Since the first report of underwater adhesive proteins of marine mussels in 1981, numerous studies have reported mussel-inspired synthetic adhesive polymers. However, none of them have developed up to human-level translational studies. Here, we report a sticky polysaccharide that effectively promotes hemostasis from animal bleeding models to first-in-human hepatectomy. We found that the hemostatic material instantly generates a barrier layer that seals hemorrhaging sites. The barrier is created within a few seconds by in situ interactions with abundant plasma proteins. Therefore, as long as patient blood contains proper levels of plasma proteins, hemostasis should always occur even in coagulopathic conditions. To date, insufficient tools have been developed to arrest coagulopathic bleedings originated from genetic disorders, chronic diseases, or surgical settings such as organ transplantations. Mussel-inspired adhesion chemistry described here provides a useful alternative to the use of fibrin glues up to a human-level biomedical application.

CTCF is crucial to the organization of mammalian genomes into loop structures. According to recent studies, the transcription apparatus is compartmentalized and concentrated at super-enhancers to form phase-separated condensates and drive the expression of cell-identity genes. However, it remains unclear whether and how transcriptional condensates are coupled to higher-order chromatin organization. Here, we show that CTCF is essential for RNA polymerase II (Pol II)-mediated chromatin interactions, which occur as hyperconnected spatial clusters at super-enhancers. We also demonstrate that CTCF clustering, unlike Pol II clustering, is independent of liquid-liquid phase-separation and resistant to perturbation of transcription. Interestingly, clusters of Pol II, BRD4, and MED1 were found to dissolve upon CTCF depletion, but were reinstated upon restoration of CTCF, suggesting a potent instructive function for CTCF in the formation of transcriptional condensates. Overall, we provide evidence suggesting that CTCF-mediated chromatin looping acts as an architectural prerequisite for the assembly of phase-separated transcriptional condensates.

Marine mussels secrete adhesive proteins to attach to solid surfaces. These proteins contain phenolic and basic amino acids exhibiting wet adhesion properties. This study used a mussel-inspired hemostatic polymer, chitosan-catechol, to treat gastrointestinal bleeding caused by endoscopic mucosal resection in a heparinized porcine model. We aimed to evaluate the hemostatic efficacy and short-term safety of this wet adhesive chitosan-catechol. We used 15 heparinized pigs. Four iatrogenic bleeding ulcers classified as Forrest Ib were created in each pig using an endoscopic mucosal resection method. One ulcer in each pig was untreated as a negative control (no-treatment group). The other three ulcers were treated with gauze (gauze group), argon plasma coagulation (APC group), and chitosan-catechol hemostatic agent (CHI-C group) each. The pigs were sacrificed on Days 1, 5, and 10, and histological examination was performed (n = 5 per day). Rapid hemostasis observed at 2 min after bleeding was 93.3% (14/15) in the CHI-C group, 6.7% (1/15) in the no-treatment group, 13.3% (2/15) in the gauze group, and 86.7% (13/15) in the APC group. No re-bleeding was observed in the CHI-C group during the entire study period. However, a few re-bleeding cases were observed on Day 1 in the no-treatment, gauze, and APC groups and on Day 5 in the gauze and APC groups. On histological analysis, the CHI-C group showed the best tissue healing among the four test groups. Considering the results, chitosan-catechol is an effective hemostatic material with reduced re-bleeding and improved healing.

Oral mucosal drug delivery systems have been developed to expedite the regeneration of oral mucosa, there are still many challenges related to residence time for drugs because the ceaseless changes of saliva, mouth movement, and involuntary swallowing prevent robust adhesion of drugs and/or drug-loaded biomaterials. Thus, it is highly desirable to develop the delivery platforms exhibiting robust, stable adhesion within oral cavities. Herein, we have developed an adhesive polysaccharide oral patch called 'Chitoral' that utilizes chemical principles shown in wet-resistant mussel adhesion. Chitoral plays an important role as an adhesive layer in wet environments. We unexpectedly found that Chitoral instantly dissolves upon contact with saliva and a labial mucous layer, and then the dissolved Chitoral compounds forms an insoluble adhesion layer with mucins at Chitoral/mucous interface nearly immediate actions. Later, Chitoral gradually converts into adhesive hydrogels by the cooperative actions of covalent crosslinking and physical entanglement. The instant, robust muco-adhesion properties of Chitoral provides long-lasting therapeutic effects of drugs resulting enhanced healing of oral ulcer. Thus, mussel-inspired, mucous-resistant adhesive platforms, Chitoral, can be a platform for oral mucosal drug delivery systems.

Owing to the destruction of ozone layer, the increased exposure to UV on the earth adversely affects not only skin diseases but also wound healing. Although the demand for sunscreens is increasing to protect the human skin from these adverse effects, commercially available sunscreens have some limitations in safety. In this study, silk fibroin (SF) composite with biocompatibility and blood coagulation activity was prepared for a highly safe sunscreen. However, the SF has a disadvantage in that it is difficult to dissolve in water. To improve the solubility of SF, butyl glycidyl ether (BGE) was reacted with the side chain of SF to prepare a freely water-soluble SF (mSF) derivative, and the phase behavior according to the mixing ratio of SF derivative and tannic acid (TA) was observed. In addition, ZnO nanoparticles were added to the mSF-TA solution to form a hydrogel through the coordination bonding. The UV blocking, hemostatic, antibacterial and antioxidant effects of the mSF/TA/ZnO composite hydrogel were evaluated, and the excellent skin compatibility of multifunctional hydrogel sunscreen was confirmed through a skin irritation test.

Gene therapy and rebalancing therapy have emerged as promising approaches for treating hemophilia A, but there are limitations, such as temporary efficacy due to individual differences. Genome editing for hemophilia has shown long-term therapeutic potential in preclinical trials. However, a cautious approach is necessary because genome editing is irreversible. Therefore, we attempted to induce low-level human factor 8 (hF8) gene knockin (KI) using 244-cis lipid nanoparticles and low-dose adeno-associated virus to minimize side effects and achieve a therapeutic threshold in hemophilia A mice. We selected the serpin family C member 1, SerpinC1, locus as a target to enable a combined rebalancing strategy with hF8 KI to augment efficacy. This strategy improved blood coagulation activity and reduced hemophilic complications without adverse effects. Furthermore, hemophilic mice with genome editing exhibit enhanced survival for 40 weeks. Here, we demonstrate an effective, safe, and sustainable treatment for hemophilia A. This study provides valuable information to establish safe and long-term genome-editing-mediated treatment strategies for treating hemophilia and other protein-deficient genetic diseases.

Bioconjugation of proteins can substantially expand the opportunities in biopharmaceutical development, however, applications are limited for the gene editing machinery despite its tremendous therapeutic potential. Here, a self-delivered nanomedicine platform based on bioorthogonal CRISPR/Cas9 conjugates, which can be armed with a chemotherapeutic drug for combinatorial therapy is introduced. It is demonstrated that multi-functionalized Cas9 with a drug and polymer can form self-condensed nanocomplexes, and induce significant gene editing upon delivery while avoiding the use of a conventional carrier formulation. It is shown that the nanomedicine platform can be applied for combinatorial therapy by incorporating the anti-cancer drug olaparib and targeting the RAD52 gene, leading to significant anti-tumor effects in BRCA-mutant cancer. The current development provides a versatile nanomedicine platform for combination treatment of human diseases such as cancer.

Cellular transformation is initiated by the activation of oncogenes and a closely associated developmental reprogramming of the epigenetic landscape. Transcription factors, regulators of chromatin states and microRNAs influence cell fates in development and stabilize the phenotypes of normal, differentiated cells and of cancer cells. The miR-302/367 cluster, predominantly expressed in human embryonic stem cells (hESs), can promote the cellular reprogramming of human and mouse cells and contribute to the generation of iPSC. We have used the epigenetic reprogramming potential of the miR-302/367 cluster to "de-program" tumor cells, that is, hift their gene expression pattern towards an alternative program associated with more benign cellular phenotypes. Induction of the miR-302/367 cluster in extensively mutated U87MG glioblastoma cells drastically suppressed the expression of transformation related proteins, for example, the reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC, and the transcription factors POU3F2, SALL2 and OLIG2, required for the maintenance of glioblastoma stem-like tumor propagating cells. It also diminished PI3K/AKT and STAT3 signaling, impeded colony formation in soft agar and cell migration and suppressed pro-inflammatory cytokine secretion. At the same time, the miR-302/367 cluster restored the expression of neuronal markers of differentiation. Most notably, miR-302/367 cluster expressing cells lose their ability to form tumors and to establish liver metastasis in nude mice. The induction of the miR-302/367 cluster in U87MG glioblastoma cells suppresses the expression of multiple transformation related genes, abolishes the tumor and metastasis formation potential of these cells and can potentially become a new approach for cancer therapy.

The inevitable bleeding and infections caused by disasters and accidents are the main causes of death owing to extrinsic trauma. Hemostatic agents are often used to quickly suppress bleeding and infection, and they can solve this problem in a short time. Silk fibroin (SF) has poor processibility in water, owing to incomplete solubility therein. In this study, aiming to overcome this disadvantage, a modified silk fibroin (SF-BGE), easily soluble in water, was prepared by introducing butyl glycidyl ether (BGE) into its side chain. Subsequently, a small amount of tannic acid (TA) was introduced to prepare an SF-BGE /TA solution, and ZnO nanoparticles (NPs) were added to the solution to form the coordination bonds between the ZnO and TA, leading to an SF-based nanocomposite hydrogel. A structural characterization of the SF-BGE, SF-BGE/TA, SF-BGE/TA/ZnO, and the coordination bonds between ZnO/TA was observed by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and the phase change was observed by rheological measurements. The pore formation of the SF-BGE/TA/ZnO hydrogel and dispersibility of ZnO were verified through energy-dispersive X-ray spectroscopy (EDS) and scanning electron microscopy (SEM). The cytocompatible and hemostatic performances of the SF-BGE/TA/ZnO NPs composite hydrogels were evaluated, and the hydrogels showed superior hemostatic and cytocompatible activities. Therefore, the SF-based nanocomposite hydrogel is considered as a promising material for hemostasis.

Biological functions depend on biomolecular assembly processes. Assemblies of lipid bilayers, actins, microtubules, or chromosomes are indispensable for cellular functions. These hierarchical assembly processes are reasonably predictable by understanding chemical structures of the defined building blocks and their interactions. However, biopigment assembly is rather fuzzy and unpredictable because a series of covalently coupled intermediates from catecholamine oxidation pathways progressively form a higher-level hierarchy. This study reports a different yet unexplored type of assembly process named "cation-π progressive assembly." We demonstrated for the first time that the cation-π is the primary mechanism for intermolecular assembly in dopamine-melanin biopigment. We also found that the self-assembled products physically grow and chemically gain new functions "progressively" over time in which cation-π plays important roles. The progressive assembly explains how biological systems produce wide spectra of pigment colors and broad wavelength absorption through energy-efficient processes. Furthermore, we also demonstrate surface-independent wettability control using cation-π progressive assembly.

One of the most commonly used drugs in chemotherapy, 5-fluorouracil (5-FU) has been shown to be effective in only 10-15% of patients with colon cancer. Thus, studies of the mechanisms affecting 5-FU sensitivity in these patients are necessary. The tumor suppressor protein p53 is a transcription factor that serves important roles in cell apoptosis by regulating the cell cycle. It has also been characterized as a key factor influencing drug sensitivity. Furthermore, accessible chromatin is a hallmark of active DNA regulatory elements and functions as a crucial epigenetic factor regulating cancer mechanisms. The present study assessed the genetic regulatory landscape in colon cancer by performing RNA sequencing and Assay for Transposase-Accessible Chromatin sequencing, and investigated the effects of 5-FU on chromatin accessibility and gene expression. Notably, while treatment with 5-FU mediated global increases in chromatin accessibility, chromatin organization in several genomic regions differed depending on the expression status of p53. Since the occupancy of p53 does not overlap with accessible chromatin regions, the 5-FU-mediated changes in chromatin accessibility were not regulated by direct binding of p53. In the p53-expressing condition, the 5-FU-mediated accessible chromatin region was primarily associated with genes encoding cell death pathways. Additionally, 5-FU was revealed to induce open chromatin conformation at regions containing binding motifs for AP-1 family transcription factors, which may drive expression of apoptosis pathway genes. In conclusion, expression of p53 may confer 5-FU sensitivity by regulating chromatin accessibility of distinct genes associated with cell apoptosis in a transcription-independent manner.

Additive manufacturing, or 3D printing, has revolutionized the way we create objects. However, its layer-by-layer process may lead to an increased incidence of local defects compared to traditional casting-based methods. Factors such as light intensity, depth of light penetration, component inhomogeneity, and fluctuations in nozzle temperature all contribute to defect formations. These defective regions can become sources of toxic component leakage, but pinpointing their locations in 3D printed materials remains a challenge. Traditional toxicological assessments rely on the extraction and subsequent exposure of living organisms to these harmful agents, thus only offering a passive detection approach. Therefore, the development of an active system to both identify and locate sources of toxicity is essential in the realm of 3D printing technologies. Herein, we introduce the use of the nematode model organism, Caenorhabditis elegans (C. elegans), for toxicity evaluation. C. elegans exhibits distinctive 'sensing' and 'locomotion' capabilities that enable it to actively navigate toward safe zones while steering clear of hazardous areas. This active behavior sets C. elegans apart from other aquatic and animal models, making it an exceptional choice for immediate and precise identification and localization of toxicity sources in 3D printed materials.

The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.