Sulfur-Containing Foldamer-Based Artificial Lithium Channels.

Unlike many other biologically relevant ions (Na+ , K+ , Ca2+ , Cl- , etc) and protons, whose cellular concentrations are closely regulated by highly selective channel proteins, Li+ ion is unusual in that its concentration is well tolerated over many orders of magnitude and that no lithium-specific channel proteins have so far been identified. While one naturally evolved primary pathway for Li+ ions to traverse across the cell membrane is through sodium channels by competing with Na+ ions, highly sought-after artificial lithium-transporting channels remain a major challenge to develop. Here we show that sulfur-containing organic nanotubes derived from intramolecularly H-bonded helically folded aromatic foldamers of 3.6 Šin hollow cavity diameter could facilitate highly selective and efficient transmembrane transport of Li+ ions, with high transport selectivity factors of 15.3 and 19.9 over Na+ and K+ ions, respectively.

Therapeutic efficacy of new botulinum toxin identified in CCUG 7968 strain.

Botulinum neurotoxin type A (BoNT/A) induces muscle atrophy by cleaving synaptosomal-associated protein 25. Thus, BoNT/A has been actively utilized for the treatment of masseter and gastrocnemius hypertrophy. In this study, INI101 toxin was newly identified from the CCUG 7968 strain, and its therapeutic efficacy was evaluated both in vitro and in vivo. The INI101 toxin showed identical genetic sequence, amino acid sequence, and protein subunit composition to BoNT/A produced from strain Hall A. Electromyography (EMG), and immunofluorescence staining demonstrated that INI101 (at 2 ~ 8 U/rat) effectively blocked the neuromuscular junction with no toxicity in a rat model. The EMG results showed INI101 toxin-induced weight loss and volume reduction of the gastrocnemius, similar to the effects of Botox® (BTX). Histological and immunofluorescence staining was consistent with this EMG result, showing that INI101 toxin caused muscle fiber reduction in the gastrocnemius. Notably, INI101 toxin diffused less into adjacent muscle tissue than BTX, indicating that INI101 toxin may reduce potential side effects due to diffusion into normal tissues. INI101 toxin isolated from the novel strain CCUG 7968 is a newly identified meaningful biopharmaceutical comparable to the conventional BoNT/A in the medical field. KEY POINTS: • Botulinum neurotoxin type A (BoNT/A, INI101) was identified from the CCUG 7968 strain. • INI101 toxin showed similar safety and therapeutic efficacy comparable to conventional BoNT/A both in vitro and in vivo. • INI101 toxin is a meaningful biopharmaceutical comparable to the conventional BoNT/A in the medical field.

Hierarchical Optimization of High-Performance Biomimetic and Bioinspired Membranes.

Biomimetic and bioinspired membranes have emerged as an innovative platform for water purification and aqueous separations. They are inspired by the exceptional water permeability (∼109 water molecules per second per channel) and perfect selectivity of biological water channels, aquaporins. However, only few successes have been reported for channel-based membrane fabrication due to inherent challenges of realizing coherence between channel design at the angstrom level and development of scalable membranes that maintain these molecular properties at practice-relevant scales. In this article, we feature recent progress toward practical biomimetic membranes, with the review organized along a hierarchical structural perspective that biomimetic membranes commonly share. These structures range from unitary pore shapes and tubular hydrophobic channel geometries to self-assembled bilayer structures and finally to macroscale membranes covering a size range from the angstrom, to the micrometer scale, and finally to the centimeter and larger scales. To maximize the advantage of water channel implementation into membranes, each feature needs to be optimized in an appropriate manner that provides a path to successful scale-up to achieve high performance in practical biomimetic and bioinspired membranes.

Improving extraction and post-purification concentration of membrane proteins.

Membrane proteins (MPs), despite being critically important drug targets for the pharmaceutical industry, are difficult to study due to challenges in obtaining high yields of functional protein. Most current extraction efforts use specialized non-ionic detergents to solubilize and stabilize MPs, with MPs being concentrated by ultrafiltration (UF). However, many detergents are retained during the UF step, which can destabilize MPs and/or interfere with their characterization. Here, we studied the influence of detergent selection on the extraction and UF-based concentration of biomedically-relevant MPs, the light-driven sodium and chloride transporters, KR2 and halorhodopsin (pHR) which are also model proteins for more complex mammalian rhodopsins. We also designed a flat-bottomed centrifugal filter that can concentrate MPs with enhanced removal of free detergents by promoting concentration polarization (CP). We tested the performance of this new filter using four commonly employed MP detergents, octyl-ß-D maltoside (OM), decyl-ß-D maltoside (DM), dodecyl-ß-D maltoside (DDM) and octyl-ß-D glucoside (OG), over a range of detergent and salt concentrations. Detergent passage is significantly higher for the flat-bottomed filter achieving up to 2-fold greater sieving of detergent in DM-solubilized pHR system due to the high degree of CP. We observe more efficient, up to 5-fold higher extraction of KR2 in the presence of a longer 12-carbon alkyl chain detergent, DDM compared to a shorter 8-carbon detergent, OM. Assuming complete binding and elution of the extracted protein, DDM-based extraction of KR2 could lead to a potential 7-fold improvement in purification yields compared to conventional methods which yield ∼1 mg MP per liter of cell culture. However, the longer chain detergents like DDM form larger micelles that are difficult to remove by UF. Thus, there exists a trade-off between choosing a detergent that will enable efficient extraction of MP while showing easier removal during subsequent UF. The extraction efficiency and UF-based separation of detergent micelles provide insights for other applications involving detergent-mediated separation/extraction.

Individualized predictions for clinical milestone in amyotrophic lateral sclerosis: A multialgorithmic approach.

Objective: The phenotypic heterogeneity and complex disease trajectory complicate the ability to predict specific clinical milestone for individual patients with amyotrophic lateral sclerosis (ALS). Here we developed individualized prediction models to estimate the time to the loss of autonomy in swallowing function. Methods: Utilizing the Pooled Resource Open-Access ALS Clinical Trials (PRO-ACT) database, we built three models of distinct time-to-event prediction algorithms: accelerated failure time (AFT), cox proportional hazard (COX) and random survival forest (RSF) for an individualized risk assessment of the swallowing milestone. The target variable was defined as the time to a decline in the ALSFRS-R swallowing item score to 1 or below, indicating a need for supplementary tube feeding. Results: Internal cross-validation revealed the median concordance index (C-index) of 0.851 (IQR, 0.842-0.859) for AFT, 0.850 (0.841-0.859) for COX and 0.846 (0.839-0.854) for RSF, and all models demonstrated good distributional calibration with predicted and observed event probabilities closely matched across different time intervals. For external validation with a registry dataset with characteristics different from PRO-ACT, the discriminative power was replicated with comparable C-indices for all models, whereas the calibration revealed a left-skewed distribution suggesting a bias towards overestimation of event probabilities in real-world data. While all models were effective at stratifying patients, the results of RSF model, unlike AFT and COX, did not match well with the KM curves of the corresponding risk groups, supporting the importance of nuanced understanding of data structure and algorithmic properties. Conclusion: Our models are implemented into a web application which could be applied to individualized counselling, management and clinical trial design for gastrostomy intervention. Further studies for model optimization will advance personalized care in patients with ALS.

Dehydrated Biomimetic Membranes with Skinlike Structure and Function.

Novel vapor-permeable materials are sought after for applications in protective wear, energy generation, and water treatment. Current impermeable protective materials effectively block harmful agents but trap heat due to poor water vapor transfer. Here we present a new class of materials, vapor permeable dehydrated nanoporous biomimetic membranes (DBMs), based on channel proteins. This application for biomimetic membranes is unexpected as channel proteins and biomimetic membranes were assumed to be unstable under dry conditions. DBMs mimic human skin's structure to offer both high vapor transport and small molecule exclusion under dry conditions. DBMs feature highly organized pores resembling sweat pores in human skin, but at super high densities (>1012 pores/cm2). These DBMs achieved exceptional water vapor transport rates, surpassing commercial breathable fabrics by up to 6.2 times, despite containing >2 orders of magnitude smaller pores (1 nm vs >700 nm). These DBMs effectively excluded model biological agents and harmful chemicals both in liquid and vapor phases, again in contrast with the commercial breathable fabrics. Remarkably, while hydrated biomimetic membranes were highly permeable to liquid water, they exhibited higher water resistances after dehydration at values >38 times that of commercial breathable fabrics. Molecular dynamics simulations support our hypothesis that dehydration induced protein hydrophobicity increases which enhanced DBM performance. DBMs hold promise for various applications, including membrane distillation, dehumidification, and protective barriers for atmospheric water harvesting materials.

Protein engineering of pores for separation, sensing, and sequencing.

Proteins are critical to cellular function and survival. They are complex molecules with precise structures and chemistries, which allow them to serve diverse functions for maintaining overall cell homeostasis. Since the discovery of the first enzyme in 1833, a gamut of advanced experimental and computational tools has been developed and deployed for understanding protein structure and function. Recent studies have demonstrated the ability to redesign/alter natural proteins for applications in industrial processes of interest and to make customized, novel synthetic proteins in the laboratory through protein engineering. We comprehensively review the successes in engineering pore-forming proteins and correlate the amino acid-level biochemistry of different pore modification strategies to the intended applications limited to nucleotide/peptide sequencing, single-molecule sensing, and precise molecular separations.

Liposome-mediated small RNA delivery to convert the macrophage polarity: A novel therapeutic approach to treat inflammatory uterine disease.

Macrophages are present in all tissues for maintaining tissue homeostasis, and macrophage polarization plays a vital role in alleviating inflammation. Therefore, specific delivery of polarization modulators to macrophages in situ is critical for treating inflammatory diseases. We demonstrate that a size-controlled miRNA-encapsulated macrophage-targeting liposomes (miR/MT-Lip) specifically targets macrophages to promote M1-to-M2 polarization conversion, alleviating inflammation without cytotoxicity. miR/MT-Lip, approximately 1.2 µm, showed excellent internalization through phagocytosis and/or macropinocytosis in macrophages. miR-10a/MT-Lip, but not scramble miR-Fluorescein amidite (FAM)/MT-Lip as control, effectively converted the polarization of lipopolysaccharide (LPS)-induced M1 macrophages to M2 in vitro. When miR-10a/MT-Lip was intravenously delivered to mice insulted with LPS for inflammation, the proportion of M2 macrophages was significantly increased without disturbing the population of other immune cells. Furthermore, scramble miR-FAM/MT-Lip was mainly detected in macrophages, but not other immune cells. When our miR/MT-Lip was administered to mice with Asherman's syndrome that suffer from infertility because of sterile uterine inflammation, macrophage-specific targeting of miR-10a/MT-Lip facilitated M1-to-M2 conversion for angiogenesis in the impaired uterus, resulting in restoration of healthy uterine conditions. The results indicate that our MT-Lip encapsulating small RNAs has excellent potential to treat various inflammatory disorders by fine-tuning macrophage polarization in vivo without any side effects.

PLGA Microspheres Containing Hydrophobically Modified Magnesium Hydroxide Particles for Acid Neutralization-Mediated Anti-Inflammation.

BACKGROUND: Poly(lactic-co-glycolic acid) (PLGA) microspheres have been actively used in various pharmaceutical formulations because they can sustain active pharmaceutical ingredient release and are easy to administer into the body using a syringe. However, the acidic byproducts produced by the decomposition of PLGA cause inflammatory reactions in surrounding tissues, limiting biocompatibility. Magnesium hydroxide (MH), an alkaline ceramic, has attracted attention as a potential additive because it has an acid-neutralizing effect. METHODS: To improve the encapsulation efficiency of hydrophilic MH, the MH particles were capped with hydrophobic ricinoleic acid (RA-MH). PLGA microspheres encapsulated with RA-MH particles were manufactured by the O/W method. To assess the in vitro cytotoxicity of the degradation products of PLGA, MH/PLGA, and RA-MH/PLGA microspheres, CCK-8 and Live/Dead assays were performed with NIH-3T3 cells treated with different concentrations of their degradation products. In vitro anti-inflammatory effect of RA-MH/PLGA microspheres was evaluated with quantitative measurement of pro-inflammatory cytokines. RESULTS: The synthesized RA-MH was encapsulated in PLGA microspheres and displayed more than four times higher loading content than pristine MH. The PLGA microspheres encapsulated with RA-MH had an acid-neutralizing effect better than that of the control group. In an in vitro cell experiment, the degradation products obtained from RA-MH/PLGA microspheres exhibited higher biocompatibility than the degradation products obtained from PLGA microspheres. Additionally, the RA-MH/PLGA microsphere group showed an excellent anti-inflammatory effect. CONCLUSION: Our results proved that RA-MH-encapsulated PLGA microspheres showed excellent biocompatibility with an anti-inflammatory effect. This technology can be applied to drug delivery and tissue engineering to treat various incurable diseases in the future.

Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin.

We report a simple and direct fluorimetric vesicle-based method for measuring the transport rate of the light-driven ions pumps as specifically applied to the chloride pump, halorhodopsin, from Natronomonas pharaonis (pHR). Previous measurements were cell-based and methods to determine average single channel permeability challenging. We used a water-in-oil emulsion method for directional pHR reconstitution into two different types of vesicles: lipid vesicles and asymmetric lipid-block copolymer vesicles. We then used stopped-flow experiments combined with fluorescence correlation spectroscopy to determine per protein Cl- transport rates. We obtained a Cl- transport rate of 442 (±17.7) Cl-/protein/s in egg phosphatidyl choline (PC) lipid vesicles and 413 (±26) Cl-/protein/s in hybrid block copolymer/lipid (BCP/PC) vesicles with polybutadine-polyethylene oxide (PB12PEO8) on the outer leaflet and PC in the inner leaflet at a photon flux of 1450 photons/protein/s. Normalizing to a per photon basis, this corresponds to 0.30 (±0.07) Cl-/photon and 0.28 (±0.04) Cl-/photon for pure PC and BCP/PC hybrid vesicles respectively, both of which are in agreement with recently reported turnover of ~500 Cl-/protein/s from flash photolysis experiments and with voltage-clamp measurements of 0.35 (±0.16) Cl-/photon in pHR-expressing oocytes as well as with a pHR quantum efficiency of ~30%.

Metal-Free Synthesis of Indolopyrans and 2,3-Dihydrofurans Based on Tandem Oxidative Cycloaddition.

The synthesis of versatile scaffold indolopyrans based on C-C radical-radical cross-coupling under metal-free conditions is described. The reaction involving single electron transfer between coupling partners followed by cage collapse allows highly selective cross-coupling while employing only equimolar amounts of coupling partners. Moreover, the mechanistic manifold was expanded for the functionalization of enamines to give the stereoselective synthesis of 2,3-dihydrofurans. This iodine-mediated oxidative coupling features mild conditions and fast reaction kinetics.