High Density Of Binding Sites Research Articles

Highly Productive C3H4/C3H6 Trace Separation by a Packing Polymorph of a Layered Hybrid Ultramicroporous Material.

Ultramicroporous materials can be highly effective at trace gas separations when they offer a high density of selective binding sites. Herein, we report that sql-NbOFFIVE-bpe-Cu, a new variant of a previously reported ultramicroporous square lattice, sql, topology material, sql-SIFSIX-bpe-Zn, can exist in two polymorphs. These polymorphs, sql-NbOFFIVE-bpe-Cu-AA (AA) and sql-NbOFFIVE-bpe-Cu-AB (AB), exhibit AAAA and ABAB packing of the sql layers, respectively. Whereas NbOFFIVE-bpe-Cu-AA (AA) is isostructural with sql-SIFSIX-bpe-Zn, each exhibiting intrinsic 1D channels, sql-NbOFFIVE-bpe-Cu-AB (AB) has two types of channels, the intrinsic channels and extrinsic channels between the sql networks. Gas and temperature induced transformations of the two polymorphs of sql-NbOFFIVE-bpe-Cu were investigated by pure gas sorption, single-crystal X-ray diffraction (SCXRD), variable temperature powder X-ray diffraction (VT-PXRD), and synchrotron PXRD. We observed that the extrinsic pore structure of AB resulted in properties with potential for selective C3H4/C3H6 separation. Subsequent dynamic gas breakthrough measurements revealed exceptional experimental C3H4/C3H6 selectivity (270) and a new benchmark for productivity (118 mmol g-1) of polymer grade C3H6 (purity >99.99%) from a 1:99 C3H4/C3H6 mixture. Structural analysis, gas sorption studies, and gas adsorption kinetics enabled us to determine that a binding "sweet spot" for C3H4 in the extrinsic pores is behind the benchmark separation performance. Density-functional theory (DFT) calculations and Canonical Monte Carlo (CMC) simulations provided further insight into the binding sites of C3H4 and C3H6 molecules within these two hybrid ultramicroporous materials, HUMs. These results highlight, to our knowledge for the first time, how pore engineering through the study of packing polymorphism in layered materials can dramatically change the separation performance of a physisorbent.

Scalable Green Synthesis of Robust Ultra-Microporous Hofmann Clathrate Material with Record C3 H6 Storage Density for Efficient C3 H6 /C3 H8 Separation.

Developing porous materials for C3 H6 /C3 H8 separation faces the challenge of merging excellent separation performance with high stability and easy scalability of synthesis. Herein, we report a robust Hofmann clathrate material (ZJU-75a), featuring high-density strong binding sites to achieve all the above requirements. ZJU-75a adsorbs large amount of C3 H6 with a record high storage density of 0.818 g mL-1 , and concurrently shows high C3 H6 /C3 H8 selectivity (54.2) at 296 K and 1 bar. Single-crystal structure analysis unveil that the high-density binding sites in ZJU-75a not only provide much stronger interactions with C3 H6 but also enable the dense packing of C3 H6 . Breakthrough experiments on gas mixtures afford both high separation factor of 14.7 and large C3 H6 uptake (2.79 mmol g-1 ). This material is highly stable and can be easily produced at kilogram-scale using a green synthesis method, making it as a benchmark material to address major challenges for industrial C3 H6 /C3 H8 separation.

Engineering Supramolecular Binding Sites in a Chemically Stable Metal-Organic Framework for Simultaneous High C2 H2 Storage and Separation.

Developing porous materials to overcome the trade-off between adsorption capacity and selectivity for C2 H2 /CO2 separation remains a challenge. Herein, we report a stable HKUST-1-like MOF (ZJU-50a), featuring large cages decorated with high density of supramolecular binding sites to achieve both high C2 H2 storage and selectivity. ZJU-50a exhibits one of the highest C2 H2 storage capacity (192 cm3 g-1 ) and concurrently high C2 H2 /CO2 selectivity (12) at 298 K and 1 bar. Single-crystal X-ray diffraction studies on gas-loaded ZJU-50a crystal unveil that the incorporated supramolecular binding sites can selectively take up C2 H2 molecule but not CO2 to result in both high C2 H2 storage and selectivity. Breakthrough experiments validated its separation performance for C2 H2 /CO2 mixtures, providing a high C2 H2 recovery capacity of 84.2 L kg-1 with 99.5 % purity. This study suggests a novel strategy of engineering supramolecular binding sites into MOFs to overcome the trade-off for this separation.

Engineering Supramolecular Binding Sites in a Chemically Stable Metal‐Organic Framework for Simultaneous High C2H2 Storage and Separation

AbstractDeveloping porous materials to overcome the trade‐off between adsorption capacity and selectivity for C2H2/CO2 separation remains a challenge. Herein, we report a stable HKUST‐1‐like MOF (ZJU‐50a), featuring large cages decorated with high density of supramolecular binding sites to achieve both high C2H2 storage and selectivity. ZJU‐50a exhibits one of the highest C2H2 storage capacity (192 cm3 g−1) and concurrently high C2H2/CO2 selectivity (12) at 298 K and 1 bar. Single‐crystal X‐ray diffraction studies on gas‐loaded ZJU‐50a crystal unveil that the incorporated supramolecular binding sites can selectively take up C2H2 molecule but not CO2 to result in both high C2H2 storage and selectivity. Breakthrough experiments validated its separation performance for C2H2/CO2 mixtures, providing a high C2H2 recovery capacity of 84.2 L kg−1 with 99.5 % purity. This study suggests a novel strategy of engineering supramolecular binding sites into MOFs to overcome the trade‐off for this separation.

Selective removal of cationic organic dyes via electrospun nanofibrous membranes derived from polyarylene ethers containing pendent nitriles and sulfonates

Nanofibrous membranes showing selective separation of certain dyes out from multiple organic mixtures are of great importance for many advanced applications. Although various strategies including mixed-matrix-membrane or elaborately designed surface modification have been reported for preparation of these advanced membranes, they are still limited in terms of tedious experimental work and questionable long-term stability. Herein, we have designed and synthesized a range of super-engineering polyarylene ethers containing pendent nucleophilic nitriles and negatively charged sulfonate groups, which have been further processed into nanofibrous membranes (NFM) via the one-step electrospinning technique. Thanks to the presence of high density of surface active binding sites, the electrospun polymeric nanofibers exhibited a high adsorption capacity of 796.25 mg/g for cationic organic dye of methylene blue. More importantly, the optimized polymeric NFM allows rapid selective removal of cationic dyes from their mixtures with other dyes molecules, and the removal efficiency of cationic dyes arrives up to 99% even after eight separation-regeneration cycles. Due to their facile fabrication, highly selective purification feature as well as good recyclability, the protocol revealed in this work would open new way for fabrication of advanced separation membranes for wastewater purification.

Fabrication of 2D nanosheet sorbents through metastable emulsion droplets and subsequent two-step grafting polymerization for efficient blood lead removal in vitro

Hemoperfusion is a powerful and yet simple method for lead poisoning treatment, but creation of safe and effective sorbents with excellent selectivity remains a real challenge. To address this, we here construct 2D nanosheet sorbents (BM-SH) through metastable emulsion droplets and subsequent two-step grafting polymerization for efficient blood lead removal in vitro. Metastable emulsion droplets endow typical nanosized sheet-like structure (thickness of 30 nm) and relatively round shape. The consecutive two grafting processes using hydroxyethyl methacrylate (HEMA) and L-cysteine monomer (D-SH) provide BM-SH with a high density of accessible binding sites towards lead ions (Pb2+). A high adsorption capacity of 390.5 mg g−1 and quick capture 97.35 % of Pb2+ within initial 10 min are obtained, surpassing most of the reported sorbents for lead removal. Besides, adsorption distribution coefficient (Kd) of BM-SH among four coexisting metal ions achieved 7792 mL g−1, showing outstanding selectivity toward Pb2+. Importantly, a possible adsorption mechanism is recognized as coordination with carboxyl, sulfydryl and imino groups from L-cysteine, and mercapto ligand as the key chelating agent may be the reason for high Pb2+ affinity. And what’s more, BM-SH displays good hemocompatibility and high efficiency of blood lead removal rate (above 86 % in vitro).

Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Tobacco Mosaic Virus Bioparticles as Enzyme Nanocarriers.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1–3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.

Selective enrichment of sialylated glycopeptides with mesoporous poly-melamine-formaldehyde (mPMF) material.

Analysis of glycoprotein sialylation is challenging due to the relatively low abundance of sialylated glycopeptides (SGPs) in complex biosamples and low signals of SGPs in mass spectrometry. In this study, a mesoporous poly-melamine-formaldehyde (mPMF) polymer was prepared and utilized as the high-efficiency sorbent for SGPs. The mPMF polymer featured high surface area (755.4m2g-1) and high density of amine and triazine functional groups. This polymer demonstrated high enrichment selectivity (resistant to 100 molarfold interference of BSA) and superior adsorption capacity (560mgg-1) for SGPs. The high performance of mPMF toward SGPs ascribes to the unique physicochemical properties of mPMF and high density of accessible binding sites for glycopeptides. Further application of mPMF to HeLa S3 cell lysate resulted in 576 characterized glycopeptides with 218 unique glycosylation sites. This finding provides a new choice of promising extraction approach for characterization of protein glycosylation. Graphical abstract A mesoporous poly-melamine-formaldehyde (mPMF) polymer was prepared and utilized as the high-efficiency enrichment sorbent for sialylated glycopeptides (SGPs).

Herpes simplex viral nucleoprotein creates a competitive transcriptional environment facilitating robust viral transcription and host shut off.

Herpes simplex virus-1 (HSV-1) replicates within the nucleus coopting the host's RNA Polymerase II (Pol II) machinery for production of viral mRNAs culminating in host transcriptional shut off. The mechanism behind this rapid reprogramming of the host transcriptional environment is largely unknown. We identified ICP4 as responsible for preferential recruitment of the Pol II machinery to the viral genome. ICP4 is a viral nucleoprotein which binds double-stranded DNA. We determined ICP4 discriminately binds the viral genome due to the absence of cellular nucleosomes and high density of cognate binding sites. We posit that ICP4's ability to recruit not just Pol II, but also more limiting essential components, such as TBP and Mediator, create a competitive transcriptional environment. These distinguishing characteristics ultimately result in a rapid and efficient reprogramming of the host's transcriptional machinery, which does not occur in the absence of ICP4.

2-(Allyloxy) methylol-12-crown-4 ether functionalized polymer brushes from porous PolyHIPE using UV-initiated surface polymerization for recognition and recovery of lithium

It is highly desirable to develop an efficient adsorbent with large capacity, high selectivity and fast kinetics to recovery virtually inexhaustible lithium ion (Li+) resources from salt lake brine. In this work, 2-(allyloxy) methylol-12-crown-4 ether (2AM12C4) functionalized polymer brushes (PVBC-g-PCE) from macroporous polymeric high internal-phase emulsion (PolyHIPE) using UV-initiated surface polymerization are synthesized for selectively recovery of Li+. PVBC-g-PCE possesses the advantages of strong physical strength, high density of accessible binding sites, and the size of highly permeatable voids and interconnecting pores is about 8.0–18 µm and 3.3–4.8 µm, respectively, which is a favorable adsorbent for specific capture Li+. PVBC-g-PCE has a maximum capacity at pH 6.0, and the pH-dependent adsorption amount suggests the grafted polymer brushes of 2AM12C4 is the driving force to enhance binding performance. PVBC-g-PCE has a fast adsorption equilibrium within 45 min, and the kinetic results are well-described by the pseudo-second-order model, indicating the specific host-guest interaction between functionalized 2AM12C4 and Li+ is the main adsorption mechanism. Moreover, the maximum monolayer binding amount of PVBC-g-PCE is 4.43 mg g−1, and the strong affinity in the presence of four kinds of interfering ions (i.e. Na+, K+, Mg2+, and Ca2+) is also observed, which can also be proved by the high relative selectivity separation coefficient (αr) between 3.16 and 7.98. It is expected that this PVBC-g-PCE with abundant easy-to-access specific recognition sites can be applied as a promising adsorbent for effective separation of Li+.

Genome-wide identification of clusters of predicted microRNA binding sites as microRNA sponge candidates.

The number of discovered natural miRNA sponges in plants, viruses, and mammals is increasing steadily. Some sponges like ciRS-7 for miR-7 contain multiple nearby miRNA binding sites. We hypothesize that such clusters of miRNA binding sites on the genome can function together as a sponge. No systematic effort has been made in search for clusters of miRNA targets. Here, we, to our knowledge, make the first genome-wide target site predictions for clusters of mature human miRNAs. For each miRNA, we predict the target sites on a genome-wide scale, build a graph with edge weights based on the pairwise distances between sites, and apply Markov clustering to identify genomic regions with high binding site density. Significant clusters are then extracted based on cluster size difference between real and shuffled genomes preserving local properties such as the GC content. We then use conservation and binding energy to filter a final set of miRNA target site clusters or sponge candidates. Our pipeline predicts 3673 sponge candidates for 1250 miRNAs, including the experimentally verified miR-7 sponge ciRS-7. In addition, we point explicitly to 19 high-confidence candidates overlapping annotated genomic sequence. The full list of candidates is freely available at http://rth.dk/resources/mirnasponge, where detailed properties for individual candidates can be explored, such as alignment details, conservation, accessibility and target profiles, which facilitates selection of sponge candidates for further context specific analysis.

Noncovalent, Electrostatic Interactions Induce Positively Cooperative Binding of Small Molecules to Alzheimer's and Parkinson's Disease-Related Amyloids.

Amyloids are self-assembled protein aggregates that represent a major hallmark of many neurologic and systemic diseases. Among the common features of amyloids is the presence of a high density of multiple binding sites for small molecule ligands, making them an attractive target for design of multimeric binding agents. Here, we demonstrate that noncovalent, intermolecular interactions between a 1:1 mixture of oppositely charged benzothiazole molecules enhances their binding to two different amyloid aggregates: Alzheimer's-related amyloid-β (Aβ) peptides or Parkinson's-related α-synuclein (αS) proteins. We show that this mixture leads to positively cooperative binding to amyloid targets, with up to 10-fold enhancement of binding compared to the uncharged parent compound. The observed enhancement of amyloid binding using noncovalent interactions was similar in magnitude to a benzothiazole dimer to aggregated Aβ. These results represent a novel strategy for designing amyloid-targeting molecules with enhanced affinity, which could aid in the development of new diagnostic or treatment strategies for amyloid-associated diseases.

Chemotactic Activity of Site-Specific Multivalent Conjugates of Stromal Cell-Derived Factor 1<i>α</i> on Branched Nanoparticles

Stromal cell-derived factor 1α (SDF1α) is a potent chemokine for the recruitment of stem cells. A challenge is to maintain its activity and control its release. In this study, we engineered a recombinant cysteine-SDF1α (cysSDF1α) protein, and performed multivalent conjugation of cysSDF1 through the maleimide functional group to two forms of branched nanoparticles: multi-arm poly (ethylene glycol) (MA-PEG) and hyaluronic acid (HA). We characterized the chemotactic activity of the conjugates, and determined how the molecular weight (MW) of MA-PEG and HA affected the chemotactic activity. CysSDF1α had similar efficiency to wild-type SDF1α in cell recruitment. Multivalent conjugation of cysSDF1α to low MW MA-PEG (~18 nm) did not significantly affect the chemotactic activity, while the conjugation of cysSDF1α to high MW MA-PEG (~72 nm) lowered the efficiency, possibly due to the larger spacing between conjugated SDF1α molecules. HA has a linear backbone and a high density of multivalent binding sites; however, the chemotactic activity of HA-linked cys-SDF1α was much lower, which further decreased with the increase of HA MW from 200 kDa (~0.78 μm) to 700 kDa (~2.7 μm). Digestion of HA into smaller fragments using hyaluronidase partially recovered the chemotactic activity of cysSDF1α, suggesting that high MW HA might exert steric hindrance for SDF1α binding to its receptors on cell surface and that HA could be used as a depot for SDF1α storage and release. These results demonstrate that multivalent conjugates of SDF1α to nanoparticles may be used to engineer SDF1α delivery for cell recruitment and tissue regeneration.

Investigating the Effect of Ligand Amount and Injected Therapeutic Activity: A Simulation Study for 177Lu-Labeled PSMA-Targeting Peptides.

In molecular radiotherapy with 177Lu-labeled prostate specific membrane antigen (PSMA) peptides, kidney and/or salivary glands doses limit the activity which can be administered. The aim of this work was to investigate the effect of the ligand amount and injected activity on the tumor-to-normal tissue biologically effective dose (BED) ratio for 177Lu-labeled PSMA peptides. For this retrospective study, a recently developed physiologically based pharmacokinetic model was adapted for PSMA targeting peptides. General physiological parameters were taken from the literature. Individual parameters were fitted to planar gamma camera measurements (177Lu-PSMA I&T) of five patients with metastasizing prostate cancer. Based on the estimated parameters, the pharmacokinetics of tumor, salivary glands, kidneys, total body and red marrow was simulated and time-integrated activity coefficients were calculated for different peptide amounts. Based on these simulations, the absorbed doses and BEDs for normal tissue and tumor were calculated for all activities leading to a maximal tolerable kidney BED of 10 Gy2.5/cycle, a maximal salivary gland absorbed dose of 7.5 Gy/cycle and a maximal red marrow BED of 0.25 Gy15/cycle. The fits yielded coefficients of determination > 0.85, acceptable relative standard errors and low parameter correlations. All estimated parameters were in a physiologically reasonable range. The amounts (for 25−29 nmol) and pertaining activities leading to a maximal tumor dose, considering the defined maximal tolerable doses to organs of risk, were calculated to be 272±253 nmol (452±420 μg) and 7.3±5.1 GBq. Using the actually injected amount (235±155 μg) and the same maximal tolerable doses, the potential improvement for the tumor BED was 1–3 fold. The results suggest that currently given amounts for therapy are in the appropriate order of magnitude for many lesions. However, for lesions with high binding site density or lower perfusion, optimizing the peptide amount and activity might improve the tumor-to-kidney and tumor-to-salivary glands BED ratio considerably.

Parkinson’s Disease: New Insights into Pathophysiology and Rehabilitative Approaches

Parkinson's disease (PD) is a multifactorial neurodegenerative disorder, but up till now, its etiology remains largely unknown. Progressive impairment of voluntary motor control, which represents the primary clinical feature of the disease, is caused by a loss of midbrain substantia nigra dopamine (DA) neurons. PD prevalence increases steadily with age. Therefore, with the aging population, we will see an increase in social and health impact of this disease. Currently, the drugs available improve physical performances and lengthen life expectancy and autonomy in the activities of daily living by reducing adverse events; it follows that they improve the quality of life of patients. However, these drugs are symptomatic and not curative. When the patient reaches the maximum allowable therapeutic dosage of levodopa, we have to face great difficulties and impotence in disease's management. Recently, the role of rehabilitation in the field of neurodegenerative diseases is becoming more and more important as it results from several studies. In this special issue, we wanted to focus our attention on the “holistic view” of the disease because we believe that only a multidisciplinary approach could help to face it in the best way. PD involves miscellaneous systems (physical, mental, and behavioral structures) affecting also the autonomy in self-care management. Furthermore, in this special issue we wanted to draw the attention to the importance of rehabilitation in PD. An amount of epidemiological evidence stressed the importance of the relationships between genetic and environmental factors. L. Polito et al. summarize evidence on gene-environment interplay in the development of PD focusing on susceptibility factors and causal genetic mutation. To the best of our knowledge, this review is the first attempt to analyze all those genetic factors that modify the impact of environmental exposure. Brilliantly, F. Magrinelli et al. contributed to examining the neuropathology, neuropharmacology, and neurophysiology of motor dysfunction of PD, with particular attention to those clinical features that impair quality of life and functional ability of patients. Starting from anatomic basis of the main circuit involved in PD, subsequently they analyzed the pathophysiology of PD motor symptoms (bradykinesia and tremor rigidity) and signs (postural balance, gait impairment, fatigue, and pain). Currently, pharmacological treatment of motor symptoms of PD depends mainly on the administration of dopaminergic drugs (levodopa and dopamine agonists) and other drugs involved in levodopa metabolism (monoamine oxidase-B inhibitors and catechol-O-methyltransferase inhibitors). Usually, in the late disease stages, PD patients treated by dopaminergic drugs display various complications, such as on-off fluctuations of Parkinsonian symptoms and dyskinesia. Unfortunately, most patients with advanced PD are unsatisfactorily controlled by the usual therapeutic approach. A. Daniele et al. examine other pharmacological targets regardless of the dopaminergic pathway. In particular, they focus on potential beneficial effects of zolpidem, a positive allosteric modulator of GABAA receptors, on the motor symptoms of PD. In fact, some studies have previously suggested that there is a high density of zolpidem binding sites in the globus pallidus and in the substantia nigra, which are the output structures of the basal ganglia. Two experimental works complete this brief special issue. The first is a case control study carried out by M. Tramontano et al. with the aim of investigating the efficacy of a blindfolded balance training in the improvement of gait parameters in people with PD compared to patients who underwent a standard physical therapy program. The second is a prospective open-label feasibility study evaluating the impact of 8-week action observation training (video-therapy) for the treatment of postural instability and balance impairment in PD patients. Although in this latter study A. Santamato et al. did not find positive results, they underline one of the most important shortcomings in PD rehabilitation field. In fact, absence of standard procedures to improve balance and posture in PD subjects makes this area a virgin field to explore. We hope that the contents of this special issue may help clinicians better understand the rationale of current pharmacological and rehabilitation strategies for PD and give viability to further studies that integrate current knowledge with innovations in the field of rehabilitation. Vincenza Frisardi Andrea Santamato Binith Cheeran

Can a Positive Allosteric Modulation of GABAergic Receptors Improve Motor Symptoms in Patients with Parkinson’s Disease? The Potential Role of Zolpidem in the Treatment of Parkinson’s Disease

At present, patients with advanced Parkinson's disease (PD) are unsatisfactorily controlled by currently used anti-Parkinsonian dopaminergic drugs. Various studies suggest that therapeutic strategies based on nondopaminergic drugs might be helpful in PD. Zolpidem, an imidazopyridine widely used as sleep inducer, shows high affinity only for GABAA receptors containing the α-1 subunit and facilitates GABAergic neurotransmission through a positive allosteric modulation of GABAA receptors. Various observations, although preliminary, consistently suggest that in PD patients zolpidem may induce beneficial (and sometimes remarkable) effects on motor symptoms even after single doses and may also improve dyskinesias. Since a high density of zolpidem binding sites is in the two main output structures of the basal ganglia which are abnormally overactive in PD (internal globus pallidus, GPi, and substantia nigra pars reticulata, SNr), it was hypothesized that in PD patients zolpidem may induce through GABAA receptors an inhibition of GPi and SNr (and, possibly, of the subthalamic nucleus also), resulting in an increased activity of motor cortical areas (such as supplementary motor area), which may give rise to improvement of motor symptoms of PD. Randomized clinical trials are needed in order to assess the efficacy, safety, and tolerability of zolpidem in treating motor symptoms of PD.

Activation of PAC1 Receptors in Rat Cerebellar Granule Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through N-Type Calcium Channels

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca2+]i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca2+ response. Preincubation of granule cells with the Ca2+ ATPase inhibitor, thapsigargin, or the d-myo-inositol 1,4,5-trisphosphate (IP3) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca2+]i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca2+ channel blocker, NiCl2, significantly attenuated the PACAP-evoked [Ca2+]i increase. Preincubation of granule neurons with the N-type Ca2+ channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca2+]i response, whereas the L-type Ca2+ channel blocker, nifedipine, and the P- and Q-type Ca2+ channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca2+]i in granule neurons, which is mediated by both mobilization of calcium from IP3-sensitive intracellular stores and activation of N-type Ca2+ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes.

Development of high-productivity, strong cation-exchange adsorbers for protein capture by graft polymerization from membranes with different pore sizes

This paper describes the surface modification of macroporous membranes using ATRP (atom transfer radical polymerization) to create cation-exchange adsorbers with high protein binding capacity at high product throughput. The work is motivated by the need for a more economical and rapid capture step in downstream processing of protein therapeutics. Membranes with three reported nominal pore sizes (0.2, 0.45, 1.0μm) were modified with poly(3-sulfopropyl methacrylate, potassium salt) tentacles, to create a high density of protein binding sites. A special formulation was used in which the monomer was protected by a crown ether to enable surface-initiated ATRP of this cationic polyelectrolyte. Success with modification was supported by chemical analysis using Fourier-transform infrared spectroscopy and indirectly by measurement of pure water flux as a function of polymerization time. Uniformity of modification within the membranes was visualized with confocal laser scanning microscopy. Static and dynamic binding capacities were measured using lysozyme protein to allow comparisons with reported performance data for commercial cation-exchange materials. Dynamic binding capacities were measured for flow rates ranging from 13 to 109 column volumes (CV)/min. Results show that this unique ATRP formulation can be used to fabricate cation-exchange membrane adsorbers with dynamic binding capacities as high as 70mg/mL at a throughput of 100CV/min and unprecedented productivity of 300mg/mL/min.

Understanding the in vivo uptake kinetics of a phosphatidylethanolamine-binding agent 99mTc-Duramycin

(99m)Tc-Duramycin is a peptide-based molecular probe that binds specifically to phosphatidylethanolamine (PE). The goal was to characterize the kinetics of molecular interactions between (99m)Tc-Duramycin and the target tissue. High level of accessible PE is induced in cardiac tissues by myocardial ischemia (30min) and reperfusion (120min) in Sprague-Dawley rats. Target binding and biodistribution of (99m)Tc-duramycin were captured using SPECT/CT. To quantify the binding kinetics, the presence of radioactivity in ischemic versus normal cardiac tissues was measured by gamma counting at 3, 10, 20, 60 and 180min after injection. A partially inactivated form of (99m)Tc-Duramycin was analyzed in the same fashion. A compartment model was developed to quantify the uptake kinetics of (99m)Tc-Duramycin in normal and ischemic myocardial tissue. (99m)Tc-duramycin binds avidly to the damaged tissue with a high target-to-background radio. Compartment modeling shows that accessibility of binding sites in myocardial tissue to (99m)Tc-Duramycin is not a limiting factor and the rate constant of target binding in the target tissue is at 2.2ml/nmol/min/g. The number of available binding sites for (99m)Tc-Duramycin in ischemic myocardium was estimated at 0.14nmol/g. Covalent modification of D15 resulted in a 9-fold reduction in binding affinity. (99m)Tc-Duramycin accumulates avidly in target tissues in a PE-dependent fashion. Model results reflect an efficient uptake mechanism, consistent with the low molecular weight of the radiopharmaceutical and the relatively high density of available binding sites. These data help better define the imaging utilities of (99m)Tc-Duramycin as a novel PE-binding agent.

Silica-supported aminoxyls as reactive materials for NOxremoval

The design of new materials for gaseous NOx (NO and NO2) removal at ambient temperature using organic active sites is reported. The materials consist of unfunctionalized silica and silica modified by immobilized aminoxyls and function via sequential processes consisting of (i) NO oxidation to NO2 and (ii) NO2 storage. NOx removal by physical mixtures of immobilized PTIO (2-phenyl-4,4,5,5-tetramethyl-imidazoline-3-oxide-1-oxyl) sites on silica as the NO oxidant and hydrated silica as the NO2 trap occurs with significant degradation of the PTIO oxidant via undesired side reactions with NO2 when NO2 adsorption sites are fewer than required for its complete removal along the packed bed. The use of packed beds with sufficient NO2 adsorption sites requires a large excess of unfunctionalized silica, because of its low surface density of geminal silanols, which are shown to be the relevant sites for NO2 storage on silica at ambient temperature based on density functional theory calculations. This PTIO degradation is circumvented by the design of NOx traps consisting of immobilized PTIO on silica as the NO oxidant and immobilized TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) on silica as an adsorbent with a high density of strong NO2 binding sites. Packed beds consisting of a 2 : 1 molar mixture of PTIO and TEMPO sites consume NOx as predicted by stoichiometry without detectable PTIO degradation and also without a contribution from geminal silanols as NO2 storage sites. This result requires that PTIO and TEMPO sites on silica render geminal silanols as inactive towards NO2 storage presumably because of the titration of these silanols by immobilized aminoxyls.