CORRIGENDUM: Correction of the ACKNOWLEDGEMENTS (Fund/Grant Support). Epigenetic modulation inhibits epithelial-mesenchymal transition-driven fibrogenesis and enhances characteristics of chemically-derived hepatic progenitors.

[This corrects the article on p. 274 in vol. 106, PMID: 38725803.].

Epigenetic modulation inhibits epithelial-mesenchymal transition-driven fibrogenesis and enhances characteristics of chemically-derived hepatic progenitors.

Purpose: One of the novel cell sources of cell-based liver regenerative medicine is human chemically-derived hepatic progenitors (hCdHs). We previously established this cell by direct hepatocyte reprogramming with a combination of small molecules (hepatocyte growth factor, A83-01, CHIR99021). However, there have been several issues concerning the cell's stability and maintenance, namely the occurrences of epithelial-mesenchymal transition (EMT) that develop fibrotic phenotypes, resulting in the loss of hepatic progenitor characteristics. These hepatic progenitor attributes are thought to be regulated by SOX9, a transcription factor essential for hepatic progenitor cells and cholangiocytes. Methods: To suppress the fibrotic phenotype and improve our long-term hCdHs culture technology, we utilized the epigenetic modulating drugs DNA methyltransferase inhibitor (5-azacytidine) and histone deacetylase inhibitor (sodium butyrate) that have been reported to suppress and revert hepatic fibrosis. To confirm the essential role of SOX9 to our cell, we used clustered regularly interspaced short palindromic repeats-interference (CRISPRi) to repress the SOX9 expression. Results: The treatment of only 5-azacytidine significantly reduces the fibrosis/mesenchymal marker and EMT-related transcription factor expression level in the early passages. Interestingly, this treatment also increased the hepatic progenitor markers expression, even during the reprogramming phase. Then, we confirmed the essential role of SOX9 by repressing the SOX9 expression with CRISPRi which resulted in the downregulation of several essential hepatic progenitor cell markers. Conclusion: These results highlight the capacity of 5-azacytidine to inhibit EMT-driven hepatic fibrosis and the significance of SOX9 on hepatic progenitor cell stemness properties.

Programmable Enzymatic Reaction Network in Artificial Cell-Like Polymersomes.

The ability to precisely control in vitro enzymatic reactions in synthetic cells plays a crucial role in the bottom-up design of artificial cell models that can recapitulate the key cellular features and functions such as metabolism. However, integration of enzymatic reactions has been limited to bulk or microfluidic emulsions without a membrane, lacking the ability to design more sophisticated higher-order artificial cell communities for reconstituting spatiotemporal biological information at multiple length scales. Herein, droplet microfluidics is utilized to synthesize artificial cell-like polymersomes with distinct molecular permeability for spatiotemporal control of enzymatic reactions driven by external signals and fuels. The presence of a competing reverse enzymatic reaction that depletes the active substrates is shown to enable demonstration of fuel-driven formation of sub-microcompartments within polymersomes as well as realization of out-of-equilibrium systems. In addition, the different permeability characteristics of polymersome membranes are exploited to successfully construct a programmable enzymatic reaction network that mimics cellular communication within a heterogeneous cell community through selective molecular transport.

Discovery of N-benzylbenzamide-based allosteric inhibitors of Aurora kinase A.

Aurora kinases (AurkA/B/C) regulate the assembly of bipolar mitotic spindles and the fidelity of chromosome segregation during mitosis, and are attractive therapeutic targets for cancers. Numerous ATP-competitive AurkA inhibitors have been developed as potential anti-cancer agents. Recently, a few allosteric inhibitors have been reported that bind to the allosteric Y-pocket within AurkA kinase domain and disrupt the interaction between AurkA and its activator TPX2. Herein we report a novel allosteric AurkA inhibitor (6h) of N-benzylbenzamide backbone. Compound 6h suppressed the both catalytic activity and non-catalytic functions of AurkA. The inhibitory activity of 6h against AurkA (IC50 = 6.50 µM) was comparable to that of the most potent allosteric AurkA inhibitor AurkinA. Docking analysis against the Y-pocket revealed important pharmacophores and interactions that were coherent with structure-activity relationship. In addition, 6h suppressed DNA replication in G1-S phase, which is a feature of allosteric inhibition of AurA. Our current study may provide a useful insight in designing potent allosteric AurkA inhibitors.

Utilization of nicking properties of CRISPR-Cas12a effector for genome editing.

The CRISPR-Cas nickase system for genome editing has attracted considerable attention owing to its safety, efficiency, and versatility. Although alternative effectors to Cas9 have the potential to expand the scope of genome editing, their application has not been optimized. Herein, we used an enhanced CRISPR-Cas12a nickase system to induce mutations by targeting genes in a human-derived cell line. The optimized CRISPR-Cas12a nickase system effectively introduced mutations into target genes under a specific directionality and distance between nickases. In particular, the single-mode Cas12a nickase system can induce the target-specific mutations with less DNA double-strand breaks. By inducing mutations in the Thymine-rich target genes in single- or dual-mode, Cas12a nickase compensates the limitations of Cas9 nickase and is expected to contribute to the development of future genome editing technologies.

Microencapsulation of alcohol solvents and high-content actives for efficient transdermal delivery.

The barrier function of the skin in effectively protecting the underlying tissue from the surrounding environment makes it challenging to achieve the efficient transdermal delivery of actives. Herein, we report on alcohol-solvent-encapsulated microcapsules to achieve enhanced skin efficacy. We show that using palm oil as the shell material allows for the microencapsulation of a broad range of alcohol solvents, including ethanol and dipropylene glycol (DPG), as well as on-demand release. Moreover, clinical trials reveal that the high-content actives in microcapsules result in enhanced skin efficacy, and the presence of DPG effectively mediates the transdermal delivery of these actives without causing any skin irritation. We envision that the alcohol-solvent microencapsulation strategy outlined in this work offers new possibilities in cosmetics, food, and drug delivery systems.

Measurement of diets that are healthy, environmentally sustainable, affordable, and equitable: A scoping review of metrics, findings, and research gaps.

Introduction: Research on the impacts of dietary patterns on human and planetary health is a rapidly growing field. A wide range of metrics, datasets, and analytical techniques has been used to explore the role of dietary choices/constraints in driving greenhouse gas (GHG) emissions, environmental degradation, health and disease outcomes, and the affordability of food baskets. Many argue that each domain is important, but few have tackled all simultaneously in analyzing diet-outcome relationships. Methods: This paper reviews studies published between January 2015 and December 2021 (inclusive) that examined dietary patterns in relation to at least two of the following four thematic pillars: (i) planetary health, including, climate change, environmental quality, and natural resource impacts, (ii) human health and disease, (iii) economic outcomes, including diet cost/affordability, and (iv) social outcomes, e.g., wages, working conditions, and culturally relevant diets. We systematically screened 2,425 publications by title and abstract and included data from 42 eligible publications in this review. Results: Most dietary patterns used were statistically estimated or simulated rather than observed. A rising number of studies consider the cost/affordability of dietary scenarios in relation to optimized environmental and health outcomes. However, only six publications incorporate social sustainability outcomes, which represents an under-explored dimension of food system concerns. Discussion: This review suggests a need for (i) transparency and clarity in datasets used and analytical methods; (ii) explicit integration of indicators and metrics linking social and economic issues to the commonly assessed diet-climate-planetary ecology relationships; (iii) inclusion of data and researchers from low- and middle-income countries; (iv) inclusion of processed food products to reflect the reality of consumer choices globally; and (v) attention to the implications of findings for policymakers. Better understanding is urgently needed on dietary impacts on all relevant human and planetary domains simultaneously.

Structural basis for CEP192-mediated regulation of centrosomal AURKA.

Aurora kinase A (AURKA) performs critical functions in mitosis. Thus, the activity and subcellular localization of AURKA are tightly regulated and depend on diverse factors including interactions with the multiple binding cofactors. How these different cofactors regulate AURKA to elicit different levels of activity at distinct subcellular locations and times is poorly understood. Here, we identified a conserved region of CEP192, the major cofactor of AURKA, that mediates the interaction with AURKA. Quantitative binding studies were performed to map the interactions of a conserved helix (Helix-1) within CEP192. The crystal structure of Helix-1 bound to AURKA revealed a distinct binding site that is different from other cofactor proteins such as TPX2. Inhibiting the interaction between Helix-1 and AURKA in cells led to the mitotic defects, demonstrating the importance of the interaction. Collectively, we revealed a structural basis for the CEP192-mediated AURKA regulation at the centrosome, which is distinct from TPX2-mediated regulation on the spindle microtubule.

Broad-temperature-range mechanically tunable hydrogel microcapsules for controlled active release.

Here, we report PNIPAm-co-PEGDA hydrogel shelled microcapsules with a thin oil layer to achieve tunable thermo-responsive release of the encapsulated small hydrophilic actives. We use a microfluidic device integrated with a temperature-controlled chamber for consistent and reliable production of the microcapsules by utilizing triple emulsion drops (W/O/W/O) with a thin oil layer as capsule templates. The interstitial oil layer between the aqueous core and the PNIPAm-co-PEGDA shell provides a diffusion barrier for the encapsulated active until the temperature reaches a critical point above which the destabilization of interstitial oil layer occurs. We find that the destabilization of the oil layer with temperature increase is caused by outward expansion of the aqueous core due to volume increase and the radial inward compression from the deswelling of the thermo-responsive hydrogel shell. The copolymerization of NIPAm with PEGDA increases the biocompatibility of the resulting microcapsule while offering the ability to alter the compressive modulus in broad ranges by simply varying crosslinker concentrations thereby to precisely tune the onset release temperature. Based on this concept, we further demonstrate that the release temperature can be enhanced up to 62 °C by adjusting the shell thickness even without varying the chemical composition of the hydrogel shell. Moreover, we incorporate gold nanorods within the hydrogel shell to spatiotemporally regulate the active release from the microcapsules by illuminating with non-invasive near infrared (NIR) light.

dCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration.

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9's specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant.

High-Precision Synthesis of RNA-Loaded Lipid Nanoparticles for Biomedical Applications.

The recent development of RNA-based therapeutics in delivering nucleic acids for gene editing and regulating protein translation has led to the effective treatment of various diseases including cancer, inflammatory and genetic disorder, as well as infectious diseases. Among these, lipid nanoparticles (LNP) have emerged as a promising platform for RNA delivery and have shed light by resolving the inherent instability issues of naked RNA and thereby enhancing the therapeutic potency. These LNP consisting of ionizable lipid, helper lipid, cholesterol, and poly(ethylene glycol)-anchored lipid can stably enclose RNA and help them release into the cells' cytosol. Herein, the significant progress made in LNP research starting from the LNP constituents, formulation, and their diverse applications is summarized first. Moreover, the microfluidic methodologies which allow precise assembly of these newly developed constituents to achieve LNP with controllable composition and size, high encapsulation efficiency as well as scalable production are highlighted. Furthermore, a short discussion on current challenges as well as an outlook will be given on emerging approaches to resolving these issues.

Spatiotemporal control of signal-driven enzymatic reaction in artificial cell-like polymersomes.

Living cells can spatiotemporally control biochemical reactions to dynamically assemble membraneless organelles and remodel cytoskeleton. Herein, we present a microfluidic approach to prepare semi-permeable polymersomes comprising of amphiphilic triblock copolymer to achieve external signal-driven complex coacervation as well as biophysical reconstitution of cytoskeleton within the polymersomes. We also show that the microfluidic synthesis of polymersomes enables precise control over size, efficient encapsulation of enzymes as well as regulation of substrates without the use of biopores. Moreover, we demonstrate that the resulting triblock copolymer-based membrane in polymersomes is size-selective, allowing phosphoenol pyruvate to readily diffuse through the membrane and induce enzymatic reaction and successive coacervation or actin polymerization in the presence of pyruvate kinase and adenosine diphosphate inside the polymersomes. We envision that the Pluronic-based polymersomes presented in this work will shed light in the design of in vitro enzymatic reactions in artificial cell-like vesicles.

Skin Penetration Enhancer-Incorporated Lipid Nanovesicles (SPE-LNV) for Skin Brightening and Wrinkle Treatment.

In this work, we utilize skin penetration enhancers (SPEs) such as ceramide and fatty acids in lipid nanovesicles to promote the transdermal delivery of active ingredients. These SPE-incorporated lipid nanovesicles (SPE-LNV) interact with the constituents of skin's outermost stratum corneum (SC) layer, enabling even niacinamide and adenosine with high water solubility to effectively permeate through, leading to enhanced skin efficacy. We demonstrate by both in vitro and in vivo skin permeation studies that the SPE-LNV formulation containing both ceramide and fatty acids (LNV-CF) exhibits deeper penetration depth and faster permeation rate compared to conventional lipid nanovesicles (LNV) without SPE as well as LNV-C with only ceramide. Moreover, in vivo clinical trials were also performed to confirm that LNV-CF most effectively mediates the delivery of niacinamide and adenosine, resulting in a substantial decrease in melanin index as well as skin wrinkle compared to the control groups. We envision that the strategy of incorporating both ceramide and fatty acids in lipid nanovesicles offers a simple and convenient route for the rapid and effective delivery of water-soluble active ingredients across the skin barrier layer.

Use of a 3D inkjet-printed model to access dust particle toxicology in the human alveolar barrier.

Fine dust particles in the air travel into our body via the airway tract and cause severe respiratory diseases. Thus, the analysis of the effects of dust particles on the respiratory system has been receiving significant research interest. However, most studies on the toxicity of dust particles involve two-dimensional (2D) cell cultures, animal models, and epidemiology. Here, we inkjet-printed a three-dimensional (3D) alveolar barrier model to study how dust particles cause respiratory diseases. The three-layered in vitro model was exposed to A2 fine test dust with varying concentrations and exposure durations. The results highlighted the destruction of the tissue architecture along with apoptosis in the bioprinted alveolar barrier. The damage at the cellular level induced an increase in the amount of pro-inflammatory cytokines secreted, followed by triggering of the signal transduction pathway and activation of transcription factors. As a consequence of the release of cytokines, the extracellular matrix was degraded, which led to the collapse of the cell structure, loss of cell polarity, and a decrease in barrier tightness. Further, the pulmonary surfactant protein-related genes in the dust-treated alveolar tissue were investigated to evaluate the possible role of dust particles in pulmonary surfactant dysfunction. This study demonstrated the use of 3D-printed tissue model to evaluate the physiological impact of fine dust particles on cytotoxicity, alveolar barrier rigidity, and surfactant secretion of an alveolar barrier.

Tailoring the Hydrophilicity for Delayed Condensation Frosting in Antifogging Coatings.

In the last few decades, numerous studies have focused on designing suitable hydrophilic materials to inhibit surface-induced fog or frost under extreme conditions. As fogging and condensation frosting on a film involves molecular interaction with water prior to forming discrete droplets on the surface, it is essential to control the extent of a film to strongly bind with water molecules for antifogging coatings. While the water contact angle measurement is commonly used to probe the hydrophilicity of a film, it oftentimes fails to predict the antifogging and antifrosting performance as this value only reflects the wettability of a given surface to water droplet. In this work, a polysaccharide-based film composed of chitosan (CHI) and carboxymethyl cellulose (CMC) is used as the model system and oligo(ethylene glycol) (OEG) moieties are additionally introduced to study the effect of OEG moieties on antifogging and condensation frosting. We show that the film containing OEG-grafted CHI exhibits excellent frost-resistant capability due to the OEG moieties in the film that serve as active sites for water molecules to strongly interact in a nonfreezable state.

Polyolefin-based electrospun fibrous matrices embedded with magnetic nanoparticles for effective removal of viscous oils.

In this work, we present a poly (ethylene-co-1-octene)-based fibrous matrix prepared via electrospinning for highly efficient removal of viscous oils. The sorbent consisting of linear low density polyethylene (LLDPE) allows selective absorption of crude oil spills at the water surface without the need for additional isolation of the matrix prior to the refining process. Moreover, the high specific pore volume of the LLDPE sorbent with uniform fibrous morphology was shown to enable the sorbent reach 81.5 ± 5.9% of its equilibrium absorption capacity within 5 min. Furthermore, magnetic nanoparticles (MNP) are incorporated into each fiber comprising the matrix to facilitate the recovery process via external magnetic field without altering the intrinsic absorption capacity. We envision that these sorbents offer a sustainable route for the quick and thorough clean-up of spilled oil due to their high absorption capacity, fast absorption rate, ease of recovery, and absence of secondary waste.

Oral delivery of stem-cell-loaded hydrogel microcapsules restores gut inflammation and microbiota.

Mesenchymal stem cells (MSCs) are an attractive candidate for the treatment of inflammatory bowel disease (IBD), but their poor delivery rate to an inflamed colon is a major factor hampering the clinical potential of stem cell therapies. Moreover, there remains a formidable hurdle to overcome with regard to survival and homing in to injured sites. Here, we develop a strategy utilizing monodisperse hydrogel microcapsules with a thin intermediate oil layer prepared by a triple-emulsion drop-based microfluidic approach as an in-situ oral delivering carrier. The oral delivery of stem-cell-loaded hydrogel microcapsules (SC-HM) enhances MSC survival and retention in the hostile stomach environment due to the intermediate oil layer and low value of the overall stiffness, facilitating programmable cell release during gastrointestinal peristalsis. SC-HM is shown to induce tissue repair, reduce the colonic macrophage infiltration responsible for the secretion of the pro-inflammatory factors, and significantly mitigate the severity of IBD in a mouse model, where MSCs released by SC-HM successfully accumulate at the colonic crypt. Moreover, a metagenomics analysis reveals that SC-HM ameliorates the dysbiosis of specific bacterial genera, including Bacteroides acidifaciens, Lactobacillus (L.) gasseri, Lactobacillus reuteri, and L. intestinalis, implying optimization of the microorganism's composition and abundance. These findings demonstrate that SC-HM is a potential IBD treatment candidate.

Highly specific chimeric DNA-RNA-guided genome editing with enhanced CRISPR-Cas12a system.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system is composed of a Cas12a effector that acts as a DNA-cleaving endonuclease and a crispr RNA (crRNA) that guides the effector to the target DNA. It is considered a key molecule for inducing target-specific gene editing in various living systems. Here, we improved the efficiency and specificity of the CRISPR-Cas12a system through protein and crRNA engineering. In particular, to optimize the CRISPR-Cas12a system at the molecular level, we used a chimeric DNA-RNA guide chemically similar to crRNA to maximize target sequence specificity. Compared with the wild-type (wt)-Cas12a system, when using enhanced Cas12a system (en-Cas12a), the efficiency and target specificity improved on average by 2.58 and 2.77 times, respectively. In our study, when the chimeric DNA-RNA-guided en-Cas12a effector was used, the gene-editing efficiency and accuracy were simultaneously increased. These findings could contribute to highly accurate genome editing, such as human gene therapy, in the near future.

Biocompatible amphiphilic Janus nanoparticles with enhanced interfacial properties for colloidal surfactants.

Emulsions in which water droplets are dispersed in fluorocarbon oil phase (W/F emulsions) serve as effective means to encapsulate bioactives and precisely execute reactions in confined space due to the gas permeability, chemical inertness, and biocompatibility offered by the continuous phase. While molecular surfactants consisting of perfluorinated polyether (PFPE) and polyethylene glycol (PEG) have been used to stabilize these emulsions, these surfactants cannot effectively prevent coalescence and cross-contamination between the neighboring droplets. Herein, we present Janus nanoparticles (F-SiO2-PEG) as biocompatible colloidal surfactants to achieve excellent stability in W/F emulsions. By utilizing monolayered wax colloidosomes as templates, we show that Janus silica nanoparticles with two distinctive surface wetting properties can be synthesized in high purity. Moreover, we demonstrate that additional PEGylation of these Janus particles allows these colloidal surfactants to strongly adhere at the W/F interface, granting excellent emulsion stability compared to the equivalent randomly functionalized nanoparticles and prevent non-specific adsorption of proteins. As the strategy outlined in this work is general, we anticipate that it can be further extended to prepare Janus particles with tailored interfacial properties for biomedical, cosmetics, and pharmaceutical applications involving emulsions.

Water-Saturated Ion Gel for Humidity-Independent High Precision Epidermal Ionic Temperature Sensor.

Although ion gels are attractive sensing materials for deformable epidermal sensors or implantable devices, their sensing performances are highly affected by environmental humidity change, so that their sensing reliability cannot be secured. This study proposes a new concept of maintaining the high-precision temperature sensing performance of highly deformable ion gel sensors. In this approach, a hydrophobic ion gel sensing layer is kept water-saturated by attaching a hydrogel layer, rather than attempting to completely block water penetration. This study performs experimental and theoretical investigation on water concentration in the ion gel, using the analysis of mass transportation at the interface of the ion gel and the hydrogel. By using the charge relaxation time of the ionic molecules, the temperature sensor is not affected by environmental humidity in the extreme range of humidity (30%-100%). This study demonstrates a highly deformable on-skin temperature sensor which shows the same performance either in water or dry state and while exercising with large strains (ε = 50%).