Simultaneous Quantitation of Amino Acid Mixtures using Clustering Agents

A method that uses the abundances of large clusters formed in electrospray ionization to determine the solution-phase molar fractions of amino acids in multi-component mixtures is demonstrated. For solutions containing either four or 10 amino acids, the relative abundances of protonated molecules differed from their solution-phase molar fractions by up to 30-fold and 100-fold, respectively. For the four-component mixtures, the molar fractions determined from the abundances of larger clusters consisting of 19 or more molecules were within 25% of the solution-phase molar fractions, indicating that the abundances and compositions of these clusters reflect the relative concentrations of these amino acids in solution, and that ionization and detection biases are significantly reduced. Lower accuracy was obtained for the 10-component mixtures where values determined from the cluster abundances were typically within a factor of three of their solution molar fractions. The lower accuracy of this method with the more complex mixtures may be due to specific clustering effects owing to the heterogeneity as a result of significantly different physical properties of the components, or it may be the result of lower S/N for the more heterogeneous clusters and not including the low-abundance more highly heterogeneous clusters in this analysis. Although not as accurate as using traditional standards, this clustering method may find applications when suitable standards are not readily available

Motor Skills Enhance Procedural Memory Formation and Protect against Age-Related Decline

The ability to consolidate procedural memories declines with increasing age. Prior knowledge enhances learning and memory consolidation of novel but related information in various domains. Here, we present evidence that prior motor experience-in our case piano skills-increases procedural learning and has a protective effect against age-related decline for the consolidation of novel but related manual movements. In our main experiment, we tested 128 participants with a sequential finger-tapping motor task during two sessions 24 hours apart. We observed enhanced online learning speed and offline memory consolidation for piano players. Enhanced memory consolidation was driven by a strong effect in older participants, whereas younger participants did not benefit significantly from prior piano experience. In a follow up independent control experiment, this compensatory effect of piano experience was not visible after a brief offline period of 30 minutes, hence requiring an extended consolidation window potentially involving sleep. Through a further control experiment, we rejected the possibility that the decreased effect in younger participants was caused by training saturation. We discuss our results in the context of the neurobiological schema approach and suggest that prior experience has the potential to rescue memory consolidation from age-related cognitive decline

Anticancer Therapeutic Alginate-Based Tissue Sealants for Lung Repair

Injury to the connective tissue that lines the lung, the pleura, or the lung itself can occur from many causes including trauma or surgery, as well as lung diseases or cancers. To address current limitations for patching lung injuries, to stop air or fluid leaks, an adherent hydrogel sealant patch system was developed, based on methacrylated alginate (AMA) and AMA dialdehyde (AMA-DA) blends, which is capable of sealing damaged tissues and sustaining physiological pressures. Methacrylation of alginate hydroxyl groups rendered the polysaccharide capable of photo-cross-linking when mixed with an eosin Y-based photoinitiator system and exposed to visible green light. Oxidation of alginate yields functional aldehyde groups capable of imine bond formation with proteins found in many tissues. The alginate-based patch system was rigorously tested on a custom burst pressure testing device. Blending of nonoxidized material with oxidized (aldehyde modified) alginates yielded patches with improved burst pressure performance and decreased delamination as compared with pure AMA. Human mesothelial cell (MeT-5A) viability and cytotoxicity were retained when cultured with the hydrogel patches. The release and bioactivity of doxorubicin-encapsulated submicrospheres enabled the fabrication of drug-eluting adhesive patches and were effective in decreasing human lung cancer cell (A549) viability

Self-Healing and Thermoresponsive Dual-Cross-Linked Alginate Hydrogels Based on Supramolecular Inclusion Complexes

β-Cyclodextrin (β-CD), with a lipophilic inner cavity and hydrophilic outer surface, interacts with a large variety of nonpolar guest molecules to form noncovalent inclusion complexes. Conjugation of β-CD onto biomacromolecules can form physically cross-linked hydrogel networks upon mixing with a guest molecule. Herein, the development and characterization of self-healing, thermoresponsive hydrogels, based on host–guest inclusion complexes between alginate-<i>graft</i>-β-CD and Pluronic F108 (poly­(ethylene glycol)-<i>b</i>-poly­(propylene glycol)-<i>b</i>-poly­(ethylene glycol)), are described. The mechanics, flow characteristics, and thermal response were contingent on the polymer concentration and the host–guest molar ratio. Transient and reversible physical cross-linking between host and guest polymers governed self-assembly, allowing flow to occur under shear stress and facilitating complete recovery of the material’s properties within a few seconds of unloading. The mechanical properties of the dual-cross-linked, multi-stimuli-responsive hydrogels were tuned as high as 30 kPa at body temperature and are advantageous for biomedical applications such as drug delivery and cell transplantation

Dual-Cross-Linked Methacrylated Alginate Sub-Microspheres for Intracellular Chemotherapeutic Delivery

Intracellular delivery vehicles comprised of methacrylated alginate (Alg-MA) were developed for the internalization and release of doxorubicin hydrochloride (DOX). Alg-MA was synthesized via an anhydrous reaction, and a mixture of Alg-MA and DOX was formed into sub-microspheres using a water/oil emulsion. Covalently cross-linked sub-microspheres were formed via exposure to green light, in order to investigate effects of cross-linking on drug release and cell internalization, compared to traditional techniques, such as ultraviolet (UV) light irradiation. Cross-linking was performed using light exposure alone or in combination with ionic cross-linking using calcium chloride (CaCl₂). Alg-MA sub-microsphere diameters were between 88 and 617 nm, and ζ-potentials were between −20 and −37 mV. Using human lung epithelial carcinoma cells (A549) as a model, cellular internalization was confirmed using flow cytometry; different sub-microsphere formulations varied the efficiency of internalization, with UV-cross-linked sub-microspheres achieving the highest internalization percentages. While blank (nonloaded) Alg-MA submicrospheres were noncytotoxic to A549 cells, DOX-loaded sub-microspheres significantly reduced mitochondrial activity after 5 days of culture. Photo-cross-linked Alg-MA sub-microspheres may be a potential chemotherapeutic delivery system for cancer treatment

Internalized FGF-2-Loaded Nanoparticles Increase Nuclear ERK1/2 Content and Result in Lung Cancer Cell Death

Innovative cancer treatments, which improve adjuvant therapy and reduce adverse events, are desperately needed. Nanoparticles provide controlled intracellular biomolecule delivery in the absence of activating external cell surface receptors. Prior reports suggest that intracrine signaling, following overexpression of basic fibroblast growth factor (FGF-2) after viral transduction, has a toxic effect on diseased cells. Herein, the research goals were to (1) encapsulate recombinant FGF-2 within stable, alginate-based nanoparticles (ABNs) for non-specific cellular uptake, and (2) determine the effects of ABN-mediated intracellular delivery of FGF-2 on cancer cell proliferation/survival. In culture, human alveolar adenocarcinoma basal epithelial cell line (A549s) and immortalized human bronchial epithelial cell line (HBE1s) internalized ABNs through non-selective endocytosis. Compared to A549s exposed to empty (i.e., blank) ABNs, the intracellular delivery of FGF-2 via ABNs significantly increased the levels of lactate dehydrogenase, indicating that FGF-2-ABN treatment decreased the transformed cell integrity. Noticeably, the nontransformed cells were not significantly affected by FGF-2-loaded ABN treatment. Furthermore, FGF-2-loaded ABNs significantly increased nuclear levels of activated-extracellular signal-regulated kinase &frac12; (ERK1/2) in A549s but had no significant effect on HBE1 nuclear ERK1/2 expression. Our novel intracellular delivery method of FGF-2 via nanoparticles resulted in increased cancer cell death via increased nuclear ERK1/2 activation