the aged to infection. Arkansas Immunology Foundation

Effects of hypoxia on the expression and function of P-gp in Caco-2 cells.

Hypoxia can alter the oral bioavailability of drugs, including various substrates (drugs) of P-glycoprotein (P-gp), suggesting that hypoxia may affect the function of P-gp in intestinal epithelial cells. Currently, Caco-2 monolayer model is the classic model for studying the function of intestinal epithelial P-gp. This study combines the Caco-2 monolayer model with hypoxia to investigate the effects of hypoxia on the expression and function of P-gp in Caco-2 cells, which helps to elucidate the mechanism of changes in drug transport on intestinal epithelial cells in high-altitude hypoxia environment. Normally cultured Caco-2 cells were cultured in 1% oxygen concentration for 24, 48, and 72 h, respectively. After the extraction of the membrane proteins, the levels of P-gp were measured by Western blotting. The hypoxia time, with the most significant change of P-gp expression, was selected as the subsequent study condition. After culturing Caco-2 cells in transwell cells for 21 days and establishing a Caco-2 monolayer model, they were divided into a normoxic control group and a hypoxic group. The normoxic control group was continuously cultured in normal condition for 72 h, while the hypoxic group was incubated for 72 h in 1% oxygen concentration. The integrity and polarability of Caco-2 cells monolayer were evaluated by transepithelial electrical resistance (TEER), apparent permeability (Papp) of lucifer yellow, the activity of alkaline phosphatase (AKP), and microvilli morphology and tight junction structure under transmission electron microscope. Then, the Papp of rhodamine 123 (Rh123), a kind of P-gp specific substrate, was detected and the efflux rate was calculated. The Caco-2 cell monolayer, culturing at plastic flasks, was incubated for 72 h in 1% oxygen concentration, the expression level of P-gp was detected. P-gp was decreased in Caco-2 cells with 1% oxygen concentration, especially the duration of 72 h (P<0.01). In hypoxic group, the TEER of monolayer was more than 400 Ω·cm2, the Papp of lucifer yellow was less than 5×10-7 cm/s, and the ratio of AKP activity between apical side and basal side was greater than 3. The establishment of Caco-2 monolayer model was successful, and hypoxia treatment did not affect the integrity and polarization state of the model. Compared with the normoxic control group, the efflux rate of Rh123 was significantly reduced in Caco-2 cell monolayer of the hypoxic group (P<0.01). Hypoxia reduced the expression of P-gp in Caco-2 cell monolayer (P<0.01). Hypoxia inhibits P-gp function in Caco-2 cells, which may be related to the decreased P-gp level.

Real-time assessment of neutrophil metabolism and oxidative burst using extracellular flux analysis.

Neutrophil responses are critical during inflammatory and infective events, and neutrophil dysregulation has been associated with poor patient outcomes. Immunometabolism is a rapidly growing field that has provided insights into cellular functions in health and disease. Neutrophils are highly glycolytic when activated, with inhibition of glycolysis associated with functional deficits. There is currently very limited data available assessing metabolism in neutrophils. Extracellular flux (XF) analysis assesses real time oxygen consumption and the rate of proton efflux in cells. This technology allows for the automated addition of inhibitors and stimulants to visualise the effect on metabolism. We describe optimised protocols for an XFe96 XF Analyser to (i) probe glycolysis in neutrophils under basal and stimulated conditions, (ii) probe phorbol 12-myristate 13-acetate induced oxidative burst, and (iii) highlight challenges of using XF technology to examine mitochondrial function in neutrophils. We provide an overview of how to analyze XF data and identify pitfalls of probing neutrophil metabolism with XF analysis. In summary we describe robust methods for assessing glycolysis and oxidative burst in human neutrophils and discuss the challenges around using this technique to assess mitochondrial respiration. XF technology is a powerful platform with a user-friendly interface and data analysis templates, however we suggest caution when assessing neutrophil mitochondrial respiration.

Mitochondrial calcium uniporter b deletion inhibits platelet function and reduces susceptibility to arterial thrombosis

BackgroundMitochondrial calcium uniporter b (MCUb) is a negative regulator of the mitochondrial calcium uniporter (MCU) and is known to limit mitochondrial calcium ion (Ca2+) uptake. The role of MCUb in platelet function remains unclear. ObjectivesUtilizing MCUb−/− mice, we examined the role of MCUb in regulating platelet function and thrombosis. MethodsPlatelet activation was evaluated in agonist-induced standardized in vitro assays. Susceptibility to arterial thrombosis was evaluated in FeCl3 injury–induced carotid artery and laser injury–induced mesenteric artery thrombosis models. The glycolytic proton efflux rate and oxygen consumption rate were measured to evaluate aerobic glycolysis. ResultsUpon stimulation, MCUb−/− platelets exhibited reduced cytoplasmic Ca2+ responses concomitant with increased mitochondrial Ca2+ uptake. MCUb−/− platelets displayed reduced agonist-induced platelet aggregation and spreading on fibrinogen and decreased α and dense-granule secretion and clot retraction. MCUb−/− mice were less susceptible to arterial thrombosis in FeCl3 injury–induced carotid and laser injury–induced mesenteric thrombosis models with unaltered tail bleeding time. In adoptive transfer experiments, thrombocytopenic hIL-4Rα/GPIbα-transgenic mice transfused with MCUb−/− platelets were less susceptible to FeCl3 injury-induced carotid thrombosis compared with hIL-4Rα/GPIbα-Tg mice transfused with wild type platelets, suggesting a platelet-specific role of MCUb in thrombosis. MCUb−/− stimulated platelets exhibited reduced glucose uptake, decreased glycolytic rate, and lowered pyruvate dehydrogenase phosphorylation, suggesting that mitochondrial Ca2+ mediates bioenergetic changes in platelets. ConclusionOur findings suggest that mitochondrial Ca2+ signaling and glucose oxidation are functionally linked in activated platelets and reveal a novel role of MCUb in platelet activation and arterial thrombosis.

The Consistence of Dynamic Contrast-Enhanced MRI and Filter-Exchange Imaging in Measuring Water Exchange Across the Blood-Brain Barrier in High-Grade Glioma.

Water exchange across blood-brain barrier (BBB) (WEXBBB ) is an emerging biomarker of BBB dysfunction with potential applications in many brain diseases. Several MRI methods have been proposed to measure WEXBBB , but evidence remains scarce whether different methods can produce comparable WEXBBB . To explore whether dynamic contrast-enhanced (DCE)-MRI and vascular water exchange imaging (VEXI) could produce comparable WEXBBB in high-grade glioma (HGG) patients. Prospective cross-sectional. 13 HGG patients (58.4 ± 9.4 years, 9 females, 4 WHO III and 9 WHO IV). A 3 T, spoiled gradient-recalled-echo DCE-MRI and VEXI containing two pulsed-gradient spin-echo blocks separated by a mixing block. The enhanced tumor and contralateral normal-appearing white matter (cNAWM) volume-of-interests (VOIs) were drew by two neuroradiologists. And whole-brain NAWM and normal-appearing gray matter (NAGM) without tumor-affected regions were segmented by automated segmentation algorithm in FSL. Student's t-test was used to evaluate parameters difference between cNAWM and tumor, NAGM and NAWM, respectively. The correlation between vascular water efflux rate constant (kbo ) from DCE-MRI and apparent exchange rate across BBB (AXRBBB ) from VEXI was evaluated by Pearson correlation. P < 0.05 was considered statistically significant. Compared with cNAWM, both kbo and AXRBBB were significantly reduced in tumor (kbo = 3.50 ± 1.18 sec-1 vs. 1.03 ± 0.75 sec-1 ; AXRBBB = 3.54 ± 1.11 sec-1 vs. 1.94 ± 1.04 sec-1 ). Both kbo and AXRBBB showed significantly higher values in NAWM than NAGM (kbo = 3.50 ± 0.59 sec-1 vs. 2.10 ± 0.56 sec-1 ; AXRBBB = 3.35 ± 0.77 sec-1 vs. 2.07 ± 0.52 sec-1 ). The VOI-averaged kbo and AXRBBB were also linearly correlated in tumor, NAWM, and NAGM (r = 0.59). DCE-MRI and VEXI showed comparable and correlated WEXBBB in HGG patients, suggesting that the consistence and reliability of these two MRI methods in measuring WEXBBB . 2. Stage 1.

Abstract 408: Novel growth hormone receptor antagonist with discrete PEGylations markedly improve response to therapy in human pancreatic and bile duct cancers in mice

Abstract Here we present a new and improved GHR antagonist candidate (compound G) with novel discrete PEGylations exhibiting marked efficacy in synergizing anti-cancer therapies. The American Cancer Society estimates the 5-year survival rates of pancreatic adenocarcinoma (PAAD) and bile duct cancer/cholangiocarcinoma (CHOL) to be a dismal 10% in 2022, indicating an urgent need for new therapeutic approaches for both of these devastating diseases. Several human cancers, including PAAD and CHOL express two validated oncological targets - the growth hormone receptor (GHR) and the insulin-like growth factor 1 receptor (IGF1R), as well as their cognate ligands growth hormone (GH) and insulin-like growth factor 1 (IGF1), respectively. Our laboratory had pioneered the discovery of pharmacologic antagonists for the growth hormone receptor (GHR), including the only approved and prescribed GHR antagonist, pegvisomant (Pfizer), prescribed for acromegaly (a condition of excess GH due to a hypersecreting pituitary adenoma). GHR antagonists not only inhibit the effects of GH but also robustly suppress the production of IGF1. The novel GHR antagonist compound G, that we present here, has a greater binding affinity than Pegvisomant to the GHR. And unlike Pegvisomant, compound G binds to the prolactin receptor as well as the GHR. A large body of research by us and others have earlier shown that the covert actions of tumoral GHR and IGF1R activation collectively drive mechanisms of therapy resistance including, but not limited to, active multidrug efflux, activation of epithelial-to-mesenchymal transition (EMT), resistance to apoptosis, increased fibrosis, increase of cancer stem cells, tumor angiogenesis and senescence. Our novel discrete PEGylated GHR antagonist, compound G, alone exhibits significant suppression of cancer cell viability, invasive potential, and drug efflux rate in PAAD and CHOL cell cultures. In Nude mice with human PAAD or CHOL xenografts, GHR inhibition by compound-G markedly sensitized the tumors towards both low and high doses of gemcitabine (for PAAD) or gemcitabine-cisplatin (for CHOL) treatments. Compound-G in combination with chemotherapy resulted in markedly reduced tumor sizes in all CHOL and PAAD mice and almost complete tumor clearance in 40% of the mice with PAAD. In the GHR antagonist treated tumors, significantly lower expression of ABC transporters (Abcb1, Abcg5, Abcc2, Abcg2), EMT mediators (Cdh2, Zeb1, Snai2, Epas1) and apoptosis marker (Bcl2) genes compared to control were observed - accounting for the molecular mechanisms underlying the observed effects. Collectively, our data provide a robust pre-clinical validation for treatment regimens combining GHR antagonist and chemotherapy leading to highly efficacious tumor clearance. Citation Format: Reetobrata Basu, John J. Kopchick. Novel growth hormone receptor antagonist with discrete PEGylations markedly improve response to therapy in human pancreatic and bile duct cancers in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 408.

Finite volume simulation of calcium distribution in a cholangiocyte cell

Cholangiocytes are the cells of the liver having a major role in the conditioning of bile used in digestion. Other functions of cholangiocytes are in apoptosis and bicarbonate secretion. The Calcium in the intracellular environment of various cells including cholangiocytes regulates a large number of functions. This regulating mechanism in cholangiocytes has been poorly understood to date. In order to analyze the calcium regulation in cholangiocyte cells, a mathematical model for a one-dimensional steady-state case is constructed in this study. This involves a non-linear reaction-diffusion equation with appropriate boundary conditions. The influx from IP$_{3}$ receptor, ryanodine receptor (RYR), and plasma membrane as well as the efflux of calcium from SERCA pump and plasma membrane have been employed in the model. The finite volume method and Newton-Raphson method have been used to solve the problem. Numerical findings have been used to examine the effects of parameters like diffusion coefficient, rate of SERCA pump efflux, buffer, and influx from plasma membrane on calcium concentration in cholangiocyte cells. The information generated from the model can be useful for understanding the mechanism of cholestatic disorders which can be further useful in the diagnosis and treatment of these disorders.

Reduced P-glycoprotein recognition of a radioiodine-labeled phosphonium cation by stilbenylation for mitochondrial membrane potential based-imaging

The accumulation of radiolabeled phosphonium cations in cells is dependent on the mitochondrial membrane potential (MMP). However, the efflux of these cations from tumor cells via P-glycoprotein (P-gp) limits their clinical application as MMP-based imaging tracers. In the present study, we designed (E)-diethyl-4-[125I]iodobenzyl-4-stilbenylphosphonium ([125I]IDESP), which contains a stilbenyl substituent, as a P-gp inhibitor to reduce P-gp recognition, and evaluated its biological properties in comparison with 4-[125I]iodobenzyl dipropylphenylphosphonium ([125I]IDPP). The in vitro cellular uptake ratio of [125I]IDESP in P-gp expressing K562/Vin cells to the parent (P-gp negative) K562 cells was significantly higher than that of [125I]IDPP. The efflux rate of [125I]IDESP was not significantly different between K562 and K562/Vin, while [125I]IDPP was rapidly effluxed from K562/Vin compared with K562, and the efflux of [125I]IDPP from K562/Vin was inhibited by the P-gp inhibitor, cyclosporine A. The cellular uptake of [125I]IDESP was well correlated with the MMP levels. These results suggested that [125I]IDESP was accumulated in cells depending on the MMP levels, without being effluxed via P-gp, while [125I]IDPP was rapidly effluxed from the cells via P-gp. Despite having suitable in vitro properties for MMP-based imaging, [125I]IDESP showed rapid blood clearance and lower tumor accumulation than [125I]IDPP. Improvement in the normal tissue distribution of [125I]IDESP is required to develop an agent for use in in vivo MMP-based tumor imaging.

Simultaneous estimation of the cellular water exchange rate, intracellular volume fraction, and longitudinal relaxation rate in cancer cells.

The purpose of the current study was to investigate the feasibility of simultaneously estimating the cellular water efflux rate ( ), intracellular longitudinal relaxation rate ( ), and intracellular volume fraction ( ) of a cell suspension using multiple samples with different gadolinium concentrations. Numerical simulation studies were conducted to assess the uncertainty in the estimation of , , and from saturation recovery data using single (SC) or multiple concentrations (MC) of gadolinium-based contrast agent (GBCA). In vitro experiments with 4 T1 murine breast cancer and SCCVII squamous cell cancer models were conducted at 11 T to compare parameter estimation using the SC protocol with that using the MC protocol. The cell lines were challenged with a Na+ /K+ -ATPase inhibitor, digoxin, to assess the treatment response in terms of , , and . Data analysis was conducted using the two-compartment exchange model for parameter estimation. The simulation study data demonstrate that the MC method, compared with the SC method, reduces the uncertainty of the estimated by decreasing the interquartile ranges from 27.3% ± 3.7% to 18.8% ± 5.1% and the median differences from ground truth from 15.0% ± 6.3% to 7.2% ± 4.2%, while estimating and simultaneously. In the cell studies, the MC method demonstrated reduced uncertainty in overall parameter estimation compared with the SC approach. MC method-measured parameter changes in cells treated with digoxin increased by 11.7% (p = 0.218) and by 5.9% (p = 0.234) for 4 T1 cells, respectively, and decreased by 28.8% (p = 0.226) and by 1.6% (p = 0.751) for SCCVII cells, respectively. did not change noticeably by the treatment. The results of this study substantiate the feasibility of using saturation recovery data of multiple samples with different GBCA concentrations for simultaneous measurement of the cellular water efflux rate, intracellular volume fraction, and intracellular longitudinal relaxation rate in cancer cells.

Comparison of quantitative parameters and radiomic features as inputs into machine learning models to predict the Gleason score of prostate cancer lesions

IntroductionThe classification of prostate cancer (PCa) lesions using Prostate Imaging Reporting and Data System (PI-RADS) suffers from poor inter-reader agreement. This study compared quantitative parameters or radiomic features from multiparametric magnetic resonance imaging (mpMRI) or positron emission tomography (PET), as inputs into machine learning (ML) to predict the Gleason scores (GS) of detected lesions for improved PCa lesion classification. Methods20 biopsy-confirmed PCa subjects underwent imaging before radical prostatectomy. A pathologist assigned GS from tumour tissue. Two radiologists and one nuclear medicine physician delineated the lesions on the mpMR and PET images, yielding 45 lesion inputs. Seven quantitative parameters were extracted from the lesions, namely T2-weighted (T2w) image intensity, apparent diffusion coefficient (ADC), transfer constant (KTRANS), efflux rate constant (Kep), and extracellular volume ratio (Ve) from mpMR images, and SUVmean and SUVmax from PET images. Eight radiomic features were selected out of 109 radiomic features from T2w, ADC and PET images. Quantitative parameters or radiomic features, with risk factors of age, prostate-specific antigen (PSA), PSA density and volume, of 45 different lesion inputs were input in different combinations into four ML models - Decision Tree (DT), Support Vector Machine (SVM), k-Nearest-Neighbour (kNN), Ensembles model (EM). ResultsSUVmax yielded the highest accuracy in discriminating detected lesions. Among the 4 ML models, kNN yielded the highest accuracies of 0.929 using either quantitative parameters or radiomic features with risk factors as input. ConclusionsML models' performance is dependent on the input combinations and risk factors further improve ML classification accuracy.

Soil Nutrient Distribution and Preferential Flow Transport Patterns in Robinia Pseudoacacia Communities of Degraded Wetlands

The Yellow River Delta wetlands in the Yellow River Delta National Nature Reserve are facing serious degradation due to water scarcity and soil salinization. This study aims to investigate the mechanism of wetland degradation by analyzing the small-scale distribution of soil nutrients and preferential flow transport patterns in the Robinia Pseudoacacia community, which is a typical vegetation community in degraded wetlands. Soil physical and chemical properties based on field staining experiments were analyzed, and indoor solute penetration experiments were conducted to investigate the distribution of soil nutrients and hydrological characteristics. The results showed that the contents of soil organic carbon, organic matter, total nitrogen, and available phosphorus decreased with increasing soil depth, with higher contents in the preferential flow area than in the matrix flow area. Soil organic carbon, organic matter, total nitrogen, total phosphorus, and available phosphorus showed positive correlations with each other, while soil pH and conductivity exhibited negative correlations with the above nutrients. The efflux rate of the Acacia community exhibited a gradual decline as soil depth increased, and the relative concentration of the solution exhibited a non-monotonic pattern of decrease, increase, and subsequent decrease with increasing soil depth. The findings could provide valuable guidance for the restoration and management of degraded wetlands in the Yellow River Delta.

Differences in CO2 Emissions on a Bare-Drained Peat Area in Sarawak, Malaysia, Based on Different Measurement Techniques

The drainage and cultivation of peatlands will lead to subsidence and mineralisation of organic matter, increasing carbon (C) loss as more CO2 is emitted. There is little information about carbon emissions from bare peat soil. A study was undertaken to measure the CO2 emissions from a logged-over peat swamp area that was purposely vegetation-free. We aimed to report CO2 emissions from a bare, drained peatland developed for an oil palm plantation. For 12 months, we used eddy covariance (EC), closed chambers, and soil subsidence measurements to derive CO2 emissions from a logged-over peat swamp area. Significant variations in the estimated soil CO2 efflux were observed in the three tested measurement techniques. The average CO2 flux rate measured by the EC technique was 4.94 ± 0.12 µmol CO2 m−2 s−1 (or 68.55 tonnes CO2 ha−1 year−1). Meanwhile, the soil CO2 efflux rate measured by the closed chamber technique was 4.19 ± 0.22 µmol CO2 m−2 s−1 (or 58.14 tonnes CO2 ha−1 year−1). Subsidence amounted to 1.9 cm year−1, corresponding to 36.12 tonnes CO2 ha−1 year−1. The estimation of the C loss was found to be highest by the EC technique, lower by the soil chamber technique, and lowest by the peat subsidence rate technique. The higher CO2 emission rate observed in the EC technique could be attributed to soil microbial respiration and decomposing woody residues in the nearby stacking rows due to the large EC footprint. It could also be affected by CO2 advection from oil palms adjacent to the study site. Despite the large differences in the CO2 emission rates by the different techniques, this study provides valuable information on the soil heterotrophic respiration of deep peat in Sarawak. Carbon emissions from a bare peat area cover only a fraction of the soil CO2 respiration component, i.e., the soil heterotrophic respiration. Further investigations are needed to determine the CO2 emissions by soil microbial activities and plant roots from other peat areas in Sarawak.

Pantothenic Acid Uptake and Metabolism by Red Blood Cells of Rats

Red blood cells (RBC) of rats and humans contain pantothenic acid (PA) derivatives, generally referred to as bound PA, which were never characterized or quantitated. This study was undertaken to define those factors that determine the rates of uptake and efflux of PA, and possibly PA derivatives, in RBC. Uptake of PA by RBC was studied by incubating RBC with different concentrations of PA (0.34–34.0 µmol/L), each containing [14C]PA (0.34 µmol/L) at 37°C for 3–360 min. The effects of Na+, energy and pH on the uptake of PA in RBC were determined in experiments varying media concentrations of Na+ (0–152 mmol/L), glucose (0 and 10 mmol/L) or pH (7.2–7.6). The release of PA from RBC was studied by incubating RBC containing [14C]PA (0.10–10.4 nmol/107 RBC, final concentration) in fresh media at 37°C for 10–240 min. Uptake and efflux of PA were found to be nonsaturable; uptake was not affected by Na+, energy or pH. Quantitative and qualitative determination of PA derivatives was by enzymatic hydrolysis of lysed RBC, followed by analysis of PA by RIA and paper chromatography. The RBC were found to contain PA, 4′-phosphopantothenic acid and pantetheine. We conclude that PA diffuses passively into and out of RBC and that RBC contain only PA, 4′-phosphopantothenic acid, pantetheine and no CoA.

Evaluation of cellular water exchange in a mouse glioma model using dynamic contrast-enhanced MRI with two flip angles

This manuscript aims to evaluate the robustness and significance of the water efflux rate constant (kio) parameter estimated using the two flip-angle Dynamic Contrast-Enhanced (DCE) MRI approach with a murine glioblastoma model at 7 T. The repeatability of contrast kinetic parameters and kio measurement was assessed by a test–retest experiment (n = 7). The association of kio with cellular metabolism was investigated through DCE-MRI and FDG-PET experiments (n = 7). Tumor response to a combination therapy of bevacizumab and fluorouracil (5FU) monitored by contrast kinetic parameters and kio (n = 10). Test–retest experiments demonstrated compartmental volume fractions (ve and vp) remained consistent between scans while the vascular functional measures (Fp and PS) and kio showed noticeable changes, most likely due to physiological changes of the tumor. The standardized uptake value (SUV) of tumors has a linear correlation with kio (R2 = 0.547), a positive correlation with Fp (R2 = 0.504), and weak correlations with ve (R2 = 0.150), vp (R2 = 0.077), PS (R2 = 0.117), Ktrans (R2 = 0.088) and whole tumor volume (R2 = 0.174). In the treatment study, the kio of the treated group was significantly lower than the control group one day after bevacizumab treatment and decreased significantly after 5FU treatment compared to the baseline. This study results support the feasibility of measuring kio using the two flip-angle DCE-MRI approach in cancer imaging.

Impact of Prenatal Exposure to Maternal Diabetes and High-Fat Diet on Postnatal Myocardial Ketone Body Metabolism in Rats.

Infants exposed to diabetic pregnancy are at higher risk of cardiomyopathy at birth and early onset cardiovascular disease (CVD) as adults. Using a rat model, we showed how fetal exposure to maternal diabetes causes cardiac disease through fuel-mediated mitochondrial dysfunction, and that a maternal high-fat diet (HFD) exaggerates the risk. Diabetic pregnancy increases circulating maternal ketones which can have a cardioprotective effect, but whether diabetes-mediated complex I dysfunction impairs myocardial metabolism of ketones postnatally remains unknown. The objective of this study was to determine whether neonatal rat cardiomyocytes (NRCM) from diabetes- and HFD-exposed offspring oxidize ketones as an alternative fuel source. To test our hypothesis, we developed a novel ketone stress test (KST) using extracellular flux analyses to compare real-time ß-hydroxybutyrate (βHOB) metabolism in NRCM. We also compared myocardial expression of genes responsible for ketone and lipid metabolism. NRCM had a dose-dependent increase in respiration with increasing concentrations of βHOB, demonstrating that both control and combination exposed NRCM can metabolize ketones postnatally. Ketone treatment also enhanced the glycolytic capacity of combination exposed NRCM with a dose-dependent increase in the glucose-mediated proton efflux rate (PER) from CO2 (aerobic glycolysis) alongside a decreased reliance on PER from lactate (anaerobic glycolysis). Expression of genes responsible for ketone body metabolism was higher in combination exposed males. Findings demonstrate that myocardial ketone body metabolism is preserved and improves fuel flexibility in NRCM from diabetes- and HFD-exposed offspring, which suggests that ketones might serve a protective role in neonatal cardiomyopathy due to maternal diabetes.

Hypothermia impairs glymphatic drainage in traumatic brain injury as assessed by dynamic contrast-enhanced MRI with intrathecal contrast.

The impact of hypothermia on the impaired drainage function of the glymphatic system in traumatic brain injury (TBI) is not understood. Male Sprague-Dawley rats undergoing controlled cortical impact injury (CCI) were subjected to hypothermia or normothermia treatment. The rats undergoing sham surgery without CCI were used as the control. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with intrathecal administration of low- and high-molecular-weight contrast agents (Gd-DTPA and hyaluronic acid conjugated Gd-DTPA) was performed after TBI and head temperature management. The semiquantitative kinetic parameters characterizing the contrast infusion and cleanout in the brain, including influx rate, efflux rate, and clearance duration, were calculated from the average time-intensity curves. The qualitative and semiquantitative results of DCE-MRI obtained from all examined perivascular spaces and most brain tissue regions showed a significantly increased influx rate and efflux rate and decreased clearance duration among all TBI animals, demonstrating a significant impairment of glymphatic drainage function. This glymphatic drainage dysfunction was exacerbated when additional hypothermia was applied. The early glymphatic drainage reduction induced by TBI and aggravated by hypothermia was linearly related to the late increased deposition of p-tau and beta-amyloid revealed by histopathologic and biochemical analysis and cognitive impairment assessed by the Barnes maze and novel object recognition test. The glymphatic system dysfunction induced by hypothermia may be an indirect alternative pathophysiological factor indicating injury to the brain after TBI. Longitudinal studies and targeted glymphatic dysfunction management are recommended to explore the potential effect of hypothermia in TBI.

Single-cell clinical chemistry: Exploring glucose uptake kinetics and HbA1c levels in individual red blood cells.

Single-molecule techniques have been a primary focus of many advances in biophysics over the last decades. However, the fact that many related technologies such as microfluidics, confocal microscopy, etc. are becoming standard laboratory tools makes it possible to pursue other goals such as single cell measurements. If single molecules are the building blocks of matter, single cells are the building blocks of biology, and the individual detection thereof offers similar benefits. Currently, most blood work is cuvette-based, averaging tens of millions of blood cells to obtain a single ensemble value. Measuring single blood cells yields a distribution of values, which provides more information about a given patient as well as how that patient may differ from the general populace. All of which is important in an era of big data which increasingly inclines medicine towards a more personalized approach. Our work focuses on the relationship between glucose, red blood cells (RBCs) and glycated hemoglobin, as quantifying the interplay of these parameters is important for a better understanding of diabetes. Using a microfluidic perfusion system allows the placement of RBCs and control over the solutions to which they are exposed. Using confocal microscopy and image analysis, we are able to observe the kinetics of fluorescently-labeled glucose passing into and out of individual RBCs and extract the relevant information, such as glucose influx and efflux rates, intracellular vs blood glucose levels, and more. Overall, we seek to quantify the relationship between bloodstream and intracellular glucose levels to inform a more personalized, and perhaps improved, diagnostic strategy for diabetes. We are also currently developing methods for quantifying %HbA1c at the single cell level utilizing Raman microscopy and mass spectrometry.

PCSK9-D374Y Suppresses Hepatocyte Migration through Downregulating Free Cholesterol Efflux Rate and Activity of Extracellular Signal-Regulated Kinase.

Proprotein convertase subtilisin/kexin type 9 can mediate the intracellular lysosomal degradation of the low-density lipoprotein receptor protein in hepatocytes and decrease the liver's ability to scavenge low-density lipoprotein cholesterol from circulation, resulting in high levels of cholesterol in the circulatory system. Current studies have primarily focused on the relationship between PCSK9 and blood lipid metabolism; however, the biological function of PCSK9 in hepatocytes is rarely addressed. In this study, we evaluate its effects in the human hepatoma carcinoma cell line HepG2, including proliferation, migration, and free cholesterol transport. PCSK9-D374Y is a gain-of-function mutation that does not affect proliferation but significantly suppresses the migration and cholesterol efflux capacity of these cells. The suppression of the transmembrane outflow of intracellular-free cholesterol regulates small G proteins and the suppression of extracellular signal-regulated kinase. In summary, PCSK9-D374Y affects hepatocyte features, including their migration and free cholesterol transport capabilities.

Noninvasive micro-test technology: monitoring ion and molecular flow in plants.

Noninvasive micro-test technology (NMT) is a novel technology to investigate the influx and/or efflux rate, direction, and concentration of selected ions and molecules in a microenvironment surrounding living cells or intact organisms. NMT measures and records the spatial electric potential differences in liquid media-surrounded living cells, tissues, and small organisms. Measurements are taken by a vibrating selective and/or specific flux sensor, combined with computerized algorithm to reveal ionic or molecular exchanges in the microenvironment with high sensitivity.

Characterization of Capecitabine Loaded Stealth Liposomes by Nuclear Magnetic Resonance

Background Liposomes are lipid aggregates with a careful choice of lipid composition and process parameters. Liposomes are formulated by optimization of process parameters of liposomes to achieve desired bioavailability and to improve stability. Objectives The main objective of the present research was to formulate colon targeting capecitabine loaded stealth liposomes characterized by using Nuclear magnetic resonance NMR techniques to determine the fractions of the drug capecitabine CAP encapsulated in the liposomal suspension.Methods Liposome properties were evaluated by NMR. 31P-NMR 31Phosphorus NMR in combination with the use of chemical shift reagents were used for the identification of lamellarity of liposomes which can have a strong effect on both encapsulation efficiency and the efflux rate of encapsulated active compounds.Results The results suggested that capecitabine remained intact during the loading process and the liposomal drug was present in a soluble form. Conclusion 31P-NMR was found to be an effective method to study the lamellarity of liposomes and to determine the distributed fractions of encapsulated capecitabine.