Measurement Of Membrane Permeability Research Articles

Charge-Dependent Crossover in Aqueous Organic Redox Flow Batteries Revealed Using Online NMR Spectroscopy.

Aqueous organic redox-flow batteries (AORFBs) are promising candidates for low-cost grid-level energy storage. However, their wide-scale deployment is limited by crossover of redox-active material through the separator membrane, which causes capacity decay. Traditional membrane permeability measurements do not capture all contributions to crossover in working batteries, including migration and changes in ion size and charge. Here we present a new method for characterizing crossover in operating AORFBs using online 1H NMR spectroscopy. By the introduction of a separate pump to decouple NMR and battery flow rates, this method opens a route to quantitative time-resolved monitoring of redox-flow batteries under real operating conditions. In this proof-of-concept study of a 2,6-dihydroxyanthraquinone (2,6-DHAQ)/ferrocyanide model system, we observed a doubling of the 2,6-DHAQ crossover during battery charging, which we attribute to migration effects. This new membrane testing methodology will advance our understanding of crossover and accelerate the development of improved redox-flow batteries.

Acceleration of Pd-V intermetallic diffusion by hydrogen

Vanadium-based membranes have great potential for hydrogen purification due to their perfect selectivity, high permeability, and relatively low cost. With appropriate surface cleaning, V efficiently permeates hydrogen at elevated temperature, but performance declines due to its affinity to absorb impurities. The application of palladium thin films maintains a clean surface that catalyzes hydrogen dissociation and recombination. Hydrogen permeation in Pd-V-Pd membranes initially reach theoretical permeability, but declines due to Pd-V interdiffusion. The objective of this work was to quantify the intermetallic diffusion process as a function of temperature and ambient. Pd-V composites were subjected to various annealing treatments and characterized using Auger electron spectroscopy, X-ray diffraction, and energy dispersive X-ray spectroscopy, as well as correlated to measurements of membrane permeability. In an inert environment Pd-V interdiffusion was observable as low as 300 °C, and the diffusion coefficient had an activation energy of 44 kJ/mol. Furthermore, the presence of hydrogen at partial pressures > 10 kPa accelerated interdiffusion six-fold at T = 400 °C. Membrane performance degraded with an activation energy 75 kJ/mol, suggesting that intermetallic diffusion leads to both a loss of catalytic activity and as well as degradation of bulk permeability. These findings provide a baseline for evaluating hydrogen permeable interdiffusion barriers to overcome these challenges.

An impedance based microfluidic sensor for evaluation of individual red blood cell solute permeability

—In this paper, we investigate a microfluidic based sensing device for cell membrane permeability measurements in real time with applications in rapid assessment of red blood cell (RBC) quality at the individual cell level. The microfluidic chip was designed with unique abilities to line up the RBCs in the centerline of the microchannel using positive dielectrophoresis (p-DEP) forces, rapid mixing of RBCs with various media (e.g. containing permeating or nonpermeating solutes) injected from different inlets to achieve high mixing efficiency. The chip detects the impedance values of the RBCs within 0.19 s from the start of mixing with other media, at ten electrodes along the length of the channel and enables time series measurements of volume change of individual cell caused by cell osmosis in anisosmotic fluids over a 0.8 s postmixing timespan. This technique enables estimating water permeability of individual cell accurately. Here we first present confirmation of a linear voltage-diameter relationship in polystyrene bead standards. Next, we show that under equilibrium conditions, the voltage-volume relationship in rat red blood cells (RBCs) is linear, corresponding to previously published Boyle van ‘t Hoff plots. Using rat cells as a model for human, we present the first measurement of water permeability in individual red blood cells and confirm that these data align with previously published population level values for human RBC. Finally, we present preliminary evidence for possible application of our device to identify individual RBCs infected with Plasmodium falciparum malaria parasites. Future developments using this device will address the use of whole blood with non-homogenous cell populations, a task currently performed by clinical Coulter counters.

Systematic comparison of unilamellar vesicles reveals that archaeal core lipid membranes are more permeable than bacterial membranes.

One of the deepest branches in the tree of life separates the Archaea from the Bacteria. These prokaryotic groups have distinct cellular systems including fundamentally different phospholipid membrane bilayers. This dichotomy has been termed the lipid divide and possibly bestows different biophysical and biochemical characteristics on each cell type. Classic experiments suggest that bacterial membranes (formed from lipids extracted from Escherichia coli, for example) show permeability to key metabolites comparable to archaeal membranes (formed from lipids extracted from Halobacterium salinarum), yet systematic analyses based on direct measurements of membrane permeability are absent. Here, we develop a new approach for assessing the membrane permeability of approximately 10 μm unilamellar vesicles, consisting of an aqueous medium enclosed by a single lipid bilayer. Comparing the permeability of 18 metabolites demonstrates that diether glycerol-1-phosphate lipids with methyl branches, often the most abundant membrane lipids of sampled archaea, are permeable to a wide range of compounds useful for core metabolic networks, including amino acids, sugars, and nucleobases. Permeability is significantly lower in diester glycerol-3-phosphate lipids without methyl branches, the common building block of bacterial membranes. To identify the membrane characteristics that determine permeability, we use this experimental platform to test a variety of lipid forms bearing a diversity of intermediate characteristics. We found that increased membrane permeability is dependent on both the methyl branches on the lipid tails and the ether bond between the tails and the head group, both of which are present on the archaeal phospholipids. These permeability differences must have had profound effects on the cell physiology and proteome evolution of early prokaryotic forms. To explore this further, we compare the abundance and distribution of transmembrane transporter-encoding protein families present on genomes sampled from across the prokaryotic tree of life. These data demonstrate that archaea tend to have a reduced repertoire of transporter gene families, consistent with increased membrane permeation. These results demonstrate that the lipid divide demarcates a clear difference in permeability function with implications for understanding some of the earliest transitions in cell origins and evolution.

Implications of Bacterial Adaptation to Phenol Degradation under Suboptimal Culture Conditions Involving Stenotrophomonas maltophilia KB2 and Pseudomonas moorei KB4

Despite the well-described abundance of phenol-degrading bacteria, knowledge concerning their degradation abilities under suboptimal conditions is still very limited and needs to be expanded. Therefore, this work aimed to study the growth and degradation potential of Stenotrophomonas maltophilia KB2 and Pseudomonas moorei KB4 strains toward phenol under suboptimal temperatures, pH, and salinity in connection with the activity of catechol dioxygenases, fatty acid profiling, and membrane permeability. The methodology used included: batch culture of bacteria in minimal medium supplemented with phenol (300 mg/L), isolating and measuring the activity of catechol 1,2- and 2,3-dioxygenases, calculating kinetic parameters, chromatographic analysis of fatty acid methyl esters (FAMEs) and determining the membrane permeability. It was established that the time of phenol utilisation by both strains under high temperatures (39 and 40 °C) proceeded 10 h; however, at the lowest temperature (10 °C), it was extended to 72 h. P. moorei KB4 was more sensitive to pH (6.5 and 8.5) than S. maltophilia KB2 and degraded phenol 5–6 h longer. Salinity also influenced the time of phenol removal. S. maltophilia KB2 degraded phenol in the presence of 2.5% NaCl within 28 h, while P. moorei KB4 during 72 h. The ability of bacteria to degrade phenol in suboptimal conditions was coupled with a relatively high activity of catechol 1,2- and/or 2,3-dioxygenases. FAME profiling and membrane permeability measurements indicated crucial alterations in bacterial membrane properties during phenol degradation leading predominantly to an increase in fatty acid saturation and membrane permeability. The obtained results offer hope for the potential use of both strains in environmental microbiology and biotechnology applications.

Characterization of Dissolution-Permeation System using Hollow Fiber Membrane Module and Utility to Predict in Vivo Drug Permeation Across BCS Classes

A dissolution-permeation system has potential to provide insight into the kinetic contributions of dissolution and permeation to overall drug absorption. The goals of the study were to characterize a dissolution-hollow fiber membrane (D-HFM) system and compare its resulting in vitro drug permeation constants (Kp′) to in vivo clinical permeation constants (kp), for four drugs in various Biopharmaceutics Classification System (BCS) classes. Model predictions for D-HFM were made based on derived mixing tank (MT) and complete radial (CRM) flow models and independent measurement of membrane permeability. Experimental D-HFM studies included donor flow rate and donor volume sensitivity studies, and drug permeation profile studies. Additionally, for the four drugs, Kp′from D-HFM system was compared to (kp) from literature, as well as Kp′ values from side-by-side diffusion cell and dissolution/Caco-2 system. Results show progressive D-HFM system development as a dissolution-permeation tool. Results indicated that D-HFM models using MT or CRM provided close agreement between predicted and observed drug permeation profiles. Drug permeation in D-HFM system was volume dependent, as predicted. Favorably, more drug permeated through the D-HFM system (10–20% in 60 min) compared to side-by-side diffusion cell (1%) and dissolution/Caco-2 system (0.1%). Kp′ from D-HFM system was also closer to in vivo kp; the two other in vitro models showed lower Kp′. Overall, studies reflect that HFM module has potential to incorporate drug permeation into the in vitro assessment of in vivo tablet and capsule performance.

Adaptation of phenol-degrading Pseudomonas putida KB3 to suboptimal growth condition: A focus on degradative rate, membrane properties and expression of xylE and cfaB genes

Detailed characterization of new Pseudomonas strains that degrade toxic pollutants is required and utterly necessary before their potential use in environmental microbiology and biotechnology applications. Therefore, phenol degradation by Pseudomonas putida KB3 under suboptimal temperatures, pH, and salinity was examined in this study. Parallelly, adaptive mechanisms of bacteria to stressful growth conditions concerning changes in cell membrane properties during phenol exposure as well as the expression level of genes encoding catechol 2,3-dioxygenase (xylE) and cyclopropane fatty acid synthase (cfaB) were determined. It was found that high salinity and the low temperature had the most significant effect on the growth of bacteria and the rate of phenol utilization. Degradation of phenol (300 mg L−1) proceeded 12-fold and seven-fold longer at 10 °C and 5% NaCl compared to the optimal conditions. The ability of bacteria to degrade phenol was coupled with a relatively high activity of catechol 2,3-dioxygenase. The only factor that inhibited enzyme activity by approximately 80% compared to the control sample was salinity. Fatty acid methyl ester (FAMEs) profiling, membrane permeability measurements, and hydrophobicity tests indicated severe alterations in bacteria membrane properties during phenol degradation in suboptimal growth conditions. The highest values of pH, salinity, and temperature led to a decrease in membrane permeability. FAME analysis showed fatty acid saturation indices and cyclopropane fatty acid participation at high temperature and salinity. Genetic data showed that suboptimal growth conditions primarily resulted in down-regulation of xylE and cfaB gene expression.

Recent Experimental Developments in Studying Passive Membrane Transport of Drug Molecules.

The ability to measure the passive membrane permeation of drug-like molecules is of fundamental biological and pharmaceutical importance. Of significance, passive diffusion across the cellular membranes plays an effective role in the delivery of many pharmaceutical agents to intracellular targets. Hence, approaches for quantitative measurement of membrane permeability have been the topics of research for decades, resulting in sophisticated biomimetic systems coupled with advanced techniques. In this review, recent developments in experimental approaches along with theoretical models for quantitative and real-time analysis of membrane transport of drug-like molecules through mimetic and living cell membranes are discussed. The focus is on time-resolved fluorescence-based, surface plasmon resonance, and second-harmonic light scattering approaches. The current understanding of how properties of the membrane and permeant affect the permeation process is discussed.

Microfluidic measurement of individual cell membrane water permeability

This paper reports a microfluidic lab-on-chip for dynamic particle sizing and real time individual cell membrane permeability measurements. To achieve this, the device measures the impedance change of individual cells or particles at up to ten time points after mixing with different media, e.g. dimethyl sulfoxide or DI water, from separate inlets. These measurements are enabled by ten gold electrode pairs spread across a 20 mm long microchannel. The device measures impedance values within 0.26 s after mixing with other media, has a detection throughput of 150 samples/second, measures impedance values at all ten electrodes at this rate, and allows tracking of individual cell volume changes caused by cell osmosis in anisosmotic fluids over a 1.3 s postmixing timespan, facilitating accurate individual cell estimates of water permeability. The design and testing were performed using yeast cells (Saccharomyces cerevisiae). The relationship between volume and impedance in both polystyrene calibration beads as well as the volume-osmolality relationship in yeast were demonstrated. Moreover, we present the first noninvasive and non-optically-based water permeability measurements in individual cells.

Scanning electrochemical microscopy for biosurface imaging

Scanning electrochemical microscopy (SECM) is a powerful technique that can provide chemical identity, quantification, and spatiotemporal information on biosurfaces. The ability of SECM for noninvasive and high-resolution electrochemical imaging has made it valuable for the study of cell phenotypes and functions. This review focuses on the latest advances of SECM technique for the biosurface imaging. The SECM measurements of different biomarkers, including oxygen consumption rate and enzyme activity of cell aggregates, redox state of cardiomyocytes, and bacterial metabolic activity, are introduced. The applicability of SECM on membrane permeability measurements, neurotransmitter measurements, and intracellular measurements is discussed.

Measurement of membrane permeability and the mitochondrial permeability transition.

The mitochondrial permeability transition (PT) is an increase in the inner membrane permeability caused by the opening of a Ca2+-activated high-conductance channel, the so-called PT pore (PTP) or mitochondrial megachannel (MMC). Recent data indicate that F-ATP synthase contributes substantially to the generation of the PTP, yet this hypothesis is the matter of controversy. In this chapter, we will describe an approach to study the pore, i.e., the evaluation of mitochondrial swelling by means of a decrease in the absorbance at 540nm. This method should be useful to resolve apparent discrepancies in the literature and help solve emerging issues on the identity of mitochondrial pores.

Effect of organic loading rate on the performance of a submerged anaerobic membrane bioreactor (SAnMBR) for malting wastewater treatment and biogas production

AbstractBACKGROUNDMost malting plants discharge their wastewater to the sewer system and have to pay high discharge fees. Malting wastewater is rich in nutrients and contains high soluble COD (mostly sugars and organic acids). Hence, it is suited for anaerobic treatment without nutrients addition and can produce biogas simultaneously. The main objective of this study was to investigate the treatability of malting wastewater in a submerged anaerobic membrane bioreactor (SAnMBR) and biogas production under mesophilic temperature conditions (36 ± 1 °C) and variable organic loading rates (OLRs).RESULTSAs the OLR was increased from 1.36 to 3.18 kg COD m‐3 d‐1, the COD removal efficiency decreased from 94.1 ± 2.5% to 90.2 ± 1.4%, the effluent COD increased from 283 ± 121 mg L‐1 to 506 ± 68 mg L‐1, and the biogas production yield decreased from 0.345 ± 0.007 to 0.308 ± 0.025 L g‐1 CODremoved. The BOD5 removal efficiency was consistently above 99%. Methane accounted for 70.9 ± 2.0% of the biogas. Membrane permeability measurements, scanning electron microscopy (SEM), and energy dispersive X‐ray (EDX) spectrometry indicated that the membrane fouling that occurred during operation of the SAnMBR could be removed by a series of physical and chemical cleaning steps.CONCLUSIONSMalting wastewater was successfully treated using a SAnMBR for the first time. The SAnMBR adapted quickly to both gradual and sudden changes in OLR. © 2017 Society of Chemical Industry

Influence of potassium humate on biochemical and agronomic attributes of bean plants grown on saline soil

ABSTRACTTo study the effect of soil application of potassium humate (KH) (0, 70 and 140 Kg ha−1) on agronomic, biochemical attributes, and yield and its components on bean (Phaseolus vulgaris L.) cultivars ‘Bronco’ grown under two sites having different level of salinity (EC = 3.00 or 5.00 ± 0.20 dS m−1). Two field trials were conducted at the Agriculture Test Station, Faculty of Agriculture, Fayoum University, in 2014 and 2015. The obtained results indicated that addition of KH increased all of the studied growth parameters, i.e. plant height, number of leaves, leaf area, stem diameter and canopy dry weight. Moreover addition of KH significantly increased membrane permeability measurements. KH significantly increased chemical composition related to salt tolerant either inorganic e.g. N, P, K, and decreased Na and Cl contents of leaves or organic components e.g. chlorophyll a + b, carotenoids, anthocyanin total phenolics, total flavonoids and proline. DPPH radical-scavenging activity and yield and its components were increased. The soil application of KH was found to be highly effective at improving the growth and yield of bean plants by alleviating the inhibitory effects of soil salinity stress.

Prevention of Synaptic Alterations and Neurotoxic Effects of PAMAM Dendrimers by Surface Functionalization.

One of the most studied nanocarriers for drug delivery are polyamidoamine (PAMAM) dendrimers. However, the alterations produced by PAMAM dendrimers in neuronal function have not been thoroughly investigated, and important aspects such as effects on synaptic transmission remain unexplored. We focused on the neuronal activity disruption induced by dendrimers and the possibility to prevent these effects by surface chemical modifications. Therefore, we studied the effects of fourth generation PAMAM with unmodified positively charged surface (G4) in hippocampal neurons, and compared the results with dendrimers functionalized in 25% of their surface groups with folate (PFO25) and polyethylene glycol (PPEG25). G4 dendrimers significantly reduced cell viability at 1 µM, which was attenuated by both chemical modifications, PPEG25 being the less cytotoxic. Patch clamp recordings demonstrated that G4 induced a 7.5-fold increment in capacitive currents as a measure of membrane permeability. Moreover, treatment with this dendrimer increased intracellular Ca2+ by 8-fold with a complete disruption of transients pattern, having as consequence that G4 treatment increased the synaptic vesicle release and frequency of synaptic events by 2.4- and 3-fold, respectively. PFO25 and PPEG25 treatments did not alter membrane permeability, total Ca2+ intake, synaptic vesicle release or synaptic activity frequency. These results demonstrate that cationic G4 dendrimers have neurotoxic effects and induce alterations in normal synaptic activity, which are generated by the augmentation of membrane permeability and a subsequent intracellular Ca2+ increase. Interestingly, these toxic effects and synaptic alterations are prevented by the modification of 25% of PAMAM surface with either folate or polyethylene glycol.

Rheological Droplet Interface Bilayers (rheo-DIBs): Probing the Unstirred Water Layer Effect on Membrane Permeability via Spinning Disk Induced Shear Stress

A new rheological droplet interface bilayer (rheo-DIB) device is presented as a tool to apply shear stress on biological lipid membranes. Despite their exciting potential for affecting high-throughput membrane translocation studies, permeability assays conducted using DIBs have neglected the effect of the unstirred water layer (UWL). However as demonstrated in this study, neglecting this phenomenon can cause significant underestimates in membrane permeability measurements which in turn limits their ability to predict key processes such as drug translocation rates across lipid membranes. With the use of the rheo-DIB chip, the effective bilayer permeability can be modulated by applying shear stress to the droplet interfaces, inducing flow parallel to the DIB membranes. By analysing the relation between the effective membrane permeability and the applied stress, both the intrinsic membrane permeability and UWL thickness can be determined for the first time using this model membrane approach, thereby unlocking the potential of DIBs for undertaking diffusion assays. The results are also validated with numerical simulations.

Multicomponent transport of alcohols in an anion exchange membrane measured by in-situ ATR FTIR spectroscopy

Multicomponent transport through membranes is encountered in many applications, including photoelectrochemical CO2 reduction devices that convert CO2 into alcohols. We report the use of in-situ ATR FTIR spectroscopy to quantify the permeability of Selemion AMV, a commonly used anion exchange membrane, to mixtures of alcohols. An in-situ ATR FTIR spectrophotometer probe inserted into a standard diffusion cell enabled straightforward measurement of membrane permeability in multicomponent transport experiments without the need to periodically remove aliquots from the diffusion cell. The solubilities of alcohols in Selemion AMV were measured using a standard sorption/desorption technique. The solution-diffusion model was used to calculate alcohol diffusivities in Selemion AMV from measured permeabilities and solubilities. The relative contributions of alcohol solubility and diffusivity to overall permeability are discussed, and changes in permeability, solubility, and diffusivity with changing composition in binary and ternary alcohol mixtures are described.

Permeability Coefficients of Lipophilic Compounds Estimated by Computer Simulations.

The ability of a drug to cross the intestine-blood barrier is a key quantity for drug design and employment and is normally quantified by the permeability coefficient P, often evaluated in the so-called Caco-2 assay. This assay is based on measuring the initial growth rate of the concentration of the drug beyond the cellular barrier but not its steady-state flux through the membrane. This might lead to confusion since, in the case of lipophilic drugs, the initial slope is strongly affected by the retention of the drug in the membrane. This effect is well known but seldom considered in the assay. Here, we exploit all-atoms molecular dynamics and bias exchange metadynamics to calculate the concentration of two lipophilic drugs across a model membrane as a function of time. This allows estimating both the steady-state flux and the initial slope of the concentration growth and comparing Caco-2 and steady-state estimates of P. We show that our computational procedure is able to reproduce the experimental values, although these may differ from the permeability coefficients by orders of magnitude. Our findings are generalized by a simplified one-dimensional model of the permeation process that may act as a roadmap to assess which measure of membrane permeability would be more appropriate and, consequently, whether retention corrections should be included in estimates based on Caco-2 assays.

Ethanol adaptation induces direct protection and cross-protection against freezing stress in Salmonella enterica serovar Enteritidis.

Salmonella enterica serovar Enteritidis (Salm.Enteritidis) encounters mild ethanol stress during its life cycle. However, adaptation to a stressful condition may affect bacterial resistance to subsequent stresses. Hence, this work was undertaken to investigate the influences of ethanol adaptation on stress tolerance of Salm.Enteritidis. Salmonella Enteritidis was subjected to different ethanol adaptation treatments (2·5-10% ethanol for 1h). Cellular morphology and tolerance to subsequent environmental stresses (15% ethanol, -20°C, 4°C, 50°C and 10% NaCl) were evaluated. It was found that 10% was the maximum ethanol concentration that allowed growth of the target bacteria. Ethanol adaptation did not cause cell-surface damage in Salm.Enteritidis as revealed by membrane permeability measurements and electron micrograph analysis. Salmonella Enteritidis adapted with 2·5-10% ethanol displayed an enhanced resistance to a 15%-ethanol challenge compared with an unchallenged control. The maximum ethanol resistance was observed when ethanol concentration used for ethanol adaptation was increased to 5·0%. Additionally, pre-adaptation to 5·0% ethanol cross-protected Salm.Enteritidis against -20°C, but not against 4°C, 50°C or 10% NaCl. Ethanol adaptation provided Salm.Enteritidis direct protection from a high level ethanol challenge and cross-protection from freezing, but not other stresses tested (low temperature, high salinity or high temperature). The results are valuable in developing adequate and efficient control measures for Salm.Enteritidis in foods.

High membrane permeability for melatonin.

The pineal gland, an endocrine organ in the brain, synthesizes and secretes the circulating night hormone melatonin throughout the night. The literature states that this hormone is secreted by simple diffusion across the pinealocyte plasma membrane, but a direct quantitative measurement of membrane permeability has not been made. Experiments were designed to compare the cell membrane permeability to three indoleamines: melatonin and its precursors N-acetylserotonin (NAS) and serotonin (5-HT). The three experimental approaches were (1) to measure the concentration of effluxing indoleamines amperometrically in the bath while cells were being dialyzed internally by a patch pipette, (2) to measure the rise of intracellular indoleamine fluorescence as the compound was perfused in the bath, and (3) to measure the rate of quenching of intracellular fura-2 dye fluorescence as indoleamines were perfused in the bath. These measures showed that permeabilities of melatonin and NAS are high (both are uncharged molecules), whereas that for 5-HT (mostly charged) is much lower. Comparisons were made with predictions of solubility-diffusion theory and compounds of known permeability, and a diffusion model was made to simulate all of the measurements. In short, extracellular melatonin equilibrates with the cytoplasm in 3.5 s, has a membrane permeability of ∼1.7 µm/s, and could not be retained in secretory vesicles. Thus, it and NAS will be "secreted" from pineal cells by membrane diffusion. Circumstances are suggested when 5-HT and possibly catecholamines may also appear in the extracellular space passively by membrane diffusion.

Effect of intercellular junction protein expression on water transport during freezing of MIN6 cells

A mouse insulinoma (MIN6) strain in which connexin expression has been inhibited by antisense technology holds promise as an experimental model system for investigating the role of gap junctions in intercellular ice propagation. However, to properly interpret measurements of intracellular ice formation kinetics, the effects of cell dehydration on cytoplasmic supercooling must be determined. Thus, the cell membrane water permeability in monolayer cultures of the antisense-transfected MIN6 strain was measured using a fluorescence quenching method. By repeating the experiments at 4°C, 12°C, 21°C, and 37°C, the activation energy for water transport was determined to be Ea=51±3kJ/mol. Although differences between membrane permeability measurements in theantisense and wild-type strains were not statistically significant, simulation of water transport during rapid freezing (130°C/min) predicted that intracellular supercooling in the genetically modified MIN6 strain may become significantly larger than the supercooling in wild-type cells at temperatures below −15°C.