Efflux Rate Research Articles

BIOM-14. TRANSMEMBRANE WATER EFFLUX RATE (KIO) IS AN MRI-DETECTABLE BIOMARKER TO EVALUATE THE EFFICIENCY OF TTFIELDS TREATMENT ON GLIOBLASTOMA

Abstract BACKGROUND Tumor Treating Fields (TTFields) has emerged as an effective treatment modality for Glioblastoma, shown to improve both OS and PFS. Yet how to monitor response to TTFields remains unclear. In this study, we proposed using transmembrane water efflux rate (kio), a non-invasive biomarker detectable in conventional MRI and reflecting cell membrane permeability, to assess the treatment efficiency of TTFields. METHODS U-87 MG human glioblastoma cell line were treated with TTFields at frequency of 200 kHz and varying field strengths of 0 V/cm, 0.8 V/cm, 1.2 V/cm and 1.6 V/cm using the inovitro system from Novocure. The measurement of kio was performed on a 0.5 T benchtop MRI system (Pure Devices GmbH) using a home-developed filter-exchange imaging (FEXI) sequence. Cell membrane morphology were quantitatively analysed using scanning electron microscopy. Patients with newly diagnosed glioblastoma (ndGBM) who underwent DCE-MRI before and after TTFields treatment were identified and the change of FEXI-kio value were analysed. RESULTS In the same frequency, the effect of TTFields on cancer cell proliferation is dependent on intensity, manifested a decrease in cell growth rate under high electric field intensity. As the TTFields electric field intensity increased, the cell membrane permeability (kio) also increased, exhibits a significant negative correlation with glioblastoma cell growth rate (p=0.031). Scanning electron microscopy reveals that TTFields can induce significant cell membrane pores, and the total surface area of membrane pores correlates significantly positively with FEXSY-kio index. DCE-MRI data obtained from 4 ndGBM before and after TTFields therapy showed that the FEXI-kio value is increased after TTFields treatment. CONCLUSION This study demonstrated that TTFields increases membrane permeability by inducing cell membrane holes, and kio is a promising non-invasive MRI biomarker to evaluate the efficiency of TTFields treatment.

Quantifying bacterial efflux within subcellular domains of Pseudomonas aeruginosa.

Molecular efflux is a mechanism through which bacteria actively expel undesirable substances. This is a crucial line of defense against toxic chemicals in harsh environments. Understanding how efflux works is critical for designing antimicrobial strategies. Though much is already known about efflux proteins, important details about the mechanisms of efflux (e.g., importance of specific subcellular domains and ejection rates) have yet to be experimentally quantified. Herein, we use the nonlinear optical technique, second harmonic light scattering, to simultaneously measure the efflux rates from the periplasm and cytosol of a Gram-negative bacterium. The influence of efflux on the uptake kinetics of a mild antimicrobial, malachite green (MG), by Pseudomonas aeruginosa was quantified. It is observed that efflux primarily occurs from the periplasm and is two orders of magnitude faster than from the cytosol. Efflux pumps activate to maintain MG concentrations in the periplasm below 1 µM, while efflux from the cytosol maintains MG concentration below 0.1 µM. Efflux pumps are shown to saturate when exogenous MG concentrations are greater than 25 µM, while the cytosol efflux function saturates at >15 µM. Finally, efflux pumps can simultaneously eject different compounds, as proven by experiments with both MG and hexane, a known effluxable compound.IMPORTANCEMolecular efflux pumps are a crucial defense mechanism that protects bacteria from an otherwise unchecked influx of toxic molecules present in the extracellular environment. The efflux functions constitute a significant hindrance to antimicrobial efficacy. While much is now known regarding the structure of these channels, knowledge of the influence of efflux in individual subcellular domains and the associated ejection rates is still lacking. Using the nonlinear optical technique, second-harmonic light scattering, we have measured the threshold concentrations for pump activation, saturation concentrations, and efflux rates from both the periplasm and cytosol in living Gram-negative bacteria. The quantified efflux data in the different subcellular compartments not only provide a clear mechanistic understanding but also are critical for developing antimicrobial strategies.

[11C]Metoclopramide PET can detect a seizure-induced up-regulation of cerebral P-glycoprotein in epilepsy patients

BackgroundP-glycoprotein (P-gp) is an efflux transporter which is abundantly expressed at the blood-brain barrier (BBB) and which has been implicated in the pathophysiology of various brain diseases. The radiolabelled antiemetic drug [11C]metoclopramide is a P-gp substrate for positron emission tomography (PET) imaging of P-gp function at the BBB. To assess whether [11C]metoclopramide can detect increased P-gp function in the human brain, we employed drug-resistant temporal lobe epilepsy (TLE) as a model disease with a well characterised, regional P-gp up-regulation at the BBB.MethodsEight patients with drug-resistant (DRE) TLE, 5 seizure-free patients with drug-sensitive (DSE) focal epilepsy, and 15 healthy subjects underwent brain PET imaging with [11C]metoclopramide on a fully-integrated PET/MRI system. Concurrent with PET, arterial blood sampling was performed to generate a metabolite-corrected arterial plasma input function for kinetic modelling. The choroid plexus was outmasked on the PET images to remove signal contamination from the neighbouring hippocampus. Using a brain atlas, 10 temporal lobe sub-regions were defined and analysed with a 1-tissue-2-rate constant compartmental model to estimate the rate constants for radiotracer transfer from plasma to brain (K1) and from brain to plasma (k2), and the total volume of distribution (VT = K1/k2).ResultsDRE patients but not DSE patients showed significantly higher k2 values and a trend towards lower VT values in several temporal lobe sub-regions located ipsilateral to the epileptic focus as compared to healthy subjects (k2: hippocampus: +34%, anterior temporal lobe, medial part: +28%, superior temporal gyrus, posterior part: +21%).Conclusions[11C]Metoclopramide PET can detect a seizure-induced P-gp up-regulation in the epileptic brain. The efflux rate constant k2 seems to be the most sensitive parameter to measure increased P-gp function with [11C]metoclopramide. Our study provides evidence that disease-induced alterations in P-gp expression at the BBB can lead to changes in the distribution of a central nervous system-active drug to the human brain, which could affect the efficacy and/or safety of drugs. [11C]Metoclopramide PET may be used to assess or predict the contribution of increased P-gp function to drug resistance and disease pathophysiology in various brain diseases.Trial registrationEudraCT 2019-003137-42. Registered 28 February 2020.

In vitro and in vivo performance of amorphous solid dispersions of ursolic acid as a function of polymer type and excipient addition.

The objective of this research was to enhance the bioavailability of ursolic acid (UA) by preparing multielement amorphous solid dispersion (ASD) systems comprising excipients. The ASDs were prepared via the solvent evaporation method, characterized by a range of techniques, and investigated with respect to permeability of human colorectal adenocarcinoma cell line (Caco-2) cells monolayers and pharmacokinetics, with comparisons made to the physical mixture and the pure drug. The (UA-choline)-Polyethylcaprolactam-polyvinyl acetate-polyethylene glycol grafted copolymer (Soluplus)-Vitamin E polyethylene glycol succinate (TPGS) ASD demonstrated superior dissolution properties compared to the corresponding binary solid dispersions and ternary solid dispersions (P<.05). The permeability studies of Caco-2 cell monolayers revealed that the ASD exhibited moderate permeability, with an efflux rate that was significantly lower than that of the UA raw material (P<.05). Pharmacokinetic studies in rats demonstrated that the oral bioavailability of the ASD was 19.0 times higher than that of UA (P<.01). The research indicated that the multielement ASD could be employed as an efficacious drug delivery system for UA. Furthermore, the Soluplus/TPGS/choline combination represents a promising candidate for the fabrication of ASDs that can load weakly acidic and poorly soluble drugs.

Copper-61 is an advantageous alternative to gallium-68 for PET imaging of somatostatin receptor-expressing tumors: a head-to-head comparative preclinical study.

Gallium-68 positron emission tomography (68Ga-PET) with the two registered somatostatin analogs, [68Ga]Ga-DOTA-Tyr3-octreotide ([68Ga]Ga-DOTA-TOC) and [68Ga]Ga-DOTA-Tyr3-octreotate ([68Ga]Ga-DOTA-TATE), where DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, is routinely used for imaging of somatostatin receptor (SST)-expressing tumors. We investigated copper-61 (61Cu) as an alternative radiometal for PET imaging of SST-expressing tumors. Compared to gallium-68, copper-61 (t1/2 = 3.33 h, E β + max = 1.22 MeV) can be produced on a large scale, enables late time point imaging, and has the therapeutic twin copper-67. Herein, DOTA-TOC and 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (NODAGA)-TOC were labeled with copper-61 and compared with the clinically used [68Ga]Ga-DOTA-TOC. [61Cu]CuCl2 was produced from an irradiated natural nickel target. DOTA-TOC and NODAGA-TOC were labeled with [61Cu]CuCl2 in ammonium acetate buffer so to achieve a reaction pH of 5-6 and a temperature of 95°C for DOTA-TOC or room temperature for NODAGA-TOC. The radioligands were evaluated head-to-head in vitro using human embryonic kidney (HEK)-SST2 cells (affinity, binding sites, cellular uptake, and efflux) and in vivo using HEK-SST2 xenografts [PET/computed tomography (CT) imaging, biodistribution, and pharmacokinetics] and compared with [68Ga]Ga-DOTA-TOC, which was prepared using a standard procedure. Dosimetry estimates were made for [61Cu]Cu-NODAGA-TOC. [61Cu]Cu-DOTA-TOC and [61Cu]Cu-NODAGA-TOC were prepared at an apparent molar activity of 25 MBq/nmol with radiochemical purities of ≥96% and ≥98%, respectively. In vitro, both presented a sub-nanomolar affinity for SST2 (IC50 = 0.23 and 0.34 nM, respectively). They were almost entirely internalized upon binding to SST2-expressing cells and had similar efflux rates at 37°C. In vivo, [61Cu]Cu-DOTA-TOC and [61Cu]Cu-NODAGA-TOC showed the same accumulation in SST2-expressing tumors. However, PET/CT images and biodistribution analyses clearly showed an unfavorable biodistribution for [61Cu]Cu-DOTA-TOC, characterized by accumulation in the liver and the abdomen. [61Cu]Cu-NODAGA-TOC displayed favorable biodistribution, comparable with [68Ga]Ga-DOTA-TOC at 1 h post-injection (p.i.). Notwithstanding, [61Cu]Cu-NODAGA-TOC showed advantages at 4 h p.i., due to the tumor retention and improved tumor-to-non-tumor ratios. The effective dose (2.41 × 10-3 mSv/MBq) of [61Cu]Cu-NODAGA-TOC, but also the dose to the other organs and the kidneys (9.65 × 10-2 mGy/MBq), suggested a favorable safety profile. Somatostatin receptor 61Cu-PET imaging not only matches the performance of 68Ga-PET at 1 h p.i. but has advantages in late-time imaging at 4 h p.i., as it provides improved tumor-to-non-tumor ratios. [61Cu]Cu-NODAGA-TOC is superior to [61Cu]Cu-DOTA-TOC in vivo. The use of the chelator NODAGA allows quantitative labeling with copper-61 at room temperature and enables the straightforward use of a kit formulation for simple manufacturing in medical centers.

Assessment of soil carbon dioxide efflux from contrasting land uses in a semi-arid savannah ecosystem, northeastern Ghana (West Africa)

Soil respiration (SR) emits a vast amount of atmospheric carbon dioxide (CO2) and contributes largely to the global greenhouse gas budget. The study assessed the dynamics of SR rates in the Vea catchment in northeastern Ghana, a sparsely gauged semi-arid savannah ecosystem characterized by distinct patterns of soil CO2 efflux. Through field measurements using soil chambers, the study quantified soil CO2 efflux rates in different land use types (woodland, cropland and grazeland), and assessed the influence of soil moisture, temperature, and soil organic carbon stocks on SR variability. The highest soil CO2 fluxes (12.97 ± 0.89 Mg CO2C ha−1 yr−1) were recorded in woodland, followed by grazeland (9.10 ± 0.42 Mg CO2C ha−1 yr−1) with cropland having the lowest rate (5.61 ± 0.29 Mg CO2C ha−1 yr−1). We recorded mean annual soil CO2 flux of 9.23 ± 0.53 Mg CO2C ha−1 yr−1 across the land use types and also observed significant seasonal and spatial variations in SR rates. The highest SR rate (220 mg CO2C m−2h−1) was recorded in the wet months (Jul-Sept and Mar-May) and the lowest rate (30 mg CO2C m−2h−1) in the dry months (Nov-Jan). For the wet season, the mean weekly soil CO2 fluxes ranged between 140 and 160 mg CO2C m−2 hr−1 as opposed to 60–75 mg CO2C m−2 hr−1 for the dry season. Seasonal and spatial variations in SR rates were largely driven by land use type, soil moisture and the interaction of soil temperature and moisture. The results underscore the importance of understanding the emission patterns from various land uses in West African savanna ecosystems to harnessing their potential for climate change mitigation.

Simulating the impact of tumor mechanical forces on glymphatic networks in the brain parenchyma.

The brain glymphatic system is currently being explored in the context of many neurological disorders and diseases, including traumatic brain injury, Alzheimer's disease, and ischemic stroke. However, little is known about the impact of brain tumors on glymphatic function. Mechanical forces generated during tumor development and growth may be responsible for compromised glymphatic transport pathways, reducing waste clearance and cerebrospinal fluid (CSF) transport in the brain parenchyma. One such force is solid stress, i.e., growth-induced forces from cell hyperproliferation and excess matrix deposition. Because there are no prior studies assessing the impact of tumor-derived solid stress on glymphatic system structure and performance in the brain parenchyma, this study serves to fill an important gap in the field. We adapted a previously developed Electrical Analog Model using MATLAB Simulink for glymphatic transport coupled with Finite Element Analysis for tumor mechanical stresses and strains in COMSOL. This allowed simulation of the impact of tumor mechanical force generation on fluid transport within brain parenchymal glymphatic units-which include perivascular spaces, astrocytic networks, interstitial spaces, and capillary basement membranes. We conducted a parametric analysis to compare the contributions of tumor size, tumor proximity, and ratio of glymphatic subunits to the stress and strain experienced by the glymphatic unit and corresponding reduction in flow rate of CSF. Mechanical stresses intensify with proximity to the tumor and increasing tumor size, highlighting the vulnerability of nearby glymphatic units to tumor-derived forces. Our stress and strain profiles reveal compressive deformation of these surrounding glymphatics and demonstrate that varying the relative contributions of astrocytes vs. interstitial spaces impact the resulting glymphatic structure significantly under tumor mechanical forces. Increased tumor size and proximity caused increased stress and strain across all glymphatic subunits, as does decreased astrocyte composition. Indeed, our model reveals an inverse correlation between extent of astrocyte contribution to the composition of the glymphatic unit and the resulting mechanical stress. This increased mechanical strain across the glymphatic unit decreases the venous efflux rate of CSF, dependent on the degree of strain and the specific glymphatic subunit of interest. For example, a 20% mechanical strain on capillary basement membranes does not significantly decrease venous efflux (2% decrease in flow rates), while the same magnitude of strain on astrocyte networks and interstitial spaces decreases efflux flow rates by 7% and 22%, respectively. Our simulations reveal that solid stress from growing brain tumors directly reduces glymphatic fluid transport, independently from biochemical effects from cancer cells. Understanding these pathophysiological implications is crucial for developing targeted interventions aimed at restoring effective waste clearance mechanisms in the brain. This study opens potential avenues for future experimental research in brain tumor-related glymphatic dysfunction.

Assessing sediment CO2 effluxes in the coastal ecosystem of North Sumatra, Indonesia

Coastal wetlands including mangrove play a vital role in regulating the local and global carbon cycle. Coastal areas contribute greatly to the carbon exchange process due to the complex interactions that occur between the atmosphere, land, and oceans. One of the important components in coastal carbon dynamics is CO2 gas exchange between soil, water and the atmosphere. This study aims to assess CO2 efflux across various land covers (namely natural mangrove, restored mangrove, and converted mangroves to oil palm and aquaculture pond) in the coastal areas of North Sumatra Province, and analyze the effect of sea tides and ebbs on the rate of CO2 efflux and their connection with the number and area of macrozoobenthos burrows. We applied direct sampling by using the static closed chamber method attached to portable CO2 analyzer. The mean of CO2 efflux in natural mangrove forest land covers was 866±585 mgCO2/m2/h during low tide conditions and 1137±792 mgCO2/m2/h during high tide conditions, followed by oil palm plantations at 760.71±341 mgCO2/m2/h, restored mangroves during low tide of 575.24±326 mgCO2/m2/h and 597.11±180 mg CO2/m2/h during high tide conditions, and the lowest was recorded in ponds at 588.55±358 mgCO2/m2/h. Further, we observed that tidal conditions affect the magnitudes of CO2 efflux in natural and restored mangrove forests, and we did not observe similar pattern in oil palms and ponds since these land covers were not influenced by regular tidal input. We also observed that no significant relationship between the number and area of macrozoobenthos burrows and CO2 efflux. Our findings suggest that CO2 effluxes in coastal wetlands are highly dynamic and presumably driven by complex factors and therefore, understanding their magnitudes and drivers requires extensive measurement covering large spatial and temporal scales.

Sweetpotato sucrose transporter IbSUT1 alters storage roots formation by regulating sucrose transport and lignin biosynthesis.

The formation and development of storage roots is the most important physiological process in sweetpotato production. Sucrose transporters (SUTs) regulate sucrose transport from source to sink organs and play important roles in growth and development of plants. However, whether SUTs involved in sweetpotato storage roots formation is so far unknown. In this study, we show that IbSUT1, a SUT, is localized to the plasma membrane. Overexpression of IbSUT1 in sweetpotato promotes the sucrose efflux rate from leaves, leading to increased sucrose levels in roots, thus induces lignin deposition in the stele, which inhibits the storage roots formation and compromises the yield. Heterologous expression of IbSUT1 in Arabidopsis significantly increases the sucrose accumulation and promotes lignification in the inflorescence stems. RNA-seq and biochemical analysis further demonstrated that IbMYB1 negatively regulates the expression of IbSUT1. Overexpression of IbMYB1 in Arabidopsis reduces the sucrose accumulation and lignification degree in the inflorescence stems. Moreover, co-overexpression of IbMYB1 and IbSUT1 restores the phenotype of lignin over-deposition in Arabidopsis. Collectively, our results reveal that IbSUT1 regulates source-sink sucrose transport and participates in the formation of sweetpotato storage roots and highlight the potential application of IbSUT1 in improving sweetpotato yield in the future.

Effects of Wildfire and Logging on Soil CO2 Efflux in Scots Pine Forests of Siberia

Wildfires and logging play an important role in regulating soil carbon fluxes in forest ecosystems. In Siberia, large areas are disturbed by fires and logging annually. Climate change and increasing anthropogenic pressure have resulted in the expansion of disturbed areas in recent decades. However, few studies have focused on the effects of these disturbances on soil CO2 efflux in the vast Siberian areas. The objective of our research was to evaluate differences in CO2 efflux from soils to the atmosphere between undisturbed sites and sites affected by wildfire and logging in Scots pine forests of southern Siberia. We examined 35 plots (undisturbed forest, burned forest, logged plots, and logged and burned plots) on six study sites in the Angara region and four sites in the Zabaikal region. Soil CO2 efflux was measured using an LI-800 infrared gas analyzer. We found that both fire and logging significantly reduced soil efflux in the first years after a disturbance due to a reduction in vegetation biomass and consumption of the forest floor. We found a substantially lower CO2 efflux in forests burned by high-severity fires (74% less compared to undisturbed forests) than in forests burned by moderate-severity (60% less) and low-severity (37% less) fires. Clearcut logging resulted in 6–60% lower soil CO2 efflux at most study sites, while multiple disturbances (logging and fire) had 48–94% lower efflux. The soil efflux rate increased exponentially with increasing soil temperature in undisturbed Scots pine forests (p &lt; 0.001) and on logged plots (p &lt; 0.03), while an inverse relationship to soil temperature was observed in burned forests (p &lt; 0.03). We also found a positive relationship (R = 0.60–0.83, p &lt; 0.001) between ground cover depth and soil CO2 efflux across all the plots studied. Our results demonstrate the importance of disturbance factors in the assessment of regional and global carbon fluxes. The drastic changes in CO2 flux rates following fire and logging should be incorporated into carbon balance models to improve their reliability in a changing environment.

Reduced copper uptake and efflux by the mussel Mytilus coruscus after Cu exposure: Implication for biomonitoring

The hard-shell mussels Mytilus coruscus have been extensively employed in pollution biomonitoring. Earlier studies indicated that metal concentrations in Mytilus coruscus may not accurately reflect the true metal contamination levels in the sampling areas, possibly due to their modified metal uptake and efflux. Given the likelihood of mussels in the field being exposed to intermittent metal contaminants, this study investigated whether different Cu pre-exposures significantly affected its uptake and efflux upon Cu exposure. We found significant reduction in Cu uptake rate constant (ku) and efflux rate constant (ke) in the mussels with varying Cu pre-exposure regimes. Specifically, the ku decreased from 1.55 ± 0.37 L g−1 d−1 in the control group to 0.65 ± 0.19 after 5 days and 0.53 ± 0.28 after 15 days of exposure to 20 μg L−1 Cu, respectively, and then was further reduced to as low as 0.096 ± 0.046 L g−1 d−1 following a 5-day exposure at 50 μg L−1 Cu. Similarly, the ke decreased from 0.18 ± 0.020 to 0.15 ± 0.015 d−1 following 5–15 days of exposure to 20 μg L−1 Cu, and further decreased to 0.081 ± 0.023 d−1 after a 5-day exposure at 50 μg L−1 Cu. Our subcellular distribution analysis underscored the critical role of the metallothionein-like protein (MTLP) fraction in modifying both Cu ku and ke during the rapid-depuration phase (ke1), whereas the metal-rich granule (MRG) fraction influenced the ke during the second depuration phase (ke2). This study demonstrated that environmental assessments utilizing biomonitoring species should consider the exposure of these organisms to ensure accurate interpretations of metal contamination in marine ecosystems and enhance the effectiveness of these species in environmental monitoring. This crucial factor is often overlooked, potentially skewing data and leading to misinterpretations of environmental health and pollution levels.

Preparation of high-performance PVDF hollow fiber microporous membranes via the c-TIPS process with water-insoluble solvent as the second solvent

Triethyl phosphate (TEP) is a promising solvent for the c-TIPS process given its high boiling point and water miscibility. However, casting solutions containing high TEP concentration generally trigger gelation, giving a dense outer surface in PVDF membrane. In this work, we fabricated PVDF hollow fiber membranes with a porous outer surface and excellent mechanical strength via a complex thermally induced phase separation (c-TIPS) method and applied them in the DCMD process. A water-insoluble solvent, dibasic ester solvents (DBEs), was used as the second solvent to modulate the phase separation process of the PVDF/TEP system. The mass transfer between the coagulation bath and the DBEs solvent was analyzed. It could decrease the polymer-solvent interactions and the rate of solvent efflux in the coagulation bath, contributing to the development of a porous membrane structure. When the weight ratio of TEP/DMA was 1, the optimized PVDF hollow fiber membrane exhibited a tensile strength of 8.34 MPa, an average pore diameter of 0.14 μm, giving the permeate flux of 18.56 ± 2.5 kg m−2 h−1 in DCMD and excellent operational stability when subjected to high brine concentration. Thus, the fabrication method shows a promising potential for tailoring the surface porosity and bulk structure. The PVDF membranes developed in this study demonstrate potential for use in membrane distillation and related applications.

Noninvasive quantification of [18F]SynVesT-1 binding using simplified reference tissue model 2.

[18F]SynVesT-1, a positron emission tomography (PET) radiotracer for the synaptic vesicle glycoprotein 2A (SV2A), demonstrates kinetics similar to [11C]UCB-J, with high brain uptake, fast kinetics fitting well with the one-tissue compartment (1TC) model, and excellent test-retest reproducibility. Challenges arise due to the similarity between k2 and [Formula: see text] (efflux rate of the reference region), when applying the simplified reference tissue model (SRTM) and related methods in [11C]UCB-J studies to accurately estimate [Formula: see text]. This study evaluated the suitability of these methods to estimate [18F]SynVesT-1 binding using centrum semiovale (CS) or cerebellum (CER) as reference regions. Seven healthy participants underwent 120-min PET scans on the HRRT scanner with [18F]SynVesT-1. Six participants underwent test and retest scans. Arterial blood sampling and metabolite analysis provided input functions for the 1TC model, serving as the gold standard for kinetic parameters values. SRTM, coupled SRTM (SRTMC) and SRTM2 estimated were applied to estimate [Formula: see text](ref: CS) and DVRCER(ref: CER) values. For SRTM2, the population average of [Formula: see text] was determined from the 1TC model applied to the reference region. Test-retest variability and minimum scan time were also calculated. The 1TC k2 (1/min) values for CS and CER were 0.031 ± 0.004 and 0.021 ± 0.002, respectively. Although SRTMC [Formula: see text] was much higher than 1TC [Formula: see text], SRTMC underestimated BPND(ref: CS) and DVRCER by an average of 3% and 1% across regions, respectively, due to similar bias in k2 and [Formula: see text] estimation. SRTM underestimated BPND(ref: CS) by an average of 3%, but with the CER as reference region, SRTM estimation was unstable and DVRCER underestimation varied by region (mean 10%). Using population average [Formula: see text] values, SRTM2 BPND and DVRCER showed the best agreement with 1TC estimates. Our findings support the use of population [Formula: see text] value in SRTM2 with [18F]SynVesT-1 for the estimation of [Formula: see text] or DVRCER, regardless of the choice of reference region.

The effects of in vitro vitamin D treatment on glycolytic reprogramming of bone marrow-derived dendritic cells from Ldlr knock-out mouse

Dendritic cells (DCs) undergo glycolytic reprogramming, a metabolic conversion process essential for their activation. Vitamin D has been reported to affect the function of DCs, but studies in metabolic diseases are insufficient. This study investigates the effects of in vitro 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment on glycolytic reprogramming of bone marrow-derived dendritic cells (BMDCs) from control, obese, and atherosclerosis mice. Six-week-old male C57BL/6J mice were fed a control diet (CON) or a Western diet (WD), and B6.129S7-Ldlrtm1Her/J mice were fed a Western diet (LDLR−/−) for 16 weeks. BMDCs were cultured in a medium containing 1,25(OH)2D3 (10 nM) for 7 days and stimulated with lipopolysaccharide (LPS, 50 ng/mL) for 24 h. In mature BMDCs, 1,25(OH)2D3 treatment decreased basal and compensatory glycolytic proton efflux rates (glycoPER), the expression of surface markers related to immune function of DCs (MHC class II, CD80, and CD86), and IL-12p70 production. In addition, mTORC1 activation and nitric oxide (NO) production were suppressed by 1,25(OH)2D3 treatment in mature BMDCs. The effect of 1,25(OH)2D3 treatment on IL-12p70 production and mTORC1 activity in the LDLR−/− group was greater than in the CON group. These findings suggest that vitamin D can affect the metabolic environment of BMDCs by regulating glycolytic reprogramming as well as by inducing tolerogenic phenotypes of DCs.

The Glucose-Glutamine Metabolic Interplay in MCF-7 Cells, a Hormone-Sensitive Breast Cancer Model.

Selective deprivation of glutamine has been shown to accelerate the generation of reactive oxygen species (ROS) and to impair the activity of a specific pentose phosphate pathway (PPP) located within the endoplasmic reticulum (ER). The consequent oxidative damage suggests that glucose flux through this reticular pathway might contribute to the redox stress of breast cancer cells. We thus evaluated whether this response is reproduced when the glutamine shortage is coupled with the glucose deprivation. Cancer growth, metabolic plasticity and redox status were evaluated under saturating conditions and after 48 h starvation (glucose 2.5 mM, glutamine 0.5 mM). The Seahorse technology was used to estimate adenosine triphosphate (ATP)-linked and ATP-independent oxygen consumption rate (OCR) as well as proton efflux rate (PER). 18F-fluoro-deoxy-glucose (FDG) uptake was evaluated through the LigandTracer device. Proliferation rate was estimated by the carboxyfluorescein-diacetate-succinimidyl ester (CFSE) staining, while cell viability by the propidium iodide exclusion assay. Starvation reduced the proliferation rate of MCF-7 cells without affecting their viability. It also decreased lactate release and PER. Overall OCR was left unchanged although ATP-synthase dependent fraction was increased under nutrient shortage. Glutaminolysis inhibition selectively impaired the ATP-independent and the oligomycin-sensitive OCR in control and starved cultures, respectively. The combined nutrient shortage decreased the cytosolic and mitochondrial markers of redox stress. It also left unchanged the expression of the reticular unfolded protein marker GRP78. By contrast, starvation decreased the expression of hexose-6P-dehydrogenase (H6PD) thus decreasing the glucose flux through the ER-PPP as documented by the profound impairment in the uptake rate of FDG. When combined with glucose deprivation, glutamine shortage does not elicit the expected enhancement of ROS generation in the studied breast cancer cell line. Combined with the decreased activity of ER-PPP, this observation suggests that glutamine interferes with the reticular glucose metabolism to regulate the cell redox balance.

Interannual variability of air-sea CO2 exchange in the Northern Yellow Sea and its underlying mechanisms

A three-dimensional (3-D) physical-biogeochemical-carbon cycle coupled model is used to investigate the interannual variability of the air-sea carbon dioxide (CO2) flux (FCO2) in the Northern Yellow Sea (NYS) from 2009 to 2018. The verification of the model indicate that the simulation results for multiple variables exhibit consistency and fit well with the observed data. The study show that although the multi-year average FCO2 in the NYS is close to the source-sink balance, there are obvious interannual differences between different years. In particular, a relatively strong source of atmospheric CO2 (1.0 mmol m–2 d–1) is exhibited in 2014, while a relatively strong sink of atmospheric CO2 (–0.7 mmol m–2 d–1) emerges in 2016. Mechanism analysis indicates that the abnormally high temperature is the main controlling factor for the relatively high CO2 efflux rate in the NYS in 2014, while the abnormally low dissolved inorganic carbon (DIC) concentration is the main factor contributing to the relatively high CO2 influx rate in 2016. Further analysis reveals that the primary reason for the low DIC concentration since the onset of winter in 2016 is the high net decrease rate of DIC in the NYS in 2015, influenced by net community production in the summer and advection processes during the autumn. The abnormally high primary production during the summer of 2015 results in the excessive reduction of DIC concentration through biological processes. In addition, due to the strong northeasterly wind event in November 2015, low-concentration-DIC water from the Yellow Sea (YS) extends into the Bohai Sea (BS). This further leads to higher DIC flux from the NYS into the BS in the upper mixed layer and increases the inflow of low-concentration-DIC water from the Southern Yellow Sea (SYS) into the NYS. These ultimately result in the abnormal reduction of DIC concentration in the upper mixed layer of the NYS during the autumn of 2015. This study enriches our understanding of interannual variability of FCO2 in the NYS, which will not only help to further reveal the variations of FCO2 under human activities and climate change, but also provide useful information for guiding the comprehensive assessment of the carbon budget.

64Cu]Cu-FAP-NOX, a N-oxalyl modified cyclic peptide for FAP PET imaging with a flexible imaging time window.

The aim of the present study was to develop a novel 64Cu-labeled cyclic peptide ([64Cu]Cu-FAP-NOX) that targets fibroblast activation protein (FAP) and may offer advantages in terms of image contrast, imaging time window, and low uptake in normal tissues. The novel cyclic peptide featuring with a N-oxalyl modified tail was constructed and conjugated to NOTA for 64Cu labeling. Biochemical and cellular assays were performed with A549.hFAP cells. The performance of [64Cu]Cu-FAP-NOX was compared to that of two established tracers ([64Cu]Cu-FAPI-04 and [68Ga]Ga-FAP-2286) and three different NOTA-conjugates in HEK-293T.hFAP xenograft mice using micro-PET imaging. Ex vivo biodistribution studies were performed to confirm the FAP specificity and to validate the PET data. Furthermore, a first-in-human study of this novel tracer was conducted on one patient with lung cancer. Compared to [64Cu]Cu-FAPI-04, [64Cu]Cu-FAP-NOX demonstrated faster and higher rates of cellular uptake and internalization in A549.hFAP cells, but lower rates of cellular efflux. All six radiotracers were rapidly taken up by the tumor within the first 4h post-injection. However, [64Cu]Cu-FAP-NOX had more intense tumor accumulation and slower washout from the target. The ratios of the tumor to normal tissue (including kidneys and muscles) increased significantly over time, with [64Cu]Cu-FAP-NOX reaching the highest ratio among all tracers. In the patient, [64Cu]Cu-FAP-NOX PET showed a comparable result to FDG PET in the primary malignant lesion while exhibiting higher uptake in pleural metastases, consistent with elevated FAP expression as confirmed by immunohistochemistry. [64Cu]Cu-FAP-NOX is a promising FAP-targeted tracer with a highly flexible imaging time window, as evidenced by preclinical evaluation encompassing biodistribution and micro-PET studies, along with a successful patient application. Furthermore, [64Cu]Cu-FAP-NOX showed enhanced image contrast and favorable pharmacokinetic properties for FAP PET imaging, warranting translation into large cohort studies.

Impact of radish seeds (Semen Raphani) on the absorption and transportation of ginsenosides in the Caco-2 cell model: a UPLC-ESI-MS analysis

This study examined the impact of Semen raphani on the absorption of ginsenosides from Panax ginseng C.A. Meyer (ginseng) using a Caco-2 cell model and Ultra-High-Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (UPLC-ESI-MS). Six primary ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rd) were quantified. Results showed that Semen Raphani increased the efflux rate of ginsenosides, particularly at higher concentrations, suggesting it inhibits their absorption. The research elucidates the intestinal absorption process of ginsenosides and the antagonistic mechanism of Semen Raphani against ginseng.

Deletion of pyruvate dehydrogenase kinases reduces susceptibility to deep vein thrombosis in mice

AbstractNeutrophils contribute to deep vein thrombosis (DVT) by releasing prothrombotic neutrophil extracellular traps (NETs). NET formation (known as NETosis) is an energy-intensive process that requires an increased rate of aerobic glycolysis. The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase complex to divert the pyruvate flux from oxidative phosphorylation toward aerobic glycolysis. Herein, we identified that the combined deletion of PDK2 and PDK4 (PDK2/4–/–) renders mice less susceptible to DVT (measured by thrombus incidence, weight, and length) in the inferior vena cava–stenosis model at day 2 after surgery. Compared with wild-type (WT) mice, the venous thrombus obtained from PDK2/4–/– mice exhibited reduced citrullinated histone content, a known marker of NETs. In line with in vivo observations, phorbol 12-myristate 13-acetate (PMA)–stimulated PDK2/4–/– neutrophils displayed reduced NETosis and secretion of cathepsin G and elastase compared with PMA-stimulated WT neutrophils. The formation of platelet aggregates mediated by PMA-stimulated PDK2/4–/– neutrophils were significantly reduced compared with PMA-stimulated WT neutrophils. Finally, PDK2/4–/– neutrophils exhibited reduced levels of intracellular Ca2+ concentration, extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation, and glycolytic proton efflux rate (a measure of aerobic glycolysis), known to facilitate NETosis. Together, these findings elucidate, to our knowledge, for the first time, the fundamental role of PDK2/4 in regulating NETosis and acute DVT.

The 14-3-3 protein nt GF14e interacts with CIPK2 and increases low potassium stress in tobacco

ABSTRACT Potassium (K+) plays a role in enzyme activation, membrane transport, and osmotic regulation processes. An increase in potassium content can significantly improve the elasticity and combustibility of tobacco and reduce the content of harmful substances. Here, we report that the expression analysis of Nt GF14e, a 14-3-3 gene, increased markedly after low-potassium treatment (LK). Then, chlorophyll content, POD activity and potassium content, were significantly increased in overexpression of Nt GF14e transgenic tobacco lines compared with those in the wild type plants. The net K+ efflux rates were severely lower in the transgenic plants than in the wild type under LK stress. Furthermore, transcriptome analysis identified 5708 upregulated genes and 2787 downregulated genes between Nt GF14e overexpressing transgenic tobacco plants. The expression levels of some potassium-related genes were increased, such as CBL-interacting protein kinase 2 (CIPK2), Nt CIPK23, Nt CIPK25, H+-ATPase isoform 2 a (AHA2a), Nt AHA4a, Stelar K+ outward rectifier 1(SKOR1), and high affinity K+ transporter 5 (HAK5). The result of yeast two-hybrid and luciferase complementation imaging experiments suggested Nt GF14e could interact with CIPK2. Overall, these findings indicate that NtGF14e plays a vital roles in improving tobacco LK tolerance and enhancing potassium nutrition signaling pathways in tobacco plants.