Hydroxyl-functionalized microporous polymer membranes with tunable para position substituent benzaldehydes for gas separation.

We report hydroxyl-functionalized microporous polymers with tunable benzaldehyde groups for gas separation membranes. These polymers were synthesized via acid-catalyzed Friedel-Crafts polycondensation. The tunability in d-spacing and fractional free volume of these polymers depends on the para position substituents (-H, -F, -Cl, and -Br) of the benzaldehyde. Specifically, the size and polarity of the para position substituent influence the polymer chain-packing structure. Consequently, the hydroxyl-functionalized microporous polymer membrane with a larger para position substituent in the benzaldehyde group exhibited improved gas permeability. This improvement is due to enhanced gas diffusivity resulting from the inefficient polymer chain-packing structure. Furthermore, these membranes demonstrated enhanced CO2 plasticization resistance, attributable to the rigid, contorted polymer structure and the hydrogen bonding interactions between hydroxyl groups. This study provides insights into the relationship between the polymer chain-packing structure, tunable para position substituents, and molecular transport.

Visualizing reactive astrocyte-neuron interaction in Alzheimer's disease using 11C-acetate and 18F-FDG.

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD). However, a clinically validated neuroimaging probe to visualize the reactive astrogliosis is yet to be discovered. Here, we show that PET imaging with 11C-acetate and 18F-fluorodeoxyglucose (18F-FDG) functionally visualizes the reactive astrocyte-mediated neuronal hypometabolism in the brains with neuroinflammation and AD. To investigate the alterations of acetate and glucose metabolism in the diseased brains and their impact on the AD pathology, we adopted multifaceted approaches including microPET imaging, autoradiography, immunohistochemistry, metabolomics, and electrophysiology. Two AD rodent models, APP/PS1 and 5xFAD transgenic mice, one adenovirus-induced rat model of reactive astrogliosis, and post-mortem human brain tissues were used in this study. We further curated a proof-of-concept human study that included 11C-acetate and 18F-FDG PET imaging analyses along with neuropsychological assessments from 11 AD patients and 10 healthy control subjects. We demonstrate that reactive astrocytes excessively absorb acetate through elevated monocarboxylate transporter-1 (MCT1) in rodent models of both reactive astrogliosis and AD. The elevated acetate uptake is associated with reactive astrogliosis and boosts the aberrant astrocytic GABA synthesis when amyloid-ß is present. The excessive astrocytic GABA subsequently suppresses neuronal activity, which could lead to glucose uptake through decreased glucose transporter-3 in the diseased brains. We further demonstrate that 11C-acetate uptake was significantly increased in the entorhinal cortex, hippocampus and temporo-parietal neocortex of the AD patients compared to the healthy controls, while 18F-FDG uptake was significantly reduced in the same regions. Additionally, we discover a strong correlation between the patients' cognitive function and the PET signals of both 11C-acetate and 18F-FDG. We demonstrate the potential value of PET imaging with 11C-acetate and 18F-FDG by visualizing reactive astrogliosis and the associated neuronal glucose hypometablosim for AD patients. Our findings further suggest that the acetate-boosted reactive astrocyte-neuron interaction could contribute to the cognitive decline in AD.

High-Loading Poly(ethylene glycol)-Blended Poly(acrylic acid) Membranes for CO2 Separation.

Poly(ethylene glycol) (PEG) is an amorphous material of interest owing to its high CO2 affinity and potential usage in CO2 separation applications. However, amorphous PEG often has a low molecular weight, making it challenging to form into the membrane. The crystalline high average molar mass poly(ethylene oxide) (PEO) cannot exhibit CO2 separation characteristics. Thus, it is crucial to employ low molecular weight PEG in high molecular weight polymers to increase the CO2 affinity for CO2 separation membranes. In this work, poly(acrylic acid) (PAA)/PEG blend membranes with a PEG-rich phase were simply fabricated by physical mixing with an ethanol solvent. The carbonyl group of the PAA and the hydroxyl group of the PEG formed a hydrogen bond. Furthermore, the thermal stability, glass transition temperature, and surface hydrophilicity of PAA/PEG blend membranes with various PEG concentrations were further characterized. The PAA/PEG(1:9) blend membrane exhibited an improved CO2 permeability of 51 Barrer with high selectivities relative to the other gas species (H2, N2, and CH4; CO2/H2 = 6, CO2/N2 = 63, CO2/CH4 = 21) at 35 °C and 150 psi owing to the enhanced CO2 affinity with the amorphous PEG-rich phase. These PAA/PEG blend membrane permeation characteristics indicate a promising prospect for CO2 capture applications.

Decreased parenchymal arteriolar tone uncouples vessel-to-neuronal communication in a mouse model of vascular cognitive impairment.

Chronic hypoperfusion is a key contributor to cognitive decline and neurodegenerative conditions, but the cellular mechanisms remain ill-defined. Using a multidisciplinary approach, we sought to elucidate chronic hypoperfusion-evoked functional changes at the neurovascular unit. We used bilateral common carotid artery stenosis (BCAS), a well-established model of vascular cognitive impairment, combined with an ex vivo preparation that allows pressurization of parenchymal arterioles in a brain slice. Our results demonstrate that mild (~ 30%), chronic hypoperfusion significantly altered the functional integrity of the cortical neurovascular unit. Although pial cerebral perfusion recovered over time, parenchymal arterioles progressively lost tone, exhibiting significant reductions by day 28 post-surgery. We provide supportive evidence for reduced adenosine 1 receptor-mediated vasoconstriction as a potential mechanism in the adaptive response underlying the reduced baseline tone in parenchymal arterioles. In addition, we show that in response to the neuromodulator adenosine, the action potential frequency of cortical pyramidal neurons was significantly reduced in all groups. However, a significant decrease in adenosine-induced hyperpolarization was observed in BCAS 14 days. At the microvascular level, constriction-induced inhibition of pyramidal neurons was significantly compromised in BCAS mice. Collectively, these results suggest that BCAS uncouples vessel-to-neuron communication-vasculo-neuronal coupling-a potential early event in cognitive decline.

Lamotrigine, an antiepileptic drug, inhibits 5-HT3 receptor currents in NCB-20 neuroblastoma cells.

Lamotrigine is an antiepileptic drug widely used to treat epileptic seizures. Using whole-cell voltage clamp recordings in combination with a fast drug application approach, we investigated the effects of lamotrigine on 5-hydroxytryptamine (5-HT)3 receptors in NCB-20 neuroblastoma cells. Co-application of lamotrigine (1~300 µM) resulted in a concentration-dependent reduction in peak amplitude of currents induced by 3 µM of 5-HT for an IC50 value of 28.2±3.6 µM with a Hill coefficient of 1.2±0.1. These peak amplitude decreases were accompanied by the rise slope reduction. In addition, 5-HT3-mediated currents evoked by 1 mM dopamine, a partial 5-HT3 receptor agonist, were inhibited by lamotrigine co-application. The EC50 of 5-HT for 5-HT3 receptor currents were shifted to the right by co-application of lamotrigine without a significant change of maximal effect. Currents activated by 5-HT and lamotrigine co-application in the presence of 1 min pretreatment of lamotrigine were similar to those activated by 5-HT and lamotrigine co-application alone. Moreover, subsequent application of lamotrigine in the presence of 5-HT and 5-hydroxyindole, known to attenuate 5-HT3 receptor desensitization, inhibited 5-HT3 receptor currents in a concentration-dependent manner. The deactivation of 5-HT3 receptor was delayed by washing with an external solution containing lamotrigine. Lamotrigine accelerated the desensitization process of 5-HT3 receptors. There was no voltage-dependency in the inhibitory effects of lamotrigine on the 5-HT3 receptor currents. These results indicate that lamotrigine inhibits 5-HT3-activated currents in a competitive manner by binding to the open state of the channels and blocking channel activation or accelerating receptor desensitization.

Vasculo-Neuronal Coupling: Retrograde Vascular Communication to Brain Neurons.

Continuous cerebral blood flow is essential for neuronal survival, but whether vascular tone influences resting neuronal function is not known. Using a multidisciplinary approach in both rat and mice brain slices, we determined whether flow/pressure-evoked increases or decreases in parenchymal arteriole vascular tone, which result in arteriole constriction and dilation, respectively, altered resting cortical pyramidal neuron activity. We present evidence for intercellular communication in the brain involving a flow of information from vessel to astrocyte to neuron, a direction opposite to that of classic neurovascular coupling and referred to here as vasculo-neuronal coupling (VNC). Flow/pressure increases within parenchymal arterioles increased vascular tone and simultaneously decreased resting pyramidal neuron firing activity. On the other hand, flow/pressure decreases evoke parenchymal arteriole dilation and increased resting pyramidal neuron firing activity. In GLAST-CreERT2; R26-lsl-GCaMP3 mice, we demonstrate that increased parenchymal arteriole tone significantly increased intracellular calcium in perivascular astrocyte processes, the onset of astrocyte calcium changes preceded the inhibition of cortical pyramidal neuronal firing activity. During increases in parenchymal arteriole tone, the pyramidal neuron response was unaffected by blockers of nitric oxide, GABAA, glutamate, or ecto-ATPase. However, VNC was abrogated by TRPV4 channel, GABAB, as well as an adenosine A1 receptor blocker. Differently to pyramidal neuron responses, increases in flow/pressure within parenchymal arterioles increased the firing activity of a subtype of interneuron. Together, these data suggest that VNC is a complex constitutive active process that enables neurons to efficiently adjust their resting activity according to brain perfusion levels, thus safeguarding cellular homeostasis by preventing mismatches between energy supply and demand. SIGNIFICANCE STATEMENT: We present evidence for vessel-to-neuron communication in the brain slice defined here as vasculo-neuronal coupling. We showed that, in response to increases in parenchymal arteriole tone, astrocyte intracellular Ca2+ increased and cortical neuronal activity decreased. On the other hand, decreasing parenchymal arteriole tone increased resting cortical pyramidal neuron activity. Vasculo-neuronal coupling was partly mediated by TRPV4 channels as genetic ablation, or pharmacological blockade impaired increased flow/pressure-evoked neuronal inhibition. Increased flow/pressure-evoked neuronal inhibition was blocked in the presence of adenosine A1 receptor and GABAB receptor blockade. Results provide evidence for the concept of vasculo-neuronal coupling and highlight the importance of understanding the interplay between basal CBF and resting neuronal activity.

Petasites japonicus Stimulates the Proliferation of Mouse Spermatogonial Stem Cells.

Oriental natural plants have been used as medical herbs for the treatment of various diseases for over 2,000 years. In this study, we evaluated the effect of several natural plants on the preservation of male fertility by assessing the ability of plant extracts to stimulate spermatogonial stem cell (SSC) proliferation by using a serum-free culture method. In vitro assays showed that Petasites japonicus extracts, especially the butanol fraction, have a significant effect on germ cells proliferation including SSCs. The activity of SSCs cultured in the presence of the Petasites japonicus butanol fraction was confirmed by normal colony formation and spermatogenesis following germ cell transplantation of the treated SSCs. Our findings could lead to the discovery of novel factors that activate SSCs and could be useful for the development of technologies for the prevention of male infertility.

Effects of paracrine factors on CD24 expression and neural differentiation of male germline stem cells.

Spermatogonial stem cells (SSCs) are adult male germ cells that develop after birth. Throughout the lifetime of an organism, SSCs sustain spermatogenesis through self-renewal and produce daughter cells that differentiate into spermatozoa. Several studies have demonstrated that SSCs can acquire pluripotency under appropriate culture conditions, thus becoming multipotent germline stem cells (mGSCs) that express markers of pluripotency in culture and form teratomas following transplantation into immunodeficient mice. In the present study, we generated neural precursor cells expressing CD24, a neural precursor marker, from pluripotent stem cell lines and demonstrated that these cells effectively differentiated along a neural lineage in vitro. In addition, we found that paracrine factors promoted CD24 expression during the neural differentiation of mGSCs. Our results indicated that the expression of CD24, enhanced by a combination of retinoic acid (RA), noggin and fibroblast growth factor 8 (FGF8) under serum-free conditions promoted neural precursor differentiation. Using a simple cell sorting method, we were able to collect neural precursor cells with the potential to differentiate from mGSCs into mature neurons and astrocytes in vitro.

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs.

Cation-selective electropreconcentration.

A cation-selective microfluidic sample preconcentration system is described. The cation sample was electropreconcentrated using a reversed-direction electroosmotic flow (EOF) and an anion-permselective filter, where an electric double layer (EDL) overlap condition existed. The anion-permselective filter between microchannels was fabricated by three different methods: 1) extending a positively charged, nanoporous, polymer membrane by photopolymerization of poly(diallyldimethylammonium chloride) (PDADMAC); 2) etching a nanochannel and then coating it with a positively-charged monomer, N-[3-(trimethoxysilyl)propyl]-N'-(4-vinylbenzyl)ethylenediamine hydrochloride (TMSVE); and, 3) etching a nanochannel and then coating it with a positively-charged, pre-formed polymer, polyE-323. The EOF direction in the microchannel was reversed by both TMSVE and polyE-323 coatings. The cation-selective preconcentration was investigated using charged fluorescent dyes and tetramethylrhodamine isothiocyanate (TRITC)-tagged peptides/proteins. The preconcentration in the three different systems was compared with respect to efficiency, dependence on buffer concentration and pH, tolerable flow rate, and sample adsorption. Both TMSVE- and polyE-323-coated nanochannels showed robust preconcentration at high flow rates, whereas the PDADMAC membrane maintained anion-permselectivity at higher buffer concentrations. The TMSVE-coated nanochannels showed a more stable preconcentration process, whereas the polyE-323-coated nanochannels showed a lower peptide sample adsorption and robust efficiency under a wide range of buffer pHs. The system described here can potentially be used for the preconcentration of cationic peptides/proteins on microfluidic devices for subsequent analyses.

Effect of sugar molecules on the cryopreservation of mouse spermatogonial stem cells.

OBJECTIVE: To study the influence of sugars and establish a serum-free freezing method for the cryopreservation of spermatogonial stem cells (SSCs). DESIGN: Animal study. SETTING: University laboratory. ANIMAL(S): C57BL/6-TgEGFP, C57BL/6 mice. INTERVENTION(S): Germ cells enriched from testis cells were frozen using standard freezing medium containing sugars, including monosaccharides, disaccharides, and trisaccharides at 50, 100, and 200 mM, respectively. To study the feasibility of establishing a serum-free freezing method, fetal bovine serum was substituted with knockout serum replacement. MAIN OUTCOME MEASURE(S): Freeze-thawed germ cells were evaluated for recovery rate, proliferation capacity, and stem cell activity after transplantation to recipient testes. RESULT(S): Supplementation of freezing medium with 200 mM disaccharide is an effective method for cryopreservation of SSCs. Trehalose is the most effective cryoprotectant among all the sugars tested and only lactose was comparable to trehalose. Our proliferation and transplantation data show that serum-free freezing can be achieved in freezing medium supplemented with 200 mM trehalose, 10% knockout serum replacement, and 10% dimethyl sulfoxide (DMSO) for cryopreservation of SSCs. CONCLUSION(S): These findings raise the possibility of effectively banking frozen SSCs from various species, including humans, in a traditional serum-free medium for germ cell research and male infertility treatments.

Cryopreservation in trehalose preserves functional capacity of murine spermatogonial stem cells.

Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.

Effects of grape seed proanthocyanidin on 5-hydroxytryptamine(3) receptors in NCB-20 neuroblastoma cells.

Proanthocyanidin is a phenolic compound present in plants, that has antioxidant, antinociceptive, anti-emetic, and neuroprotective properties. We investigated the actions of proanthocyanidin from grape seeds on 5-hydroxytryptamine (5-HT)(3) receptors in NCB-20 neuroblastoma cells using a whole-cell voltage clamp technique. Co-treatment of proanthocyanidin (0.3-100 µg/ml) and 3 µM 5-HT (near EC(50)) produced a slight inhibition of 5-HT-induced inward peak current (I(5-HT)) in NCB-20 cells, but pretreatment with proanthocyanidin for 30 s before application of 5-HT induced a much larger inhibition of I(5-HT) in an irreversible, concentration- and time-dependent manner (IC(50)=6.5±0.4 µg/ml, Hill coefficient=2.5±0.1). Proanthocyanidin also produced a concentration-dependent inhibition of currents induced by 30 µM 5-HT, near-maximal concentration (IC(50)=22.1±0.4 µg/ml, Hill coefficient=2.4±0.1). High concentrations (≧30 µg/ml) of proanthocyanidin caused a concentration-dependent inhibition of the activation and desensitization of currents induced by 30 µM 5-HT. Further studies showed that pretreatment of 20 µg/ml proanthocyanidin caused not only a rightward shift of the dose-response curve for 5-HT (EC(50) shift from 2.7±0.4 to 6.2±0.5 µM), but also a decreased E(max) (inhibition by 37.5±1.3%). The proanthocyanidin-induced inhibition of 5-HT(3) receptors did not show a significant difference within the testing holding potential ranges (-50-+30 mV). These results suggest that proanthocyanidin inhibits 5-HT(3) receptor function in NCB-20 cells in a noncompetitive mode, and that this inhibitory effect of proanthocyanidin probably contributes to the pharmacological actions of proanthocyanidin.

Enrichment of testicular gonocytes and genetic modification using lentiviral transduction in pigs.

Gonocytes are long-lived primary germ cells that reside in the center of seminiferous cords until differentiation into spermatogonia that drive spermatogenesis. In pigs, gonocytes have research value in the production of transgenic offspring through germline modification and transplantation. However, the rarity of pig gonocytes has raised the need for an efficient isolation method. Therefore, in this study we use components of extracellular matrix, laminin, fibronectin, and collagen type IV and their derivative, gelatin, to establish a negative selection system for functionally viable gonocytes in neonatal pig. We then demonstrate functional analysis with genetic modification using lentiviral transduction and successfully transplant the donor gonocytes, which colonized the seminiferous tubules of the recipient mouse. The most effective selection method was established by sequential use of laminin and gelatin, in which the purity of gonocytes was 80% and the recovery rate of gonocytes was 78%. The selected gonocytes were labeled with fluorescent dye PKH26 and transplanted into busulfan-treated immunodeficient mouse testes. The fluorescent gonocytes colonized the recipient testes, and the resultant germ cell colonies were visible up to 4 mo after transplantation. When gonocytes were transplanted after transduction with an enhanced green fluorescent protein marker gene using lentiviral vectors, the transduced germ cell colonies were visible up to 6 mo and displayed an estimated transduction efficiency of 11.1%. These results can be applied and extended to isolate and enrich gonocytes of other species for in vitro and in vivo studies and to assist in genetic modification of male germline stem cells of livestock species.

Direct effects of riluzole on 5-hydroxytryptamine (5-HT)3 receptor-activated ion currents in NCB-20 neuroblastoma cells.

The pharmacological action of riluzole, a drug that has been approved as a neuroprotective agent for the treatment of amyotrophic lateral sclerosis, has not yet been established. We examined the effects of riluzole on 5-hydroxytryptamine (5-HT)3) receptors in NCB-20 neuroblastoma cells using the whole-cell voltage clamp technique combined with a fast drug application method. Co-application of riluzole (1 - 300 microM, 5 s) produced a dose-dependent reduction in peak amplitudes and in the rise slope of the currents induced by 2 microM 5-HT. In addition, 5-HT3-mediated currents evoked by dopamine, a partial 5-HT3-receptor agonist, were inhibited by riluzole co-application. These inhibitory effects were clearly shown at low concentrations of 5-HT. The decay time constants of the receptor desensitization and deactivation were also significantly attenuated by riluzole. G-protein inhibitors (pertussis toxin and guanosine 5'-[beta-thio] diphosphate) did not completely block these inhibitory actions of riluzole. These results indicate that riluzole inhibits 5-HT3-induced ion currents directly by slowing channel activation in NCB-20 neuroblastoma cells.

Effect of 5-hydroxyindole on ethanol potentiation of 5-hydroxytryptamine (5-HT)3 receptor-activated ion current in NCB-20 neuroblastoma cells.

We examined the effect of 5-hydroxyindole (5-HI) on the potentiation of 5-hydroytryptamine (5-HT)(3) receptor function by ethanol (EtOH) so as to study whether EtOH potentiates channel function through increasing activation or blocking desensitization. We measured 5-HT(3) receptor current using a whole-cell voltage clamp technique with a method of rapid drug application in NCB-20 neuroblastoma cells. The 5-HI, an agent that block receptor desensitization, increased the decay time constant (tau), not the peak of 5-HT(3) receptor-mediated currents induced by 10 microM 5-HT. EtOH did not change the peak amplitude and tau of the current induced by 10 microM 5-HT. Coapplication of EtOH and 5-HI with 5-HT caused no increase in the peak currents, but tau was further increased. Therefore, a further block in desensitization could be induced by 5-HI, despite the presence of EtOH. These results indicate that EtOH potentiates 5-HT(3) receptor function, with these effects due at least in part by increasing channel activation rather than by blocking desensitization.