Defining developmental trajectories of prosensory cells in human inner ear organoids at single-cell resolution.

The inner ear sensory epithelia contain mechanosensitive hair cells and supporting cells. Both cell types arise from SOX2-expressing prosensory cells, but the mechanisms underlying the diversification of these cell lineages remain unclear. To determine the transcriptional trajectory of prosensory cells, we established a SOX2-2A-ntdTomato human embryonic stem cell line using CRISPR/Cas9, and performed single-cell RNA-sequencing analyses with SOX2-positive cells isolated from inner ear organoids at various time points between differentiation days 20 and 60. Our pseudotime analysis suggests that vestibular type II hair cells arise primarily from supporting cells, rather than bi-fated prosensory cells in organoids. Moreover, ion channel- and ion-transporter-related gene sets were enriched in supporting cells versus prosensory cells, whereas Wnt signaling-related gene sets were enriched in hair cells versus supporting cells. These findings provide valuable insights into how prosensory cells give rise to hair cells and supporting cells during human inner ear development, and may provide a clue to promote hair cell regeneration from resident supporting cells in individuals with hearing loss or balance disorders.

New Method to Probe the Surface Properties of Polymer Thin Films by Two-Dimensional (2D) Inverse Gas Chromatography (iGC).

Polymer-based functional surface coatings are extensively used in advanced technologies, including optics, energy, and environmental applications. Surface thermodynamic properties profoundly impact the molecular interactions that control interfacial behaviors, such as adhesion and wettability, which in turn dictate coating processes and performance. Conventionally, contact angle measurements are used to assess the surface energy of polymer films and coatings, where the wettability of a surface is assessed using probe fluids (liquid drops). However, contact angle measurement oftentimes can be nontrivial due to the roughness or chemical heterogeneity of the solid surface, as well as the potential for the liquid drop to swell or even dissolve the material being measured. Alternatively, inverse gas chromatography (iGC) is a versatile technique to measure surface thermodynamics and Lewis acid-base properties while also providing environmental control such as temperature and humidity. Despite these benefits, the application of iGC has been limited to powders or fibers, while the direct measurement of supported thin films or coatings is still a nascent area of research. This creates a challenge when using iGC as a comprehensive platform for measuring the physicochemical properties of solid surfaces. Here, we demonstrate how to effectively use iGC to characterize the surface energy of supported polymer thin films by using a two-dimensional (2D) film holder and modifying operational controls, such as the concentration range of the injected gas probe molecules. This enables the precise control of surface coverage required for analyzing samples having minimal surface area, such as thin films. Poly(methyl methacrylate) (PMMA) was employed as a benchmark to determine suitable iGC parameters and to validate our approach on polymer thin films. The seminal work presented here expands the capability of state-of-the-art iGC to embrace supported thin films (2D iGC) that could either be smooth or display texture/roughness (patterned films) as well as coatings with heterogeneous chemical/structural composition.

Thickness Dependent CO2 Adsorption of Poly(ethyleneimine) Thin Films for Direct Air Capture.

Mesoporous silica impregnated with polyethyleneimine (PEI) has been shown to be a suitable material for the direct air capture (DAC) of CO2. Factors such as CO2 concentration, temperature, and amine loading impact overall capture capacity and amine efficiency by altering diffusional resistance and reaction kinetics. When studied in the impregnated 3-dimensional sorbent material, internal diffusion impacts the evaluation of the reaction kinetics at the air/amine interface. In this work, we designed a novel tandem quartz crystal microbalance with dissipation (QCM-D) and polarization modulation infrared reflective absorption spectroscopy (PM-IRRAS) instrument. CO2 adsorption kinetics of the PEI-based amine layer in a 2-dimensional geometry were studied at a variety of film thicknesses (10 nm to 100 nm), temperatures (25 °C to 80 °C), and CO2 concentrations (5 % and 0.04 % by mole fraction). Total CO2 capture capacity increased with film thickness but decreased amine efficiency, as additional diffusional resistance for thicker films limits access to available amine sites. The capture capacity of thick films (>50 nm) is shown to be limited by amine availability, while capture of thin films (<50 nm) is limited by CO2 availability. A 50 nm PEI film was shown to be optimal for capture of 0.04 % (400 ppm) CO2. The adsorption profiles for these conditions were fitted to pseudo-first order and Avrami fractional order models. The reaction process switches between a diffusion limited reaction to a kinetic limited reaction at 80 °C when using 5 % CO2 and 55 °C when using 0.04 % CO2. These results offer accurate analysis of adsorption of CO2 at the air/amine interface of PEI films which can be used for the design of future sorbent materials.

Trace Water Effects on Crystalline 1-Ethyl-3-methylimidazolium Acetate.

Spontaneous room-temperature crystallization of 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) was observed upon removal of trace water. Sample purity was confirmed using analytical nuclear magnetic resonance spectroscopy to ensure that trace water or other contaminants did not produce this observation. Raman spectroscopy and simultaneous quartz crystal microbalance/infrared spectroscopy measurements were used to study molecular reorganization during crystallization and decrystallization using trace water in the form of atmospheric moisture. These experimental results were supplemented with density functional theory calculations that indicate imidazolium cation ring stacking and side chain clustering with an exclusive arrangement of the acetate anion in the cation ring plane upon water removal. Crystal structure formation was confirmed using two-dimensional wide-angle X-ray scattering. This natural crystallization is attributed to the removal of trace water over extended periods of time and calls attention to the molecular-level role of water in the structure of hygroscopic ionic liquid systems.

Generating high-fidelity cochlear organoids from human pluripotent stem cells.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.

100th Anniversary of Macromolecular Science Viewpoint: Integrated Membrane Systems.

Membranes fabricated from self-assembled materials are one recent example of how polymer science has been leveraged to advance membrane technology. Due to their well-defined nanostructures, the performance of membranes made from these materials is pushing the boundaries of size-selective filtration. Still, there remains a need for higher performance and more selective membranes. The advent of functional membrane platforms that rely on mechanisms beyond steric hindrance (e.g., charge-selective membranes and membrane sorbents) is one approach to realize improved solute-solute selectivity and further advance membrane technology. To date, the lab-scale demonstration of these platforms has often relied on fabrication schemes that require extended processing times. However, in order to translate lab-scale demonstrations to larger-scale implementation, it is critical that the rate of the functionalization scheme is reconciled with membrane manufacturing rates. In this viewpoint, it is postulated that substrates lined by reactive moieties that are amenable to postfabrication modification would enable the production of membranes with controlled nanostructures while providing access to a diverse array of pore wall chemistries. A comparison of reaction and manufacturing rates suggests that mechanisms that exhibit second-order reaction rate constants of at least 1 M-1 s-1 are needed for roll-to-roll processing. Furthermore, for mechanisms that exhibit rate constants greater than 300 M-1 s-1, it may be possible to integrate multiple functional domains over the membrane surface such that useful properties emerge. These multifunctional systems can expand the capabilities of membranes when the patterned chemistries interact at the heterojunctions between domains (e.g., Janus and charge-patterned mosaic membranes) or if they exhibit cooperative responses to external operating conditions (e.g., membrane pumps).

Dual-Functional Nanofiltration Membranes Exhibit Multifaceted Ion Rejection and Antifouling Performance.

Charged functional groups are often incorporated onto the surface of nanofiltration (NF) membranes to facilitate the selective rejection of multivalent ions over monovalent ions. However, since fouling-resistant surfaces tend to be electrically neutral, the incorporation of charged functionality exacerbates membrane fouling. Multifunctional Janus membrane architectures, which incorporate chemically distinct domains over their cross section, provide a strategy for balancing the competing demands associated with making fouling-resistant, ion rejecting NF membranes. Here, through the controlled exposure of poly(trifluoroethyl methacrylate-co-oligo-(ethylene glycol) methyl ether methacrylate-co-(3-azido-2-hydroxypropyl methacrylate)) copolymer substrates to a series of reactive solutions containing alkyne-terminated molecules, the process for creating dual-functional membranes by using the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction was analyzed. Under the appropriate conditions, the CuAAC reaction propagates into the copolymer substrate as a front. This phenomenon results in a process for creating layered domains of distinct functionality whereby the distribution of antifouling zwitterionic moieties and ion rejecting sulfonate moieties can be modified by manipulating the exposure time. The ion rejection and fouling propensity for a family of dual-functional membranes was examined. For short initial reaction times, which introduced a thin antifouling layer on top of an ion rejection layer, the rejection of 1 mM K2SO4, 87%, was comparable to the value for full charge control membranes, 90%. Moreover, when exposed to a fouling solution containing bovine serum albumin (BSA), these dual-functional membranes exhibited an 18% decline in normalized flux and recovered 99% of their flux upon rinsing with water. In comparison, the full charge membranes exhibited a 44% decline in normalized flux and recovered 65% of their flux upon washing. As such, the results demonstrate that the controlled functionalization process reported here is capable of balancing antifouling and ion rejection capabilities. Furthermore, the versatile nature of the click chemistry mechanism at the center of this process offers a means by which to design and fabricate multifunctional membranes for numerous future applications.

Interfacial Junctions Control Electrolyte Transport through Charge-Patterned Membranes.

Distinct transport mechanisms emerge when nanostructured substrates are patterned with multiple chemistries. For example, charge-patterned mosaic membranes possess surfaces functionalized with discrete domains of both positive and negative charge. These oppositely charged domains provide pathways for both the cation and anion from a dissolved salt to permeate through the membrane without violating the macroscopic constraint of electroneutrality. Here, by systematically varying the geometry and size of the charge pattern, we elucidate the molecular interactions that promote the transport of salts under the action of pressure-driven flow. For patterns that consist of equivalent areal coverages of positively charged and negatively charged domains, the effects of the geometric parameters were encapsulated in a single variable, the interfacial packing density, that quantified the fraction of the membrane surface covered by junctions between oppositely charged domains. Experimentally, the transport of symmetric electrolytes (i.e., KCl and MgSO4) increased with the value of the interfacial packing density, while the interfacial packing density did not significantly affect the transport of asymmetric electrolytes (i.e., K2SO4 and MgCl2). Simulations of the electrical potential near the membrane surface demonstrate that for symmetric electrolytes the structural charge heterogeneity reduces the barrier to ion partitioning, thereby promoting salt transport through the membranes. For asymmetric electrolytes, the charge heterogeneity skews the local availability of ions from the stoichiometric ratio of the salt, thus hindering salt transport. These findings demonstrate the promise of accessing transport mechanisms, which could find utility in a diverse range of chemical separations and sensing applications, through chemical patterning of membranes.

Exercise effects stress-induced analgesia and spatial learning in rats.

Previous studies indicated that intensity level may be a determining factor in the beneficial or detrimental effects of exercise on spatial memory, as chronic low-intensity level exercise appears to enhance learning and memory which stressful situations may impair. This study examines the effects of different intensity levels of acute exercise (treadmill running) on spatial memory in rats. Using the Morris water maze, spatial learning was measured in animals exposed to treadmill running at low- (20-22 m/min for 25 min daily) and high-intensity (25 m/min for 25 min daily) levels of exercise. A stress control using an electric foot shock was used to examine if the high-intensity exercise was sufficient to serve as a stressor. Stress level was estimated by examining tail flick latencies as a measure of stress-induced analgesia. The results indicate that high-intensity exercise at a level that may not induce an analgesic state is sufficient to impair early acquisition of spatial learning. However, with additional trials, all animals are capable of learning the task. Acute exposure to the electric foot shock impaired learning in the Morris water maze. Surprisingly, across all studies, there was a significantly higher analgesic state post-swim as compared to pre-swim. The results indicate that irrespective of stress level prior to water maze testing, swimming in the Morris water maze repeatedly for short durations of time is enough to induce an analgesic state.

Effects of Ginkgo biloba administered after spatial learning on water maze and radial arm maze performance in young adult rats.

Ginkgo biloba is reported to improve learning and memory in animals. However, many studies do not directly test the effects of Ginkgo on memory because the drug is administered during the learning phase of the experiments. In this study, we examined the effect of 10 mg/kg, 20 mg/kg, or 40 mg/kg G. biloba extract on spatial memory by administering the drug in the interval between training and testing. Rats were tested for long-term reference memory retention in the radial arm maze and in the Morris water maze during daily probe trials in which the hidden platform was removed. G. biloba had no effect on reference memory in either the water maze or radial arm maze. To test short-term working spatial memory using the radial arm maze, animals were removed after receiving the reward from 4 of the 8 arms and were returned to complete the maze 2 h later. While Ginkgo had no effect on working memory, over time animals exposed to Ginkgo learned task better than control animals. Thus, Ginkgo appears to enhance neither short-term working memory nor long-term reference memory, but it may promote learning of spatial information.

Rats recovering from unilateral barrel-cortex ischemia are capable of completing a whisker-dependent task using only their affected whiskers.

Rats use their vibrissae for a variety of exploratory tasks including location of objects and discrimination of texture. This study examines recovery in vibrissal function following a unilateral ischemic injury to the somatosensory cortex. Vibrissal function was examined in adult food-restricted rats performing on a two-texture discrimination device. Animals were trained and tested until the criteria of >80% correct choices was demonstrated on three consecutive days. Ischemic rats were constrained to use the affected whiskers by clipping the ipsilateral vibrissae. One group was tested after ischemia, a second group was trained before ischemia and then tested, and a third group was pre-trained and received whisker stimulation and tested post-ischemia. Nai;ve animals recovering from ischemia took longer to reach criteria than intact or unilateral trimmed control animals. Pre-trained animals with compression ischemia receiving whisker stimulation with sucrose water completed the task to criteria in the fewest number of trials. The results indicate that recovery of vibrissal function occurs following a unilateral ischemic injury. Histological analysis in animals without whisker stimulation indicates that the number of normal appearing cortical barrels following ischemia was inversely correlated to the number of trials needed to complete the behavioral task. This suggests that the natural recovery of the ability to discriminate textures is related to the degree of damage to the barrel cortex. The relationship between cortical barrels and behavioral recovery did not hold for the ischemic animals receiving whisker stimulation. This latter group demonstrated recovery despite marked anatomical lesions suggesting that the intervention influenced reorganization.

Ginkgo biloba promotes short-term retention of spatial memory in rats.

This study examines possible interactions between exposure to Ginkgo biloba extract and enriched environments on the acquisition and retention of spatial learning following massed and spaced trials. After 4 weeks of exposure to either ginkgo or vehicle, 8-week-old rats were tested using a Morris Water Maze in either massed or spaced trials. While ginkgo did not have an effect on maze acquisition or long-term retention, it did promote short-term retention of spatial memory. Following reversal training, ginkgo promoted short-term retention for two groups but impaired retention for a third. These results suggest that ginkgo has powerful effects on short-term retention that vary with training conditions.

Anatomical but not functional recovery from a sciatic nerve crush is enhanced by treatment with testosterone.

PURPOSE: Until recently, there has been a limited amount of research comparing functional and anatomical recovery following nerve injury. Previous studies emphasizing anatomical recovery (such as axonal number) have shown that testosterone promotes regeneration in crushed and transected nerves. The purpose of this study was to assess the effect of testosterone on the functional recovery of the sciatic nerve follow-ing a unilateral crush injury. METHODS: Young adult male Sprague-Dawley rats were injected daily with either 500 micro g testosterone proprionate or vehicle alone. The recovery course was followed for six weeks using functional and behavioral testing. Behavioral tests included a footprint gait analysis (as a measure of motor function), response to a skin pinch, and warm water withdrawal (measures of sensory function). RESULTS: Immediately following surgery, all tests indicated complete denervation to the leg distal to the crush site. Anatomical analysis revealed a 22 % increase in the number of axons in testosterone treated animals at 6 weeks post-crush, but no indication of differences in functional recovery. The results of behavioral testing indicated only minor differences in functional recovery as a result of testosterone treatment. CONCLUSION: The results indicate the need for a detailed comparison between anatomical regeneration and functional recovery. An increase in axon number alone may not be an accurate indicator of successful regeneration.