A triplex for intestinal protection: Neurons, microbes, and goblet cells.

Diverse intestinal components (e.g., gut-associated neurons, immune cells, gut microbes, and epithelium) are intimately intertwined with each other to maintain homeostasis in the gut. In a recent issue of Cell, Zhang et al. (2022) and Yang et al. (2022) present complementary studies uncovering interactions between nociceptor neurons, gut epithelium, and the microbiome to protect intestinal tissue from inflammation.

Universal recording of immune cell interactions in vivo.

Immune cells rely on transient physical interactions with other immune and non-immune populations to regulate their function1. To study these 'kiss-and-run' interactions directly in vivo, we previously developed LIPSTIC (labelling immune partnerships by SorTagging intercellular contacts)2, an approach that uses enzymatic transfer of a labelled substrate between the molecular partners CD40L and CD40 to label interacting cells. Reliance on this pathway limited the use of LIPSTIC to measuring interactions between CD4+ T helper cells and antigen-presenting cells, however. Here we report the development of a universal version of LIPSTIC (uLIPSTIC), which can record physical interactions both among immune cells and between immune and non-immune populations irrespective of the receptors and ligands involved. We show that uLIPSTIC can be used, among other things, to monitor the priming of CD8+ T cells by dendritic cells, reveal the steady-state cellular partners of regulatory T cells and identify germinal centre-resident T follicular helper cells on the basis of their ability to interact cognately with germinal centre B cells. By coupling uLIPSTIC with single-cell transcriptomics, we build a catalogue of the immune populations that physically interact with intestinal epithelial cells at the steady state and profile the evolution of the interactome of lymphocytic choriomeningitis virus-specific CD8+ T cells in multiple organs following systemic infection. Thus, uLIPSTIC provides a broadly useful technology for measuring and understanding cell-cell interactions across multiple biological systems.

A neural circuit mechanism for mechanosensory feedback control of ingestion.

Mechanosensory feedback from the digestive tract to the brain is critical for limiting excessive food and water intake, but the underlying gut-brain communication pathways and mechanisms remain poorly understood1-12. Here we show that, in mice, neurons in the parabrachial nucleus that express the prodynorphin gene (hereafter, PBPdyn neurons) monitor the intake of both fluids and solids, using mechanosensory signals that arise from the upper digestive tract. Most individual PBPdyn neurons are activated by ingestion as well as the stimulation of the mouth and stomach, which indicates the representation of integrated sensory signals across distinct parts of the digestive tract. PBPdyn neurons are anatomically connected to the digestive periphery via cranial and spinal pathways; we show that, among these pathways, the vagus nerve conveys stomach-distension signals to PBPdyn neurons. Upon receipt of these signals, these neurons produce aversive and sustained appetite-suppressing signals, which discourages the initiation of feeding and drinking (fully recapitulating the symptoms of gastric distension) in part via signalling to the paraventricular hypothalamus. By contrast, inhibiting the same population of PBPdyn neurons induces overconsumption only if a drive for ingestion exists, which confirms that these neurons mediate negative feedback signalling. Our findings reveal a neural mechanism that underlies the mechanosensory monitoring of ingestion and negative feedback control of intake behaviours upon distension of the digestive tract.

Predictive High-Throughput Platform for Dual Screening of mRNA Lipid Nanoparticle Blood-Brain Barrier Transfection and Crossing.

Lipid nanoparticle (LNP)-mediated nucleic acid therapies, including mRNA protein replacement and gene editing therapies, hold great potential in treating neurological disorders including neurodegeneration, brain cancer, and stroke. However, delivering LNPs across the blood-brain barrier (BBB) after systemic administration remains underexplored. In this work, we engineered a high-throughput screening transwell platform for the BBB (HTS-BBB), specifically optimized for screening mRNA LNPs. Unlike most transwell assays, which only assess transport across an endothelial monolayer, HTS-BBB simultaneously measures LNP transport and mRNA transfection of the endothelial cells themselves. We then use HTS-BBB to screen a library of 14 LNPs made with structurally diverse ionizable lipids and demonstrate it is predictive of in vivo performance by validating lead candidates for mRNA delivery to the mouse brain after intravenous injection. Going forward, this platform could be used to screen large libraries of brain-targeted LNPs for a range of protein replacement and gene editing applications.

Assessment of skin sensitization potential of zinc oxide, aluminum oxide, manganese oxide, and copper oxide nanoparticles through the local lymph node assay: 5-bromo-deoxyuridine flow cytometry method.

The advent of nanotechnology has significantly spurred the utilization of nanoparticles (NPs) across diverse sectors encompassing industry, agriculture, engineering, cosmetics, and medicine. Metallic oxides including zinc oxide (ZnO), copper oxide (CuO), manganese oxide (Mn2O3), and aluminum oxide (Al2O3), in their NP forms, have become prevalent in cosmetics and various dermal products. Despite the expanding consideration of these compounds for dermal applications, their potential for initiating skin sensitization (SS) has not been comprehensively examined. An in vivo assay, local lymph node assay: 5-bromo-2-deoxyuridine-flow cytometry method (LLNA: BrdU-FCM) recognized as an alternative testing method for screening SS potential was used to address these issues. Following the OECD TG 442B guidelines, NPs suspensions smaller than 50 nm size were prepared for ZnO and Al2O3 at concentrations of 10, 25, and 50%, and Mn2O3 and CuO at concentrations of 5, 10, and 25%, and applied to the dorsum of each ear of female BALB/c mice on a daily basis for 3 consecutive days. Regarding the prediction of test substance to skin sensitizer if sensitization index (SI)≥2.7, all 4 NPs were classified as non-sensitizing. The SI values were below 2.06, 1.33, 1.42, and 0.99 for ZnO, Al2O3, Mn2O3, and CuO, respectively, at all test concentrations. Although data presented were negative with respect to adverse SS potential for these 4 NPs, further confirmatory tests addressing other key events associated with SS adverse outcome pathway need to be carried out to arrive at an acceptable conclusion on the skin safety for both cosmetic and dermal applications.

Nanotherapeutics for immune network modulation in tumor microenvironments.

Immunotherapy has shown promise in cancer treatment, and is thus drawing increasing interest in this field. While the standard chemotherapy- and/or radiotherapy-based cancer treatments aim to directly kill cancer cells, immunotherapy uses host immune cell surveillance to fight cancer. In the tumor environment, there is a close relationship between tumor cells and the adjacent immune cells, which are largely suppressed by cancer-related regulation of immune checkpoints, immune-suppressive cytokines, and metabolic factors. The immune modulators currently approved for cancer treatment remain limited by issues with dose tolerance and insufficient efficacy. Researchers have developed and tested various nano-delivery systems with the goal of improving the treatment outcome of these drugs. By encapsulating immune modulators in particles and directing their tissue accumulation, some such systems have decreased immune-related toxicity while sharpening the antitumor response. Surface-ligand modification of nanoparticles has allowed drugs to be delivered to specific immune cells types. Researchers have also studied strategies for depleting or reprogramming the immune-suppressive cells to recover the immune environment. Combining a nanomaterial with an external stimulus has been used to induce immunogenic cell death; this favors the inflammatory environment found in tumor tissues to promote antitumor immunity. The present review covers the most recent strategies aimed at modulating the tumor immune environment, and discusses the challenges and future perspectives in developing nanoparticles for cancer immunotherapy.

Crystal structures of human NSDHL and development of its novel inhibitor with the potential to suppress EGFR activity.

NAD(P)-dependent steroid dehydrogenase-like (NSDHL), an essential enzyme in human cholesterol synthesis and a regulator of epidermal growth factor receptor (EGFR) trafficking pathways, has attracted interest as a therapeutic target due to its crucial relevance to cholesterol-related diseases and carcinomas. However, the development of pharmacological agents for targeting NSDHL has been hindered by the absence of the atomic details of NSDHL. In this study, we reported two X-ray crystal structures of human NSDHL, which revealed a detailed description of the coenzyme-binding site and the unique conformational change upon the binding of a coenzyme. A structure-based virtual screening and biochemical evaluation were performed and identified a novel inhibitor for NSDHL harboring suppressive activity towards EGFR. In EGFR-driven human cancer cells, treatment with the potent NSDHL inhibitor enhanced the antitumor effect of an EGFR kinase inhibitor. Overall, these findings could serve as good platforms for the development of therapeutic agents against NSDHL-related diseases.

Rational Design of a DNA-Scaffolded High-Affinity Binder for Langerin.

Binders of langerin could target vaccines to Langerhans cells for improved therapeutic effect. Since langerin has low affinity for monovalent glycan ligands, highly multivalent presentation has previously been key for targeting. Aiming to reduce the amount of ligand required, we rationally designed molecularly defined high-affinity binders based on the precise display of glycomimetic ligands (Glc2NTs) on DNA-PNA scaffolds. Rather than mimicking langerin's homotrimeric structure with a C3-symmetric scaffold, we developed readily accessible, easy-to-design bivalent binders. The method considers the requirements for bridging sugar binding sites and statistical rebinding as a means to both strengthen the interactions at single binding sites and amplify the avidity enhancement provided by chelation. This gave a 1150-fold net improvement over the affinity of the free ligand and provided a nanomolar binder (IC50 =300 nM) for specific internalization by langerin-expressing cells.

A Liposomal Platform for Delivery of a Protein Antigen to Langerin-Expressing Cells.

The skin is an attractive site for vaccination and harbors a dense network of Langerhans cells that are the prime target for antigen delivery approaches in the epidermis. While specific targeting of Langerhans cells has been shown to elicit the necessary T-cell response using antibody-based delivery approaches, the targeted administration of particulate antigens in the form of nanoparticle-based vaccine formulations has been challenging. We previously reported on a specific targeting ligand for human Langerin, a C-type lectin expressed on Langerhans cells. This ligand is presented on liposomes and renders them highly specific for the uptake by Langerhans cells. Here we show a detailed study of the uptake and intracellular routing of the particles in model cell lines by confocal and live cell imaging as well as flow cytometric assays. Liposomes are internalized into early endosomal compartments and accumulate in late endosomes and lysosomes, shortly followed by a release of the cargo. Furthermore, we show the encapsulation of protein antigens and their delivery to cell lines and primary human Langerhans cells. These data further support the applicability of the targeted liposomal particles for protein vaccine applications.

Oligosaccharides Self-Assemble and Show Intrinsic Optical Properties.

Self-assembling peptides and oligonucleotides have given rise to synthetic materials with several applications in nanotechnology. Aggregation of synthetic oligosaccharides into well-defined architectures has not been reported even though natural polysaccharides, such as cellulose and chitin, are key structural components of biomaterials. Here, we report that six synthetic oligosaccharides, ranging from dimers to hexamers, self-assemble into nanostructures of varying morphologies and emit within the visible spectrum in an excitation-dependent manner. Well-defined differences in chain length, monomer modification, and aggregation methods yield glycomaterials with distinct shapes and properties. The excitation-dependent fluorescence in a broad range within the visible spectrum illustrates their potential for use in optical devices and imaging applications. We anticipate that our systematic approach of studying well-defined synthetic oligosaccharides will form the foundation of our understanding of carbohydrate interactions in nature.

Staphylococcus aureus-mimetic control of antibody orientation on nanoparticles.

We designed a bacterio-mimetic nanoparticle that can noncovalently control the orientation of attached antibodies. Liposomes with Fc-binding peptide (FcBP), formulated using FcBP-conjugated PEGylated lipid, were used as model nanoparticles. Compared with control nanoparticles surface-modified with antibody covalently attached via maleimide functional groups (Mal-NPs), FcBP-capped nanoparticles (FcBP-NPs) exhibited greater binding affinity to the target protein. Human epidermal growth factor receptor 2 (HER2)-specific antibody-modified FcBP-NPs (HER2/FcBP-NPs) showed 5.3-fold higher binding affinity to HER2 than isotype IgG antibody-modified NPs, and 2.6-fold higher affinity compared with anti-HER2 antibody-conjugated Mal-NPs. Cellular uptake of HER2/FcBP-NPs in HER2-positive cells was significantly higher than that of other formulations. The biodistribution of HER2/FcBP-NPs was higher than that of antibody-conjugated NPs in HER2-positive tumor tissues, but not in HER2-negative tumors. Our findings suggest the potential of bacteriomimetic nanoparticles for controlling the orientation of antibody attachment. These nanoparticles may have diverse applications in nanomedicine, including drug delivery, molecular imaging, and diagnosis.

Multiscale Modulation of Nanocrystalline Cellulose Hydrogel via Nanocarbon Hybridization for 3D Neuronal Bilayer Formation.

Bacterial biopolymers have drawn much attention owing to their unconventional three-dimensional structures and interesting functions, which are closely integrated with bacterial physiology. The nongenetic modulation of bacterial (Acetobacter xylinum) cellulose synthesis via nanocarbon hybridization, and its application to the emulation of layered neuronal tissue, is reported. The controlled dispersion of graphene oxide (GO) nanoflakes into bacterial cellulose (BC) culture media not only induces structural changes within a crystalline cellulose nanofibril, but also modulates their 3D collective association, leading to substantial reduction in Young's modulus (≈50%) and clear definition of water-hydrogel interfaces. Furthermore, real-time investigation of 3D neuronal networks constructed in this GO-incorporated BC hydrogel with broken chiral nematic ordering revealed the vertical locomotion of growth cones, the accelerated neurite outgrowth (≈100 µm per day) with reduced backward travel length, and the efficient formation of synaptic connectivity with distinct axonal bifurcation abundancy at the ≈750 µm outgrowth from a cell body. In comparison with the pristine BC, GO-BC supports the formation of well-defined neuronal bilayer networks with flattened interfacial profiles and vertical axonal outgrowth, apparently emulating the neuronal development in vivo. We envisioned that our findings may contribute to various applications of engineered BC hydrogel to fundamental neurobiology studies and neural engineering.

Therapeutic gene editing: delivery and regulatory perspectives.

Gene-editing technology is an emerging therapeutic modality for manipulating the eukaryotic genome by using target-sequence-specific engineered nucleases. Because of the exceptional advantages that gene-editing technology offers in facilitating the accurate correction of sequences in a genome, gene editing-based therapy is being aggressively developed as a next-generation therapeutic approach to treat a wide range of diseases. However, strategies for precise engineering and delivery of gene-editing nucleases, including zinc finger nucleases, transcription activator-like effector nuclease, and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-associated nuclease Cas9), present major obstacles to the development of gene-editing therapies, as with other gene-targeting therapeutics. Currently, viral and non-viral vectors are being studied for the delivery of these nucleases into cells in the form of DNA, mRNA, or proteins. Clinical trials are already ongoing, and in vivo studies are actively investigating the applicability of CRISPR/Cas9 techniques. However, the concept of correcting the genome poses major concerns from a regulatory perspective, especially in terms of safety. This review addresses current research trends and delivery strategies for gene editing-based therapeutics in non-clinical and clinical settings and considers the associated regulatory issues.

NeuO: a fluorescent chemical probe for live neuron labeling.

To address existing limitations in live neuron imaging, we have developed NeuO, a novel cell-permeable fluorescent probe with an unprecedented ability to label and image live neurons selectively over other cells in the brain. NeuO enables robust live neuron imaging and isolation in vivo and in vitro across species; its versatility and ease of use sets the basis for its development in a myriad of neuronal targeting applications.

Influence of ionizable lipid tail length on lipid nanoparticle delivery of mRNA of varying length.

RNA-based therapeutics have gained traction for the prevention and treatment of a variety of diseases. However, their fragility and immunogenicity necessitate a drug carrier. Lipid nanoparticles (LNPs) have emerged as the predominant delivery vehicle for RNA therapeutics. An important component of LNPs is the ionizable lipid (IL), which is protonated in the acidic environment of the endosome, prompting cargo release into the cytosol. Currently, there is growing evidence that the structure of IL lipid tails significantly impacts the efficacy of LNP-mediated mRNA translation. Here, we optimized IL tail length for LNP-mediated delivery of three different mRNA cargos. Using C12-200, a gold standard IL, as a model, we designed a library of ILs with varying tail lengths and evaluated their potency in vivo. We demonstrated that small changes in lipophilicity can drastically increase or decrease mRNA translation. We identified that LNPs formulated with firefly luciferase mRNA (1929 base pairs) and C10-200, an IL with shorter tail lengths than C12-200, enhance liver transfection by over 10-fold. Furthermore, different IL tail lengths were found to be ideal for transfection of LNPs encapsulating mRNA cargos of varying sizes. LNPs formulated with erythropoietin (EPO), responsible for stimulating red blood cell production, mRNA (858 base pairs), and the C13-200 IL led to EPO translation at levels similar to the C12-200 LNP. The LNPs formulated with Cas9 mRNA (4521 base pairs) and the C9-200 IL induced over three times the quantity of indels compared with the C12-200 LNP. Our findings suggest that shorter IL tails may lead to higher transfection of LNPs encapsulating larger mRNAs, and that longer IL tails may be more efficacious for delivering smaller mRNA cargos. We envision that the results of this project can be utilized as future design criteria for the next generation of LNP delivery systems for RNA therapeutics.

Optimized microfluidic formulation and organic excipients for improved lipid nanoparticle mediated genome editing.

mRNA-based gene editing platforms have tremendous promise in the treatment of genetic diseases. However, for this potential to be realized in vivo, these nucleic acid cargos must be delivered safely and effectively to cells of interest. Ionizable lipid nanoparticles (LNPs), the most clinically advanced non-viral RNA delivery system, have been well-studied for the delivery of mRNA but have not been systematically optimized for the delivery of mRNA-based CRISPR-Cas9 platforms. In this study, we investigated the effect of microfluidic and lipid excipient parameters on LNP gene editing efficacy. Through in vitro screening in liver cells, we discovered distinct trends in delivery based on phospholipid, cholesterol, and lipid-PEG structure in LNP formulations. Combination of top-performing lipid excipients produced an LNP formulation that resulted in 3-fold greater gene editing in vitro and facilitated 3-fold greater reduction of a therapeutically-relevant protein in vivo relative to the unoptimized LNP formulation. Thus, systematic optimization of LNP formulation parameters revealed a novel LNP formulation that has strong potential for delivery of gene editors to the liver to treat metabolic disease.

Information transfer in mammalian glycan-based communication.

Glycan-binding proteins, so-called lectins, are exposed on mammalian cell surfaces and decipher the information encoded within glycans translating it into biochemical signal transduction pathways in the cell. These glycan-lectin communication pathways are complex and difficult to analyze. However, quantitative data with single-cell resolution provide means to disentangle the associated signaling cascades. We chose C-type lectin receptors (CTLs) expressed on immune cells as a model system to study their capacity to transmit information encoded in glycans of incoming particles. In particular, we used nuclear factor kappa-B-reporter cell lines expressing DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), macrophage C-type lectin (MCL), dectin-1, dectin-2, and macrophage-inducible C-type lectin (MINCLE), as well as TNFαR and TLR-1&2 in monocytic cell lines and compared their transmission of glycan-encoded information. All receptors transmit information with similar signaling capacity, except dectin-2. This lectin was identified to be less efficient in information transmission compared to the other CTLs, and even when the sensitivity of the dectin-2 pathway was enhanced by overexpression of its co-receptor FcRγ, its transmitted information was not. Next, we expanded our investigation toward the integration of multiple signal transduction pathways including synergistic lectins, which is crucial during pathogen recognition. We show how the signaling capacity of lectin receptors using a similar signal transduction pathway (dectin-1 and dectin-2) is being integrated by compromising between the lectins. In contrast, co-expression of MCL synergistically enhanced the dectin-2 signaling capacity, particularly at low-glycan stimulant concentration. By using dectin-2 and other lectins as examples, we demonstrate how signaling capacity of dectin-2 is modulated in the presence of other lectins, and therefore, the findings provide insight into how immune cells translate glycan information using multivalent interactions.

Application of stacked autoencoder for identification of bone fracture.

This study presents a stacked autoencoder (SAE)-based assessment method which is one of the unsupervised learning schemes for the investigation of bone fracture. Relatively accurate health monitoring of bone fracture requires considering physical interactions among tissue, muscle, wave propagation and boundary conditions inside the human body. Furthermore, the investigation of fracture, crack and healing process without state-of-the-art medical devices such as CT, X-ray and MRI systems is challenging. To address these issues, this study presents the SAE method that incorporates bilateral symmetry of the human legs and low-frequency transverse vibration. To verify the presented method, several examples are employed with plastic pipes, cadaver legs and human legs. Virtual spectrograms, created by applying a short-time Fourier transform to the differences in vibration responses, are employed for image-based training in SAE. The virtual spectrograms are then classified and the fine-tuning is also carried out to increase the accuracy. Moreover, a confusion matrix is employed to evaluate classification accuracy and training validity.

On-demand delivery of protein drug from 3D-printed implants.

Here, we constructed 3D-printed multiunit implants to enable remote light-controlled protein drug delivery in a spatiotemporal manner. Multiunit implants were designed to be 3D printed using polycaprolactone, lauric acid, and melanin as a matrix, and a polycaprolactone scaffold as a multiunit divider. As a model drug, insulin was loaded to each unit of the implant. The 3D printing yielded a rectangular matrix with multiunit sectors segregated by polycaprolactone lanes. Irradiation with near infrared light (NIR) triggered controlled release of insulin from the irradiated locus: Upon NIR irradiation, heat generated from the melanin melted the polycaprolactone/lauric acid matrix to release insulin from the scaffold. In the absence of melanin in the matrix, the implant did not show NIR-responsive insulin release. When lauric acid was absent from the matrix, the NIR-irradiated unit did not undergo dismantling. When the insulin-loaded multiunit implant was applied to a mouse diabetic model and irradiated with NIR, repetitive insulin release resulted in an efficient decrease of the blood glucose level over multiple days. Together, these results suggest that 3D printing technology-based multi-dosing of insulin on demand can enable convenient treatment of diabetes through external NIR irradiation, potentially avoiding the pain and discomfort of repeated insulin injections.

Lipid nanoparticle-mediated CRISPR/Cas9 gene editing and metabolic engineering for anticancer immunotherapy.

Metabolic engineering of the tumor microenvironment has emerged as a new strategy. Lactate dehydrogenase A (LDHA) is a prominent target for metabolic engineering. Here, we designed a cationic lipid nanoparticle formulation for LDHA gene editing. The plasmid DNA delivery efficiency of our lipid nanoparticle formulations was screened by testing the fluorescence of lipid nanoparticles complexed to plasmid DNA encoding green fluorescence protein (GFP). The delivery efficiency was affected by the ratios of three components: a cationic lipid, cholesterol or its derivative, and a fusogenic lipid. The lipid nanoparticle designated formulation F3 was complexed to plasmid DNA co-encoding CRISPR-associated protein 9 and LDHA-specific sgRNA, yielding the lipoplex, pCas9-sgLDHA/F3. The lipoplex including GFP-encoding plasmid DNA provided gene editing in HeLa-GFP cells. Treatment of B16F10 tumor cells with pCas9-sgLDHA/F3 yielded editing of the LDHA gene and increased the pH of the culture medium. pCas9-sgLDHA/F3 treatment activated the interferon-gamma and granzyme production of T cells in culture. In vivo, combining pCas9-sgLDHA/F3 with immune checkpoint-inhibiting anti-PD-L1 antibody provided a synergistic antitumor effect and prolonged the survival of tumor model mice. This study suggests that combining metabolic engineering of the tumor microenvironment with immune checkpoint inhibition could be a valuable antitumor strategy.