A continuous flow bioreactor system for high-throughput hyperpolarized metabolic flux analysis.

Hyperpolarized carbon-13 labeled compounds are increasingly being used in medical MR imaging (MRI) and MR imaging (MRI) and spectroscopy (MRS) research, due to its ability to monitor tissue and cell metabolism in real-time. Although radiological biomarkers are increasingly being considered as clinical indicators, biopsies are still considered the gold standard for a large variety of indications. Bioreactor systems can play an important role in biopsy examinations because of their ability to provide a physiochemical environment that is conducive for therapeutic response monitoring ex vivo. We demonstrate here a proof-of-concept bioreactor and microcoil receive array setup that allows for ex vivo preservation and metabolic NMR spectroscopy on up to three biopsy samples simultaneously, creating an easy-to-use and robust way to simultaneously run multisample carbon-13 hyperpolarization experiments. Experiments using hyperpolarized [1-13C]pyruvate on ML-1 leukemic cells in the bioreactor setup were performed and the kinetic pyruvate-to-lactate rate constants ( k PL ) extracted. The coefficient of variation of the experimentally found k PL s for five repeated experiments was C V = 35 % . With this statistical power, treatment effects of 30%-40% change in lactate production could be easily differentiable with only a few hyperpolarization dissolutions on this setup. Furthermore, longitudinal experiments showed preservation of ML-1 cells in the bioreactor setup for at least 6 h. Rat brain tissue slices were also seen to be preserved within the bioreactor for at least 1 h. This validation serves as the basis for further optimization and upscaling of the setup, which undoubtedly has huge potential in high-throughput studies with various biomarkers and tissue types.

Formate Metabolism in Shigella flexneri and Its Effect on HeLa Cells at Different Stages during the Infectious Process.

Shigellosis caused by Shigella is one of the most important foodborne illnesses in global health, but little is known about the metabolic cross talk between this bacterial pathogen and its host cells during the different stages of the infection process. A detailed understanding of the metabolism can potentially lead to new drug targets remedying the pressing problem of antibiotic resistance. Here, we use stable isotope-resolved metabolomics as an unbiased and fast method to investigate how Shigella metabolizes 13C-glucose in three different environments: inside the host cells, adhering to the host cells, and alone in suspension. We find that especially formate metabolism by bacteria is sensitive to these different environments. The role of formate in pathogen metabolism is sparsely described in the literature compared to the roles of acetate and butyrate. However, its metabolic pathway is regarded as a potential drug target due to its production in microorganisms and its absence in humans. Our study provides new knowledge about the regulatory effect of formate. Bacterial metabolism of formate is pH dependent when studied alone in culture medium, whereas this effect is less pronounced when the bacteria adhere to the host cells. Once the bacteria are inside the host cells, we find that formate accumulation is reduced. Formate also affects the host cells resulting in a reduced infection rate. This was correlated to an increased immune response. Thus, intriguingly formate plays a double role in pathogenesis by increasing the virulence of Shigella and at the same time stimulating the immune response of the host. IMPORTANCE Bacterial infection is a pressing societal concern due to development of resistance toward known antibiotics. Central carbon metabolism has been suggested as a potential new target for drug development, but metabolic changes upon infection remain incompletely understood. Here, we used a cellular infection model to study how the bacterial pathogen Shigella adapts its metabolism depending on the environment starting from the extracellular medium until Shigella successfully invaded and proliferated inside host cells. The mixed-acid fermentation of Shigella was the major metabolic pathway during the infectious process, and the glucose-derived metabolite formate surprisingly played a divergent role in the pathogen and in the host cell. Our data show reduced infection rate when both host cells and bacteria were treated with formate, which correlated with an upregulated immune response in the host cells. The formate metabolism in Shigella thus potentially provides a route toward alternative treatment strategies for Shigella prevention.

Trade-off between preamplifier noise figure and decoupling in MRI detectors.

PURPOSE: There is a limit to the maximum achievable preamplifier decoupling. In many cases, this level is not enough. To overcome this limit, the preamplifier noise figure can be compromised for further decoupling increase. This is useful in flexible MRI arrays where ensuring coil insensitivity to changes in other array elements is a challenge. METHODS: This work establishes the relation between the preamplifier noise figure and preamplifier decoupling using closed-form equations. These equations allow the evaluation of preamplifier decoupling properties and benchmark different preamplifiers against each other. The method to design the corresponding decoupling networks is described. The derived generalized design equations, which are not limited to 50 Ω pre-matched preamplifiers, greatly improve design flexibility and enable use of new amplifiers in MRI detectors. RESULTS: Using the method, the decoupling properties of three preamplifiers are studied. For demonstration, the coil decoupling is further increased by 10.8 dB using one of the preamplifiers. The noise figure is sacrificed by 0.5 dB, which is predicted by equations and verified experimentally. Although examples are shown for 3 T systems at 32.13 MHz and 127.7 MHz, the approach and equations apply to any field strength and nucleus. CONCLUSION: Preamplifier decoupling can be improved beyond what is possible by traditional approaches. The derived design equations cover a wide range of cases, including inductive coils and self-resonant low-impedance and high-impedance coils.

A cryogenic 14-channel 13 C receiver array for 3T human head imaging.

PURPOSE: This article presents a novel 14-channel receive-only array for 13 C human head imaging at 3 T that explores the SNR gain by operating at cryogenic temperature cooled by liquid nitrogen. METHODS: Cryostats are developed to evaluate single-coil bench SNR performance and cool the 14-channel array with liquid nitrogen while having enough thermal insulation between the coils and the sample. The temperature distribution for the coil array is measured. Circuits are adapted to the -189°C environment and implemented in the 14-channel array. 13 C images are acquired with the array at cryogenic and room temperature in a 3T scanner. RESULTS: Compared with room temperature, the array at cryogenic temperature provides 27%-168% SNR improvement over all voxels and 47% SNR improvement near the image center. The measurements show a decrease of the element noise correlation at cryogenic temperature. CONCLUSION: It is demonstrated that higher SNR can be achieved by cryogenically cooling the 14-channel array. A cryogenic array suitable for clinical imaging can be further developed on the array proposed. The cryogenic coil array is most likely suited for scenarios in which high SNR deep in a head and decent SNR on the periphery are required.

Reverse dynamic nuclear polarisation for indirect detection of nuclear spins close to unpaired electrons.

Polarisation transfer schemes and indirect detection are central to magnetic resonance. Using the trityl radical OX063 and a pulse electron paramagnetic resonance spectrometer operating in the Q-band (35 GHz, 1.2 T), we show here that it is possible to use pulsed dynamic nuclear polarisation (DNP) to transfer polarisation from electrons to protons and back. The latter is achieved by first saturating the electrons and then simply using a reverse DNP step. A variable mixing time between DNP and reverse DNP allows us to investigate the decay of polarisation on protons in the vicinity of the electrons. We qualitatively investigate the influence of solvent deuteration, temperature, and electron concentration. We expect reverse DNP to be useful in the investigation of nuclear spin diffusion and envisage its use in electron-nuclear double-resonance (ENDOR) experiments.

Metabolism of hyperpolarised [1-13 C]pyruvate in awake and anaesthetised rat brains.

The use of hyperpolarised 13 C pyruvate for nononcological neurological applications has not been widespread so far, possibly due to delivery issues limiting the visibility of metabolites. First proof-of-concept results have indicated that metabolism can be detected in human brain, and this may supersede the results obtained in preclinical settings. One major difference between the experimental setups is that preclinical MRI/MRS routinely uses anaesthesia, which alters both haemodynamics and metabolism. Here, we used hyperpolarised [1-13 C]pyruvate to compare brain metabolism in awake rats and under isoflurane, urethane or medetomidine anaesthesia. Spectroscopic [1-13 C]pyruvate time courses measured sequentially showed that pyruvate-to-bicarbonate and pyruvate-to-lactate labelling rates were lower in isoflurane animals than awake animals. An increased bicarbonate-to-lactate ratio was observed in the medetomidine group compared with other groups. The study shows that hyperpolarised [1-13 C]pyruvate experiments can be performed in awake rats, thus avoiding anaesthesia-related issues. The results suggest that haemodynamics probably dominate the observed pyruvate-to-metabolite labelling rates and area-under-time course ratios of referenced to pyruvate. On the other hand, the results obtained with medetomidine suggest that the ratios are also modulated by the underlying cerebral metabolism. However, the ratios between intracellular metabolites were unchanged in awake compared with isoflurane-anaesthetised rats.

Classification and biomarker identification of prostate tissue from TRAMP mice with hyperpolarized 13C-SIRA.

Hyperpolarized 13C isotope resolved spectroscopy boosts NMR signal intensity, which improves signal detection and allows metabolic fluxes to be analyzed. Such hyperpolarized flux data may offer new approaches to tissue classification and biomarker identification that could be translated in vivo. Here we used hyperpolarized stable isotope resolved analysis (SIRA) to measure metabolite specific 13C isotopic enrichments in the central carbon metabolism of mouse prostate. Prostate and tumor tissue samples were acquired from transgenic adenocarcinomas of the mouse prostate (TRAMP) mice. Before euthanasia, mice were injected with [U-13C]glucose intraperitoneally (i.p.). Polar metabolite extracts were prepared, and hyperpolarized 1D-13C NMR spectra were obtained from normal prostate (n = 19) and cancer tissue (n = 19) samples. Binary classification and feature analysis was performed to make a separation model and to investigate differences between samples originating from normal and cancerous prostate tissue, respectively. Hyperpolarized experiments were carried out according to a standardized protocol, which showed a high repeatability (CV = 15%) and an average linewidth in the 1D-13C NMR spectra of 2 ± 0.5 Hz. The resolution of the hyperpolarized 1D-13C spectra was high with little signal overlap in the carbonyl region and metabolite identification was easily accomplished. A discrimination with 95% success rate could be made between samples originating from TRAMP mice prostate and tumor tissue based on isotopomers from uniquely identified metabolites. Hyperpolarized 13C-SIRA allowed detailed metabolic information to be obtained from tissue specimens. The positional information of 13C isotopic enrichments lead to easily interpreted features responsible for high predictive classification of tissue types. This analytical approach has matured, and the robust experimental protocols currently available allow systematic tracking of metabolite flux ex vivo.

Metabolic contrast agents produced from transported solid 13C-glucose hyperpolarized via dynamic nuclear polarization.

Magnetic Resonance Imaging combined with hyperpolarized 13C-labelled metabolic contrast agents produced via dissolution Dynamic Nuclear Polarization can, non-invasively and in real-time, report on tissue specific aberrant metabolism. However, hyperpolarization equipment is expensive, technically demanding and needs to be installed on-site for the end-user. In this work, we provide a robust methodology that allows remote production of the hyperpolarized 13C-labelled metabolic contrast agents. The methodology, built on photo-induced thermally labile radicals, allows solid sample extraction from the hyperpolarization equipment and several hours' lifetime of the 13C-labelled metabolic contrast agents at appropriate storage/transport conditions. Exemplified with [U-13C, d7]-D-glucose, we remotely produce hyperpolarized 13C-labelled metabolic contrast agents and generate above 10,000-fold liquid-state Magnetic Resonance signal enhancement at 9.4 T, keeping on-site only a simple dissolution device.

Three-element matching networks for receive-only MRI coil decoupling.

PURPOSE: Preamplifier decoupling is useful for minimizing interaction between MRI array elements. The purpose of this work is to propose a general approach to designing networks for preamplifier decoupling while keeping the number of elements to a minimum. The approach is applicable to arbitrary impedance preamplifiers and arbitrary coil impedances. METHODS: Closed form design equations for decoupling networks are derived based on maximum decoupling and minimum preamplifier noise conditions. The analytical solutions are verified using numerical simulations. Design examples at 32.1, 64, 128, and 298 MHz are shown. One of the examples is realized on a test bench. The fabricated circuit is tested for decoupling and minimum noise properties. RESULTS: The design equations are verified numerically and experimentally. The fabricated network demonstrates 30.7 dB of decoupling and minimum output noise at the design frequency. CONCLUSION: The design equations lead to four alternative network solutions. Each network is realized as a T-shape or Π-shape three elements circuit topology. All four networks are identical in performance providing minimum amplifier noise and maximum decoupling for a given preamplifier and coil combination. An MRI array designer can choose any solution out of four. The considerations for choosing the most practical solution are given. The presented method enables the use of arbitrary impedance preamplifiers or transistors (not necessary 50 Ω) and provides the most compact design possible (with the least number of components), which is particularly useful in multi-element systems.

Hyperpolarization via dissolution dynamic nuclear polarization: new technological and methodological advances.

Dissolution-DNP is a method to boost liquid-state NMR sensitivity by several orders of magnitude. The technique consists in hyperpolarizing samples by solid-state dynamic nuclear polarization at low temperature and moderate magnetic field, followed by an instantaneous melting and dilution of the sample happening inside the polarizer. Although the technique is well established and the outstanding signal enhancement paved the way towards many applications precluded to conventional NMR, the race to develop new methods allowing higher throughput, faster and higher polarization, and longer exploitation of the signal is still vivid. In this work, we review the most recent advances on dissolution-DNP methods trying to overcome the original technique's shortcomings. The review describes some of the new approaches in the field, first, in terms of sample formulation and properties, and second, in terms of instrumentation.

[68Ga]Ga-NODAGA-E[(cRGDyK)]2 PET and hyperpolarized [1-13C] pyruvate MRSI (hyperPET) in canine cancer patients: simultaneous imaging of angiogenesis and the Warburg effect.

PURPOSE: Cancer has a multitude of phenotypic expressions and identifying these are important for correct diagnosis and treatment selection. Clinical molecular imaging such as positron emission tomography can access several of these hallmarks of cancer non-invasively. Recently, hyperpolarized magnetic resonance spectroscopy with [1-13C] pyruvate has shown great potential to probe metabolic pathways. Here, we investigate simultaneous dual modality clinical molecular imaging of angiogenesis and deregulated energy metabolism in canine cancer patients. METHODS: Canine cancer patients (n = 11) underwent simultaneous [68Ga]Ga-NODAGA-E[(cRGDyK)]2 (RGD) PET and hyperpolarized [1-13C]pyruvate-MRSI (hyperPET). Standardized uptake values and [1-13C]lactate to total 13C ratio were quantified and compared generally and voxel-wise. RESULTS: Ten out of 11 patients showed clear tumor uptake of [68Ga]Ga-NODAGA-RGD at both 20 and 60 min after injection, with an average SUVmean of 1.36 ± 0.23 g/mL and 1.13 ± 0.21 g/mL, respectively. A similar pattern was seen for SUVmax values, which were 2.74 ± 0.41 g/mL and 2.37 ± 0.45 g/mL. The [1-13C]lactate generation followed patterns previously reported. We found no obvious pattern or consistent correlation between the two modalities. Voxel-wise tumor values of RGD uptake and lactate generation analysis revealed a tendency for each canine cancer patient to cluster in separated groups. CONCLUSION: We demonstrated combined imaging of [68Ga]Ga-NODAGA-RGD-PET for angiogenesis and hyperpolarized [1-13C]pyruvate-MRSI for probing energy metabolism. The results suggest that [68Ga]Ga-NODAGA-RGD-PET and [1-13C]pyruvate-MRSI may provide complementary information, indicating that hyperPET imaging of angiogenesis and energy metabolism is able to aid in cancer phenotyping, leading to improved therapy planning.

13C Dynamic Nuclear Polarization using SA-BDPA at 6.7 T and 1.1 K: Coexistence of Pure Thermal Mixing and Well-Resolved Solid Effect.

SA-BDPA is a water-soluble, narrow-line width radical previously used for dynamic nuclear polarization (DNP) signal enhancement in solid-state magic angle spinning NMR spectroscopy. Here, we report the first study using SA-BDPA under dissolution DNP conditions (6.7 T and 1.15 K). Longitudinal-detected (LOD)-electron spin resonance (ESR) and 13C DNP measurements were performed on samples containing 8.4 M [13C]urea dissolved in 50:50 water:glycerol (v/v) doped with either 60 or 120 mM SA-BDPA. Two distinct DNP mechanisms, both "pure" thermal mixing and a well-resolved solid effect could clearly be identified. The radical's ESR line width (30-40 MHz), broadened predominantly by dipolar coupling, excluded any contribution from the cross effect. Microwave frequency modulation increased the enhancement by DNP at the lower radical concentration but not at the higher radical concentration. These results are compared to data acquired with trityl radical AH111501, highlighting the unusual 13C DNP properties of SA-BDPA.

Multi-site benchmarking of clinical 13C RF coils at 3T.

A quality assurance protocol for RF coils is proposed, which can be used for volume (Tx/Rx) and surface (Rx) coils. Following this protocol, a benchmarking of seven coils (from three different MR sites) dedicated to 13C MRI at 3T is reported. Coil performance is particularly important for 3T MRI at the 13C frequency, since the coil-to-sample noise ratio is typically high. The coils are evaluated experimentally using the proposed protocol based on MR spectroscopic imaging performed with two different phantoms: one head-shaped, and one with cylindrical shape and nearly twice the volume of the first one. To achieve an unbiased SNR comparison of volume and array coils, coil combination was done using sensitivity profiles extracted from the data. SNR, noise correlation matrices and example g-factor maps are reported. For globally calibrated, equal excitation angles, the measured SNR shows large differences for the volume coils of up to 115% at the phantom center for a head phantom. The arrays show lower differences in superficial SNR. The sample surface depth at which the volume coils outperform the arrays is estimated to 7 cm, and SNR furthest away from the coil surface is 28% lower for the best array compared to the best volume coil. A broad set of coils for 13C at 3T have been benchmarked. The results reported, and the method used to benchmark them, should guide the 13C community to choose the most suitable coil for a given experiment.

Stable isotope resolved metabolomics classification of prostate cancer cells using hyperpolarized NMR data.

Metabolic fingerprinting is a strong tool for characterization of biological phenotypes. Classification with machine learning is a critical component in the discrimination of molecular determinants. Cellular activity can be traced using stable isotope labelling of metabolites from which information on cellular pathways may be obtained. Nuclear magnetic resonance (NMR) spectroscopy is, due to its ability to trace labelling in specific atom positions, a method of choice for such metabolic activity measurements. In this study, we used hyperpolarization in the form of dissolution Dynamic Nuclear Polarization (dDNP) NMR to measure signal enhanced isotope labelled metabolites reporting on pathway activity from four different prostate cancer cell lines. The spectra have a high signal-to-noise, with less than 30 signals reporting on 10 metabolic reactions. This allows easy extraction and straightforward interpretation of spectral data. Four metabolite signals selected using a Random Forest algorithm allowed a classification with Support Vector Machines between aggressive and indolent cancer cells with 96.9% accuracy, -corresponding to 31 out of 32 samples. This demonstrates that the information contained in the few features measured with dDNP NMR, is sufficient and robust for performing binary classification based on the metabolic activity of cultured prostate cancer cells.

Creating a clinical platform for carbon-13 studies using the sodium-23 and proton resonances.

PURPOSE: Calibration of hyperpolarized 13 C-MRI is limited by the low signal from endogenous carbon-containing molecules and consequently requires 13 C-enriched external phantoms. This study investigated the feasibility of using either 23 Na-MRI or 1 H-MRI to calibrate the 13 C excitation. METHODS: Commercial 13 C-coils were used to estimate the transmit gain and center frequency for 13 C and 23 Na resonances. Simulations of the transmit B1 profile of a Helmholtz loop were performed. Noise correlation was measured for both nuclei. A retrospective analysis of human data assessing the use of the 1 H resonance to predict [1-13 C]pyruvate center frequency was also performed. In vivo experiments were undertaken in the lower limbs of 6 pigs following injection of hyperpolarized 13 C-pyruvate. RESULTS: The difference in center frequencies and transmit gain between tissue 23 Na and [1-13 C]pyruvate was reproducible, with a mean scale factor of 1.05179 ± 0.00001 and 10.4 ± 0.2 dB, respectively. Utilizing the 1 H water peak, it was possible to retrospectively predict the 13 C-pyruvate center frequency with a standard deviation of only 11 Hz sufficient for spectral-spatial excitation-based studies. CONCLUSION: We demonstrate the feasibility of using the 23 Na and 1 H resonances to calibrate the 13 C transmit B1 using commercially available 13 C-coils. The method provides a simple approach for in vivo calibration and could improve clinical workflow.

Detection of lentiviral suicide gene therapy in C6 rat glioma using hyperpolarised [1-13 C]pyruvate.

Hyperpolarised [1-13 C]pyruvate MRI has shown promise in monitoring therapeutic efficacy in a number of cancers including glioma. In this study, we assessed the pyruvate response to the lentiviral suicide gene therapy of herpes simplex virus-1 thymidine kinase with the prodrug ganciclovir (HSV-TK/GCV) in C6 rat glioma and compared it with traditional MR therapy markers. Female Wistar rats were inoculated with 106 C6 glioma cells. Treated animals received intratumoural lentiviral HSV-TK gene transfers on days 7 and 8 followed by 2-week GCV therapy starting on day 10. Animals were repeatedly imaged during therapy using volumetric MRI, diffusion and relaxation mapping, as well as metabolic [1-13 C]pyruvate MRS imaging. Survival (measured as time before animals reached a humane endpoint and were euthanised) was assessed up to day 30 posttherapy. HSV-TK/GCV gene therapy lengthened the median survival time from 12 to 25 days. This was accompanied by an apparent tumour growth arrest, but no changes in diffusion or relaxation parameters in treated animals. The metabolic response was more evident in the case-by-case analysis than in the group-level analysis. Treated animals also showed a 37 ± 15% decrease (P < 0.05, n = 5) in lactate-to-pyruvate ratio between therapy weeks, whereas a 44 ± 18% increase (P < 0.05, n = 6) was observed in control animals. Hyperpolarised [1-13 C]pyruvate MRI can offer complementary metabolic information to traditional MR methods to give a more comprehensive picture of the slowly developing gene therapy response. This may benefit the detection of the successful therapy response in patients.

Hyperpolarized water through dissolution dynamic nuclear polarization with UV-generated radicals.

In recent years, hyperpolarization of water protons via dissolution Dynamic Nuclear Polarization (dDNP) has attracted increasing interest in the magnetic resonance community. Hyperpolarized water may provide an alternative to Gd-based contrast agents for angiographic and perfusion Magnetic Resonance Imaging (MRI) examinations, and it may report on chemical and biochemical reactions and proton exchange while perfoming Nuclear Magnetic Resonance (NMR) investigations. However, hyperpolarizing water protons is challenging. The main reason is the presence of radicals, required to create the hyperpolarized nuclear spin state. Indeed, the radicals will also be the main source of relaxation during the dissolution and transfer to the NMR or MRI system. In this work, we report water magnetizations otherwise requiring a field of 10,000 T at room temperature on a sample of pure water, by employing dDNP via UV-generated, labile radicals. We demonstrate the potential of our methodology by acquiring a 15N spectrum from natural abundance urea with a single scan, after spontaneous magnetization transfer from water protons to nitrogen nuclei.

Autonomous cryogenic RF receive coil for 13 C imaging of rodents at 3 T.

PURPOSE: To develop an autonomous, in-bore, MR-compatible cryostat cooled with liquid nitrogen that provides full-day operation, and to demonstrate that the theoretical signal-to-noise benefit can be achieved for 13 C imaging at 3 T (32.13 MHz). METHODS: The cryogenic setup uses a vacuum-insulated fiberglass cryostat, which indirectly cools a cold finger where the RF coil is attached. The cryostat was evacuated before use and had a reservoir of liquid nitrogen for full-day operation. A 30 × 40 mm2 copper coil was mounted inside the cryostat with a 3-mm distance to the sample. Two examples of in vivo experiments of rat brain metabolism after a hyperpolarized [1-13 C]pyruvate injection are reported. RESULTS: A coil Q-factor ratio of Q88K /Q290K = 550/280 was obtained, and the theoretical SNR enhancement was verified with MR measurements. We achieved a coil temperature of 88 K and a preamplifier temperature of 77 K. A 2-fold overall SNR enhancement was achieved, compared with the best case at room temperature. The thermal performance of the coil was adequate for in vivo experiments, with an autonomy of 5 hours consuming 6 L of LN2 , extendable to over 12 hours by LN2 refilling. CONCLUSION: Cryogenic surface coils can be highly beneficial for 13 C imaging, provided that the coil-to-sample distance remains short. An autonomous, in-bore cryostat was developed that achieved the theoretical improvement in SNR.

Rapid zero-trans kinetics of Cs+ exchange in human erythrocytes quantified by dissolution hyperpolarized 133Cs+ NMR spectroscopy.

Transmembrane flux of Cs+ (a K+ congener) was measured in human red blood cells (RBCs; erythrocytes) on the 10-s time scale. This is the first report on dissolution dynamic nuclear polarization (dDNP) nuclear magnetic resonance (NMR) spectroscopy with this nuclide in mammalian cells. Four technical developments regularized sample delivery and led to high quality NMR spectra. Cation-free media with the Piezo1 (mechanosensitive cation channel) activator yoda1 maximized the extent of membrane transport. First-order rate constants describing the fluxes were estimated using a combination of statistical methods in Mathematica, including the Markov chain Monte Carlo (MCMC) algorithm. Fluxes were in the range 4-70 µmol Cs+ (L RBC)-1 s-1; these are smaller than for urea, but comparable to glucose. Methodology and analytical procedures developed will be applicable to transmembrane cation transport studies in the presence of additional Piezo1 effectors, to other cellular systems, and potentially in vivo.