Aquaporin-1 Facilitates Transmesothelial Water Permeability: In Vitro and Ex Vivo Evidence and Possible Implications in Peritoneal Dialysis

We previously showed that mesothelial cells in human peritoneum express the water channel aquaporin 1 (AQP1) at the plasma membrane, suggesting that, although in a non-physiological context, it may facilitate osmotic water exchange during peritoneal dialysis (PD). According to the three-pore model that predicts the transport of water during PD, the endothelium of peritoneal capillaries is the major limiting barrier to water transport across peritoneum, assuming the functional role of the mesothelium, as a semipermeable barrier, to be negligible. We hypothesized that an intact mesothelial layer is poorly permeable to water unless AQP1 is expressed at the plasma membrane. To demonstrate that, we characterized an immortalized cell line of human mesothelium (HMC) and measured the osmotically-driven transmesothelial water flux in the absence or in the presence of AQP1. The presence of tight junctions between HMC was investigated by immunofluorescence. Bioelectrical parameters of HMC monolayers were studied by Ussing Chambers and transepithelial water transport was investigated by an electrophysiological approach based on measurements of TEA+ dilution in the apical bathing solution, through TEA+-sensitive microelectrodes. HMCs express Zo-1 and occludin at the tight junctions and a transepithelial vectorial Na+ transport. Real-time transmesothelial water flux, in response to an increase of osmolarity in the apical solution, indicated that, in the presence of AQP1, the rate of TEA+ dilution was up to four-fold higher than in its absence. Of note, we confirmed our data in isolated mouse mesentery patches, where we measured an AQP1-dependent transmesothelial osmotic water transport. These results suggest that the mesothelium may represent an additional selective barrier regulating water transport in PD through functional expression of the water channel AQP1.

Site correlations, capacitance, and polarizability from protein protonisation fluctuations

We generalize the Kirkwood-Shumaker theory of protonisation fluctuation for an anisotropic distribution of dissociable charges on a globular protein. The fluctuations of the total charge and the total dipole moment, in contrast to their average values, depend on the same proton occupancy correlator, thus exhibiting a similar dependence also on the solution pH. This has important consequences for the Kirkwood-Shumaker interaction and its dependence on the bathing solution conditions.

Ab Initio Electronic Dielectric “Constant” of Proteins: A Baseline for Electrostatic Interaction in Biomolecular Systems

<p>The protein dielectric constant reflects the molecular heterogeneity of the proteins and can be decomposed into different components depending on the size, structure, composition, locality, and environment of the protein in general. The long history of its computation and measurement attest to the vital importance of electrostatic interactions in protein physics that engendered diverse theoretical approaches based often on scattered methodologies with various adjustable parameters. We present a new robust computational method anchored in rigorous <i>ab initio</i> quantum mechanical calculation of explicit atomistic models, without any indeterminate parameters to compute and gain insight into the <i>electronic component</i> of the static dielectric constants of small proteins under different conditions. We implement the new methodology to the 20 canonical amino acids individually, a polypeptide RGD-4C (1FUV) in different environments, and the SD1 domain in the Spike protein of SARS-COV-2. The calculated electronic dielectric constants for 1FUV and SD1 in vacuum are 28.06 and 50.02 respectively. They decrease in the presence of aqueous bathing solution.</p>

Ab Initio Electronic Dielectric “Constant” of Proteins: A Baseline for Electrostatic Interaction in Biomolecular Systems

<p>The protein dielectric constant reflects the molecular heterogeneity of the proteins and can be decomposed into different components depending on the size, structure, composition, locality, and environment of the protein in general. The long history of its computation and measurement attest to the vital importance of electrostatic interactions in protein physics that engendered diverse theoretical approaches based often on scattered methodologies with various adjustable parameters. We present a new robust computational method anchored in rigorous <i>ab initio</i> quantum mechanical calculation of explicit atomistic models, without any indeterminate parameters to compute and gain insight into the <i>electronic component</i> of the static dielectric constants of small proteins under different conditions. We implement the new methodology to the 20 canonical amino acids individually, a polypeptide RGD-4C (1FUV) in different environments, and the SD1 domain in the Spike protein of SARS-COV-2. The calculated electronic dielectric constants for 1FUV and SD1 in vacuum are 28.06 and 50.02 respectively. They decrease in the presence of aqueous bathing solution.</p>

Hyperthermia, bladder pressure, and intravesical drug delivery.

469 Background: Little is known about the pharmacokinetics of intravesical chemotherapies. Various parameters can be altered including temperature, dwell time, drug concentration, and bladder pressure. Here, we hypothesize that increasing bladder pressure during instillation will improve drug delivery. Methods: An ex-vivo porcine model was developed to evaluate determinants of drug penetration into the bladder wall. Porcine bladders were suspended in isotonic saline at 37°C with a three-way Foley catheter in the bladder. Temperature probes were positioned in the extravesical bathing solution, bladder lumen, and sutured to the detrusor to ensure maintenance of desired temperatures. 2g gemcitabine in 100mL normal saline was heated to 43°C and circulated through the bladder using the Combat Bladder Recirculation System. Bladder pressures were monitored throughout each trial. After 60 minutes of dwell time, rapid dissection was performed to obtain full-thickness bladder samples from the bladder dome, posterior wall, trigone, and left and right lateral walls. Tissue was homogenized and liquid chromatography with tandem mass spectrometry (LC/MS/MS) was performed to measure gemcitabine concentration within the bladder wall. Linear regression and Pearson correlation were performed to determine the association between mean bladder pressure during instillation and drug concentration within the bladder wall. Multiple linear regression was conducted to control for bladder location and thickness. Results: Gemcitabine concentration within the bladder wall was measured 25 times across five trials. Mean gemcitabine concentration within bladder wall was 3.68 mg/g (sd 1.35). Pressure ranged from 149.8 mmHg to 277.7 mmHg (mean 194.8, sd 22.0). On univariate analysis, higher pressure was associated with increased gemcitabine concentration within the bladder wall (correlation = 0.49, p = 0.013). This result persisted after adjusting for bladder location (ß = 0.49, p = 0.006) and thickness (ß = 0.70, p = 0.03). Unstandardized regression coefficient in each of the models was 0.099 (mmHg x g)/mg, demonstrating that for each pressure increase of 10mmHg there was an associated increase in gemcitabine concentration of approximately 1 mg/g (Table). Conclusions: Data suggest that bladder pressure dramatically improves the extent of gemcitabine penetration into the bladder wall. Future research is needed to evaluate the therapeutic effect of increased gemcitabine delivery to target tissue in patients with bladder cancer. [Table: see text]

Dye inhibition of phosphate transport in everted duodenal sacs of mice

Phenol Red has been widely used to test kidney function in man. Using the simple, everted gut sac technique has been observed to inhibit the phosphate transport by phenol red in the mouse intestine. We wanted to see if other similar organic anions are able to inhibit the phosphate transport across the mouse intestine. Both uptake and release of phosphate by the everted duodenal sacs of mice are inhibited by phenol red, bromocresol green and bromophenol blue. At the highest dose all the dyes were able to inhibit both influx and efflux significantly. Loss of phosphate from bathing solution is taken as influx and the gain of phosphate by the solution within the sac is taken efflux. At higher dosages a trend of increase in E/I% was noted. At the highest dose all the dyes were able to increase this parameter significantly over the control. Influx appears to be the primary process to be affected. Possible use of phenol red, on account of its safety in humans, as a hypophosphatemic agent is suggested.

Radiation-Activated Pre-Differentiated Retinal Tissue Monitored by Acoustic Wave Biosensor

A thickness-shear mode acoustic wave biosensor operated within a flow-through system was used to examine the response of mouse retinal tissue to radiation. Control experiments conducted with respect to exposure of the bare gold electrodes of the device under various conditions of light intensity and bathing solution yielded reversible changes in resonant frequency (Fs) and motional resistance (Rm). The magnitude of transient changes was proportional to light intensity, but independent of solution type. These alterations in acoustic parameters were ascribed to acoustic coupling phenomena at the electrode-to-liquid interface. Pre-differentiated retina from mouse samples deposited on the thickness shear mode (TSM) electrode exposed to a high light intensity condition also exhibited reversible changes in both Fs and Rm, compared to control experiments involving a coating used to attach the tissue to the electrode. In this case, the radiation-instigated reversible responses for both acoustic parameters exhibited a reduction in magnitude. The changes are ascribed to the alteration in viscoelasticity of the retinal matrix on the TSM electrode surface. The precise biophysical mechanism responsible for the changes in Fs and Rm remains a challenge, given the complex make up of retinal tissue.

Water Influx through the Wetted Surface of a Sweet Cherry Fruit: Evidence for an Associated Solute Efflux

Sweet cherries are susceptible to rain-cracking. The fruit skin is permeable to water, but also to solutes. The objectives of this study were to (1) establish whether a solute efflux occurs when a sweet cherry fruit is incubated in water; (2) identify the solutes involved; (3) identify the mechanism(s) of efflux; and (4) quantify any changes in solute efflux occurring during development and storage. Solute efflux was gravimetrically measured in wetted fruit as the increasing dry mass of the bathing solution, and anthocyanin efflux was measured spectrophotometrically. Solute and anthocyanin effluxes from a wetted fruit and water influx increased with time. All fluxes were higher for the cracked than for the non-cracked fruit. The effluxes of osmolytes and anthocyanins were positively correlated. Solute efflux depended on the stage of development and on the cultivar. In ‘Regina’, the solute efflux was lowest during stage II (25 days after full bloom (DAFB)), highest for mid-stage III (55 DAFB), and slightly lower at maturity (77 DAFB). In contrast with ‘Regina’, solute efflux in ‘Burlat’ increased continuously towards maturity, being 4.8-fold higher than in ‘Regina’. Results showed that solute efflux occurred from wetted fruit. The gravimetrically determined water uptake represents a net mass change—the result of an influx minus a solute efflux.

Fengycin induces ion channels in lipid bilayers mimicking target fungal cell membranes

The one-sided addition of fengycin (FE) to planar lipid bilayers mimicking target fungal cell membranes up to 0.1 to 0.5 μM in the membrane bathing solution leads to the formation of well-defined and well-reproducible single-ion channels of various conductances in the picosiemens range. FE channels were characterized by asymmetric conductance-voltage characteristic. Membranes treated with FE showed nonideal cationic selectivity in potassium chloride bathing solutions. The membrane conductance induced by FE increased with the second power of the lipopeptide aqueous concentration, suggesting that at least FE dimers are involved in the formation of conductive subunits. The pore formation ability of FE was not distinctly affected by the molecular shape of membrane lipids but strongly depended on the presence of negatively charged species in the bilayer. FE channels were characterized by weakly pronounced voltage gating. Small molecules known to modify the transmembrane distribution of electrical potential and the lateral pressure profile were used to modulate the channel-forming activity of FE. The observed effects of membrane modifiers were attributed to changes in lipid packing and lipopeptide oligomerization in the membrane.