Biphasic water-oil systems for functional augmentation of probiotic Lactobacillus acidophilus nanoencapsulated in luteolin-Fe3+ shells.

Single-cell nanoencapsulation (SCNE) has great potential in the enhancement of therapeutic effects of probiotic microbes. However, the material scope has been limited to water-soluble compounds to avoid non-biocompatible organic solvents that are harmful to living cells. In this work, the SCNE of probiotic Lactobacillus acidophilus with water-insoluble luteolin and Fe3+ ions is achieved by the vortex-assisted, biphasic water-oil system. The process creates L. acidophilus nanoencapsulated in the luteolin-Fe3+ shells that empower the cells with extrinsic properties, such as resistance to lysozyme attack, anti-ROS ability, and α-amylase-inhibition activity, as well as sustaining viability under acidic conditions. The proposed protocol, embracing water-insoluble flavonoids as shell components in SCNE, will be an advanced add-on to the chemical toolbox for the manipulation of living cells at the single-cell level.

MolNet_Equi: A Chemically Intuitive, Rotation-Equivariant Graph Neural Network.

Although deep-learning (DL) models suggest unprecedented prediction capabilities in tackling various chemical problems, their demonstrated tasks have so far been limited to the scalar properties including the magnitude of vectorial properties, such as molecular dipole moments. A rotation-equivariant MolNet_Equi model, proposed in this paper, understands and recognizes the molecular rotation in the 3D Euclidean space, and exhibits the ability to predict directional dipole moments in the rotation-sensitive mode, as well as showing superior performance for the prediction of scalar properties. Three consecutive operations of molecular rotation R M ${\left(R\left(M\right)\right)}$ , dipole-moment prediction φ µ R M ${\left({\phi{} }_{\mu }\left(R\left(M\right)\right)\right)}$ , and dipole-moment inverse-rotation R - 1 φ µ R M ${\left({R}^{-1}\left({\phi{} }_{\mu }\left(R\left(M\right)\right)\right)\right)}$ do not alter the original prediction of the total dipole moment of a molecule φ µ M ${\left({\phi{} }_{\mu }\right(M\left)\right)}$ , assuring the rotational equivariance of MolNet_Equi. Furthermore, MolNet_Equi faithfully predicts the absolute direction of dipole moments given molecular poses, albeit the model has been trained only with the information on dipole-moment magnitudes, not directions. This work highlights the potential of incorporating fundamental yet crucial chemical rules and concepts into DL models, leading to the development of chemically intuitive models.

A Micrometric Transformer: Compositional Nanoshell Transformation of Fe3+ -Trimesic-Acid Complex with Concomitant Payload Release in Cell-in-Catalytic-Shell Nanobiohybrids.

Nanoencapsulation of living cells within artificial shells is a powerful approach for augmenting the inherent capacity of cells and enabling the acquisition of extrinsic functions. However, the current state of the field requires the development of nanoshells that can dynamically sense and adapt to environmental changes by undergoing transformations in form and composition. This paper reports the compositional transformation of an enzyme-embedded nanoshell of Fe3+ -trimesic acid complex to an iron phosphate shell in phosphate-containing media. The cytocompatible transformation allows the nanoshells to release functional molecules without loss of activities and biorecognition, while preserving the initial shell properties, such as cytoprotection. Demonstrations include the lysis and killing of Escherichia coli by lysozyme, and the secretion of interleukin-2 by Jurkat T cells in response to paracrine stimulation by antibodies. This work on micrometric Transformers will benefit the creation of cell-in-shell nanobiohybrids that can interact with their surroundings in active and adaptive ways.

Tandem-biocatalysis reactors constructed by topological evolution of CaCO3 particles into hollow metal hydroxide spheres.

Despite remarkable advances in the design and synthesis of hollow inorganic spheres (HISs), the harsh synthetic conditions have precluded the applications of HISs to biochemical and biological fields. Herein we report a biocompatible strategy for synthesizing metal hydroxide HISs (MH-HISs) by simply mixing CaCO3 particles with metal ions in water. The ion-exchange reaction between Ca2+ and metal ions leads to the structural and chemical evolution from solid CaCO3 particles to hollow MH-HISs via core-shell and yolk-shell structures, while enabling the encapsulation of enzymes to the shells without loss of catalytic activities. The biocompatible protocol makes multienzymatic cascade reactions achievable, with great recyclability due to mechanical durability of MH-HISs.

In vitro induction of in vivo-relevant stellate astrocytes in 3D brain-derived, decellularized extracellular matrices.

In vitro fabrication of 3D cell culture systems that could provide in vivo tissue-like, structural, and biochemical environments to neural cells is essential not only for fundamental studies on brain function and behavior, but also for tissue engineering and regenerative medicine applicable to neural injury and neurodegenerative diseases. In particular, for astrocytes-which actively respond to the surroundings and exhibit varied morphologies based on stimuli (e.g., stiffness and chemicals) in vitro, as well as physiological or pathological conditions in vivo-it is crucial to establish an appropriate milieu in in vitro culture platforms. Herein, we report the induction of in vivo-relevant, stellate-shaped astrocytes derived from cortices of Rattus norvegicus by constructing the 3D cell culture systems of brain-derived, decellularized extracellular matrices (bdECMs). The bdECM hydrogels were mechanically stable and soft, and the bdECM-based 3D scaffolds supplied biochemically active environments that astrocytes could interact with, leading to the development of in vivo-like stellate structures. In addition to the distinct morphology with actively elongated endfeet, the astrocytes, cultured in 3D bdECM scaffolds, would have neurosupportive characteristics, indicated by the accelerated neurite outgrowth in the astrocyte-conditioned media. Furthermore, next-generation sequencing showed that the gene expression profiles of astrocytes cultured in bdECMs were significantly different from those cultured on 2D surfaces. The stellate-shaped astrocytes in the bdECMs were analyzed to have reached a more mature state, for instance, with decreased expression of genes for scaffold ECMs, actin filaments, and cell division. The results suggest that the bdECM-based 3D culture system offers an advanced platform for culturing primary cortical astrocytes and their mixtures with other neural cells, providing a brain-like, structural and biochemical milieu that promotes the maturity and in vivo-like characteristics of astrocytes in both form and gene expression. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrices (dECMs) have emerged as strong candidates for the construction of three-dimensional (3D) cell cultures in vitro, owing to the potential to provide native biochemical and physical environments. In this study, we fabricated hydrogels of brain-derived dECMs (bdECMs) and cultured primary astrocytes within the bdECM hydrogels in a 3D context. The cultured astrocytes exhibited a stellate morphology distinct from conventional 2D cultures, featuring tridimensionally elongated endfeet. qRT-PCR and NGS-based transcriptomic analyses revealed gene expression patterns indicative of a more mature state, compared with the 2D culture. Moreover, astrocytes cultured in bdECMs showed neurosupportive characteristics, as demonstrated by the accelerated neurite outgrowth in astrocyte-conditioned media. We believe that the bdECM hydrogel-based culture system can serve as an in vitro model system for astrocytes and their coculture with other neural cells, holding significant potential for neural engineering and therapeutic applications.

Fugetaxis of Cell-in-Catalytic-Coat Nanobiohybrids in Glucose Gradients.

Manipulation and control of cell chemotaxis remain an underexplored territory despite vast potential in various fields, such as cytotherapeutics, sensors, and even cell robots. Herein is achieved the chemical control over chemotactic movement and direction of Jurkat T cells, as a representative model, by the construction of cell-in-catalytic-coat structures in single-cell nanoencapsulation. Armed with the catalytic power of glucose oxidase (GOx) in the artificial coat, the nanobiohybrid cytostructures, denoted as Jurkat[Lipo_GOx] , exhibit controllable, redirected chemotactic movement in response to d-glucose gradients, in the opposite direction to the positive-chemotaxis direction of naïve, uncoated Jurkat cells in the same gradients. The chemically endowed, reaction-based fugetaxis of Jurkat[Lipo_GOx] operates orthogonally and complementarily to the endogenous, binding/recognition-based chemotaxis that remains intact after the formation of a GOx coat. For instance, the chemotactic velocity of Jurkat[Lipo_GOx] can be adjusted by varying the combination of d-glucose and natural chemokines (CXCL12 and CCL19) in the gradient. This work offers an innovative chemical tool for bioaugmenting living cells at the single-cell level through the use of catalytic cell-in-coat structures.

Neutralization of Cannabidiol Neurotoxicity in Neuron-Astrocyte Sandwich Coculture.

Cannabidiol (CBD), a main nonpsychoactive phytocannabinoid in the Cannabis genus, has been in the limelight for its potential health benefits in various neurological diseases. However, the safety issue of CBD in the nervous system has not been settled fully, while CBD has been reported to have mild side effects including dizziness and somnolence. In this work, a platform of neuron-astrocyte sandwich coculture to investigate the neurotoxicity of CBD, as well as the neuronal responses to CBD, in a more in vivo relevant mode is constructed. CBD (15 and 30 µm) causes the viability decrease, along with morphological damage, in the neuron-alone culture, whereas its neurotoxic effects are significantly attenuated by the supports of astrocytes in the neuron-astrocyte coculture. In addition, it is found that CBD-induced increase of intracellular Ca2+ concentration and depolarization of mitochondrial membrane potential, via activation of transient receptor potential vanilloid 1, are noticeably ameliorated by coculturing neurons with astrocytes. This work provides crucial information in the development of CBD as therapeutics for neurological disorders, as well as in a fundamental understanding of how CBD works in the nervous system.

A Natural Virucidal and Microbicidal Spray Based on Polyphenol-Iron Sols.

Numerous disinfection methods have been developed to reduce the transmission of infectious diseases that threaten human health. However, it still remains elusively challenging to develop eco-friendly and cost-effective methods that deactivate a wide range of pathogens, from viruses to bacteria and fungi, without doing any harm to humans or the environment. Herein we report a natural spraying protocol, based on a water-dispersible supramolecular sol of nature-derived tannic acid (TA) and Fe3+, which is easy-to-use and low-cost. Our formulation effectively deactivates viruses (influenza A viruses, SARS-CoV-2, and human rhinovirus) as well as suppressing the growth and spread of pathogenic bacteria (Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, and Acinetobacter baumannii) and fungi (Pleurotus ostreatus and Trichophyton rubrum). Its versatile applicability in a real-life setting is also demonstrated against microorganisms present on the surfaces of common household items (e.g., air filter membranes, disposable face masks, kitchen sinks, mobile phones, refrigerators, and toilet seats).

Vortex-assisted, nanoarchitectonic manipulation of microparticles with flavonoid-Fe3+ complex in biphasic water-oil systems.

Coordination-driven self-assembly of metal-ligand complexes is a powerful nanoarchitectonic tool for particle engineering, but its usability is limited when using two immiscible coating components. This paper reports that simple vortexing of a biphasic system of Fe3+ ions in water and flavonoids in oil forms nanoshells on individual particles, thereby enabling the utilization of water-insoluble ligands as coating materials. Mechanistic studies suggest that the biphasic mass-transfer equilibrium of flavonoid-Fe3+ species controls the shell formation, with the oil phase acting as a reservoir of coating precursors for continuous coating. The versatility and convenience of our method expand the chemical toolbox for modulating particle-material interfaces.

Cytoprotection of Probiotic Lactobacillus acidophilus with Artificial Nanoshells of Nature-Derived Eggshell Membrane Hydrolysates and Coffee Melanoidins in Single-Cell Nanoencapsulation.

One-step fabrication method for thin films and shells is developed with nature-derived eggshell membrane hydrolysates (ESMHs) and coffee melanoidins (CMs) that have been discarded as food waste. The nature-derived polymeric materials, ESMHs and CMs, prove highly biocompatible with living cells, and the one-step method enables cytocompatible construction of cell-in-shell nanobiohybrid structures. Nanometric ESMH-CM shells are formed on individual probiotic Lactobacillus acidophilus, without any noticeable decrease in viability, and the ESMH-CM shells effectively protected L. acidophilus in the simulated gastric fluid (SGF). The cytoprotection power is further enhanced by Fe3+-mediated shell augmentation. For example, after 2 h of incubation in SGF, the viability of native L. acidophilus is 30%, whereas nanoencapsulated L. acidophilus, armed with the Fe3+-fortified ESMH-CM shells, show 79% in viability. The simple, time-efficient, and easy-to-process method developed in this work would contribute to many technological developments, including microbial biotherapeutics, as well as waste upcycling.

MolNet: A Chemically Intuitive Graph Neural Network for Prediction of Molecular Properties.

Most graph neural networks (GNNs) in deep-learning chemistry collect and update atom and molecule features from the fed atom (and, in some cases, bond) features, basically based on the two-dimensional (2D) graph representation of 3D molecules. However, the 2D-based models do not faithfully represent 3D molecules and their physicochemical properties, exemplified by the overlooked field effect that is a "through-space" effect, not a "through-bond" effect. We propose a GNN model, denoted as MolNet, which accommodates the 3D non-bond information in a molecule, via a noncovalent adjacency matrix A ‾ , and also bond-strength information from a weighted bond matrix B . Comparative studies show that MolNet outperforms various baseline GNN models and gives a state-of-the-art performance in the classification task of BACE dataset and regression task of ESOL dataset. This work suggests a future direction for the construction of deep-learning models that are chemically intuitive and compatible with the existing chemistry concepts and tools.

Cell-in-Catalytic-Shell Nanoarchitectonics: Catalytic Empowerment of Individual Living Cells by Single-Cell Nanoencapsulation.

Cell-in-shell biohybrid structures, synthesized by encapsulating individual living cells with exogenous materials, have emerged as exciting functional entities for engineered living materials, with emergent properties outside the scope of biochemical modifications. Artificial exoskeletons have, to date, provided physicochemical shelters to the cells inside in the first stage of technological development, and further advances in the field demand catalytically empowered, cellular hybrid systems that augment the biological functions of cells and even introduce completely new functions to the cells. This work describes a facile and generalizable strategy for empowering living cells with extrinsic catalytic capability through nanoencapsulation of living cells with a supramolecular metal-organic complex of Fe3+ and benzene-1,3,5-tricarboxylic acid (BTC). A series of enzymes are embedded in situ, without loss of catalytic activity, in the Fe3+ -BTC shells, not to mention the superior characteristics of cytocompatible and rapid shell-forming processes. The nanoshell enhances the catalytic efficiency of multienzymatic cascade reactions by confining reaction intermediates to its internal voids and the nanoencapsulated cells acquire exogenous biochemical functions, including enzymatic cleavage of lethal octyl-ß-d-glucopyranoside into d-glucose, with autonomous cytoprotection. The system will provide a versatile, nanoarchitectonic tool for interfacing biological cells with functional materials, especially for catalytic bioempowerment of living cells.

In Vitro Studies on Therapeutic Effects of Cannabidiol in Neural Cells: Neurons, Glia, and Neural Stem Cells.

(‒)-Cannabidiol (CBD) is one of the major phytocannabinoids extracted from the Cannabis genus. Its non-psychoactiveness and therapeutic potential, partly along with some anecdotal-if not scientific or clinical-evidence on the prevention and treatment of neurological diseases, have led researchers to investigate the biochemical actions of CBD on neural cells. This review summarizes the previously reported mechanistic studies of the CBD actions on primary neural cells at the in vitro cell-culture level. The neural cells are classified into neurons, microglia, astrocytes, oligodendrocytes, and neural stem cells, and the CBD effects on each cell type are described. After brief introduction on CBD and in vitro studies of CBD actions on neural cells, the neuroprotective capability of CBD on primary neurons with the suggested operating actions is discussed, followed by the reported CBD actions on glia and the CBD-induced regeneration from neural stem cells. A summary section gives a general overview of the biochemical actions of CBD on neural cells, with a future perspective. This review will provide a basic and fundamental, but crucial, insight on the mechanistic understanding of CBD actions on neural cells in the brain, at the molecular level, and the therapeutic potential of CBD in the prevention and treatment of neurological diseases, although to date, there seem to have been relatively limited research activities and reports on the cell culture-level, in vitro studies of CBD effects on primary neural cells.

Enzyme-Mediated Kinetic Control of Fe3+-Tannic Acid Complexation for Interface Engineering.

Supramolecular self-assembly of Fe3+ and tannic acid (TA) has received great attention in the fields of materials science and interface engineering because of its exceptional surface coating properties. Although advances in coating strategies often suggest that kinetics in the generation of interface-active Fe3+-TA species is deeply involved in the film formation, there is no acceptable elucidation for the coating process. In this work, we developed the enzyme-mediated kinetic control of Fe2+ oxidation to Fe3+ in a Fe2+-TA complex in the iron-gall-ink-revisited coating method. Specifically, hydrogen peroxide, produced in the glucose oxidase (GOx)-catalyzed reaction of d-glucose, accelerated Fe2+ oxidation, and the optimized kinetics profoundly facilitated the film formation to be about 9 times thicker. We also proposed a perspective considering the coating process as nucleation and growth. From this viewpoint, the kinetics in the generation of interface-active Fe3+-TA species should be optimized because it determines whether the interface-active species forms a film on the substrate (i.e., heterogeneous nucleation and film growth) or flocculates in solution (i.e., homogeneous nucleation and particle growth). Moreover, GOx was concomitantly embedded into the Fe3+-TA films with sustained catalytic activities, and the GOx-mediated coating system was delightfully adapted to catalytic single-cell nanoencapsulation.

Rotational Variance-Based Data Augmentation in 3D Graph Convolutional Network.

This work proposes the data augmentation by molecular rotation, with consideration that the protein-ligand binding events are rotation-variant. As a proof-of-concept, known active (i. e., 1-labeled) ligands to human ß-secretase 1 (BACE-1) are rotated for the generation of 0-labeled data, and the rotation-dependent prediction accuracy of 3D graph convolutional network (3DGCN) is investigated after data augmentation. The data augmentation makes the orientation-recognizing ability of 3DGCN improved significantly in the classification task for BACE-1/ligand binding. Furthermore, the data-augmented 3DGCN has a capability for predicting active ligands from a candidate dataset, via improved performance of orientation recognition, which would be applied to virtual drug screening and discovery.

A Decade of Advances in Single-Cell Nanocoating for Mammalian Cells.

Strategic advances in the single-cell nanocoating of mammalian cells have noticeably been made during the last decade, and many potential applications have been demonstrated. Various cell-coating strategies have been proposed via adaptation of reported methods in the surface sciences and/or materials identification that ensure the sustainability of labile mammalian cells during chemical manipulation. Here an overview of the methodological development and potential applications to the healthcare sector in the nanocoating of mammalian cells made during the last decade is provided. The materials used for the nanocoating are categorized into polymers, hydrogels, polyphenolic compounds, nanoparticles, and minerals, and the corresponding strategies are described under the given set of materials. It also suggests, as a future direction, the creation of the cytospace system that is hierarchically composed of the physically separated but mutually interacting cellular hybrids.

Neuroprotective Effect of Cannabidiol Against Hydrogen Peroxide in Hippocampal Neuron Culture.

Introduction: Reports on the neurotoxic and neuroprotective effects of cannabidiol (CBD) have not been in complete accord, showing different and somewhat contradictory results depending upon the brain cell types and experimental conditions employed. This work systematically examines the neuroprotective capability of CBD against oxidative stress (i.e., hydrogen peroxide [H2O2]) as well as its toxicity profile in the in vitro culture platform of primary hippocampal neurons. Materials and Methods: The low cell-density (100 neurons per mm2) culture was used for analyzing the viability and morphology of neurons at a single-cell level with a confocal laser-scanning microscope (CLSM). Primary neurons were obtained from the hippocampal tissues of embryonic day-18 (E18) Sprague-Dawley rat pups and treated with CBD (0.1-100 µM) and/or H2O2 (0.1-50 µM) at 1 DIV (days in vitro). Results: The lethal concentration 50 (LC50) value (the concentration causing 50% cell death) of CBD was calculated to be 9.85 µM after 24 h of incubation, and that of H2O2 was 2.46 µM under the same conditions. The neuroprotection ratio of CBD against H2O2 ([H2O2]=10 µM) was 2.40 with 5 µM of CBD, increasing the cell viability to 57% from 24%. The CLSM analysis suggested that the cell-death mechanisms were different for CBD and H2O2, and CBD did not completely rescue the morphological alterations of primary hippocampal neurons caused by H2O2, such as neurite degeneration, at least in the in vitro neuron culture. Conclusion: Although CBD showed both neurotoxic and neuroprotective effects on hippocampal neurons in the in vitro setting, the use of low-concentrated (i.e., 5 µM) CBD, not causing toxic effects on the neurons, significantly rescued the neurons from the oxidative stress (H2O2), confirming its neuroprotection capability.

Inexpensive water soluble methyl methacrylate-functionalized hydroxyphthalimide: variations of the mycophenolic acid core for selective live cell imaging of free cysteine.

Evident from numerous studies, cysteine plays a crucial role in cellular function. Reactions with analyte also enables for molecular recognition to adhere to molecular therapeutic potential; integration between synthetic probes therefore allows for a potentially deep therapy-related interogation of biological systems (theranostics). The development of molecular cysteine probes with extremely accurate detection is still a key challenge for the field. The development of water-soluble organic molecular fluorescent probes able to efficiently distinguish common biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) by chemical recognition means i.e. by (binding, cleavage) in biological systems is a greatly sought research challenge due to the similarity of the small sulfhydryl-containing species. Herein, we have developed a water-soluble and highly cell viable fluorescent organic molecule (log P = 0.82) for the selective detection of cysteine. The probe (Myco-Cys) shows a "turn-on" response with the cleavage ester linkage of the methacrylate as cysteine is encountered in solution. The probe shows strong fluorescence enhancement (16.5-fold) when treated with Cys (1 equiv., 10 µM) compared to closely related species such as amino acids, including HCy/GSH, and the limit of detection was determined as 45.0 nM. DFT calculations helped confirm the photomechanism of Myco-Cys. Furthermore, the sensing ability of the probe was demonstrated by living cell assays through the use of confocal fluorescence microscopy. Myco-Cys could selectively detect cysteine among biothiols. Myco-Cys was able to monitor the cysteine level, apart from the oxidative stress present in the form of H2O2 in A549 cells.

Neuronal Migration on Silicon Microcone Arrays with Different Pitches.

Neuronal migration is a complicated but fundamental process for proper construction and functioning of neural circuits in the brain. Many in vivo studies have suggested the involvement of environmental physical features of a neuron in its migration, but little effort has been made for the in vitro demonstration of topography-driven neuronal migration. This work investigates migratory behaviors of primary hippocampal neurons on a silicon microcone (SiMC) array that presents 14 different pitch domains (pitch: 2.5-7.3 µm). Neuronal migration becomes the maximum at the pitch of around 3 µm, with an upper migration threshold of about 4 µm. Immunocytochemical studies indicate that the speed and direction of migration, as well as its probability of occurrence, are correlated with the morphology of the neuron, which is dictated by the pitch and shape of underlying SiMC structures. In addition to the effects on neuronal migration, the real-time imaging of migrating neurons on the topographical substrate reveals new in vitro modes of neuronal migration, which have not been observed on the conventional flat culture plate, but been suggested by in vivo studies.

Layer-wise relevance propagation of InteractionNet explains protein-ligand interactions at the atom level.

Development of deep-learning models for intermolecular noncovalent (NC) interactions between proteins and ligands has great potential in the chemical and pharmaceutical tasks, including structure-activity relationship and drug design. It still remains an open question how to convert the three-dimensional, structural information of a protein-ligand complex into a graph representation in the graph neural networks (GNNs). It is also difficult to know whether a trained GNN model learns the NC interactions properly. Herein, we propose a GNN architecture that learns two distinct graphs-one for the intramolecular covalent bonds in a protein and a ligand, and the other for the intermolecular NC interactions between the protein and the ligand-separately by the corresponding covalent and NC convolutional layers. The graph separation has some advantages, such as independent evaluation on the contribution of each convolutional step to the prediction of dissociation constants, and facile analysis of graph-building strategies for the NC interactions. In addition to its prediction performance that is comparable to that of a state-of-the art model, the analysis with an explainability strategy of layer-wise relevance propagation shows that our model successfully predicts the important characteristics of the NC interactions, especially in the aspect of hydrogen bonding, in the chemical interpretation of protein-ligand binding.