Integrative Single-Cell RNA-Seq and ATAC-Seq Analysis of Mouse Corneal Epithelial Cells.

Purpose: Corneal epithelial homeostasis is maintained by coordinated gene expression across distinct cell populations, but the gene regulatory programs underlying this cellular diversity remain to be characterized. Here we applied single-cell multi-omics analysis to delineate the gene regulatory profile of mouse corneal epithelial cells under normal homeostasis. Methods: Single cells isolated from the cornea epithelium (with marginal conjunctiva) of adult mice were subjected to scRNA-seq and scATAC-seq using the 10×Genomics platform. Cell types were clustered by the graph-based visualization method uniform manifold approximation and projection and unbiased computational informatics analysis. The scRNA-seq and scATAC-seq datasets were integrated following the integration pipeline described in ArchR and Seurat. Results: We characterized diverse corneal epithelial cell types based on gene expression signatures and chromatin accessibility. We found that cell type-specific accessibility regions were mainly located at distal regions, suggesting essential roles of distal regulatory elements in determining corneal epithelial cell diversity. Trajectory analyses revealed a continuum of cell state transition and higher coordination between transcription factor (TF) motif accessibility and gene expression during corneal epithelial cell differentiation. By integrating transcriptomic and chromatin accessibility analysis, we identified cell type-specific and shared gene regulation programs. We also uncovered critical TFs driving corneal epithelial cell differentiation, such as nuclear factor I (NFI) family members, Rarg, Elf3. We found that nuclear factor-κB (NF-κB) family members were positive TFs in limbal cells and some superficial cells, but they were involved in regulating distinct biological processes. Conclusions: Our study presents a comprehensive gene regulatory landscape of mouse cornea epithelial cells, and provides valuable foundations for future investigation of corneal epithelial homeostasis in the context of cornea pathologies and regenerative medicine.

Assessing the effects of aging on the liver endothelial cell landscape using single-cell RNA sequencing.

Endothelial cell (EC) function declines with age and contributes to the development of many vascular-related disease processes. Currently, the effects of aging on the molecular regulatory mechanisms of liver ECs have not been fully elucidated. Here, we employed single-cell RNA sequencing to map the transcriptome of ECs and analyzed their relationship with aging. We identified 8 different EC subtypes, interestingly, 2 of which were specially expressed in aged mice ECs namely aged capillary ECs (Aged ECs) and pro-inflammation capillary ECs (Proinfla.ECs). Double immunostaining for an EC marker (Cd31) and a marker of these specialized EC phenotypes confirmed the single-cell RNA sequencing data. Gene ontology analysis revealed that Aged ECs and Proinfla.ECs were associated with inflammatory response. Then we found that liver proliferating capillary ECs (Prolife.ECs) were most affected by senescence. Single-cell transcript analysis suggests that Prolife.ECs and angiogenic capillary ECs may form a poor microenvironment that promotes angiogenesis and tumorigenesis. Pseudo-temporal trajectories revealed that Prolife.ECs have different differentiation pathways in young and aged mice. In aged mice, Prolife.ECs could specifically differentiate into an unstable state, which was mainly composed of angiogenic capillary ECs. Intercellular communication revealed inflammatory activation in old group. Overall, this work compared the single-cell RNA profiles of liver ECs in young and aged mice. These findings provide a new insight into liver aging and its molecular mechanisms, and further exploration of Aged ECs and Proinfla.ECs may help to elucidate the molecular mechanisms associated with senescence.

The Function of the Mutant p53-R175H in Cancer.

Wild-type p53 is known as "the guardian of the genome" because of its function of inducing DNA repair, cell-cycle arrest, and apoptosis, preventing the accumulation of gene mutations. TP53 is highly mutated in cancer cells and most TP53 hotspot mutations are missense mutations. Mutant p53 proteins, encoded by these hotspot mutations, lose canonical wild-type p53 functions and gain functions that promote cancer development, including promoting cancer cell proliferation, migration, invasion, initiation, metabolic reprogramming, angiogenesis, and conferring drug resistance to cancer cells. Among these hotspot mutations, p53-R175H has the highest occurrence. Although losing the transactivating function of the wild-type p53 and prone to aggregation, p53-R175H gains oncogenic functions by interacting with many proteins. In this review, we summarize the gain of functions of p53-R175H in different cancer types, the interacting proteins of p53-R175H, and the downstream signaling pathways affected by p53-R175H to depict a comprehensive role of p53-R175H in cancer development. We also summarize treatments that target p53-R175H, including reactivating p53-R175H with small molecules that can bind to p53-R175H and alter it into a wild-type-like structure, promoting the degradation of p53-R175H by targeting heat-shock proteins that maintain the stability of p53-R175H, and developing immunotherapies that target the p53-R175H-HLA complex presented by tumor cells.

Preliminary study on mechanical characteristics of maxillofacial soft and hard tissues for virtual surgery.

PURPOSE: Virtual surgery system can provide us a realistic and immersive training environment, in which haptic force-feedback gives operators 'touching feeling.' Appropriate deformation models of soft and hard tissues are required for the achievement of real-time haptic feedback. To improve accuracy of modeling and haptic feedback simulation for maxillofacial virtual surgery, mechanical characteristics of soft and hard tissues should be explored. METHODS: Craniofacial soft tissues from one male and female cadavers were divided into two layers: skin and muscle. Maxillofacial tissues were divided into frontal, chin, temporalis, masseter regions. Insertion and cutting process were conducted using VMX42 5-axis linkage system and recorded by piezoelectric dynamometer. Maximum stiffness values were analyzed, and insertion curves before puncture were fitted using a polynomial model. Elasticity modulus and hardness of maxillofacial hard tissues were measured and analyzed using Berkovich nanoindentation. RESULTS: Tissues in different maxillofacial regions, as well as from different layers (skin and muscle), displayed various mechanical performance. Maximum stiffness values and cutting force of soft tissues in male and female had significant difference. The third-order polynomial was demonstrated to fit the insertion curves well before puncture. Furthermore, elasticity modulus and hardness of enamel were significantly greater than that of zygoma, maxilla and mandible. CONCLUSION: Mechanical properties of hard tissues are relatively stable, which can be applied in virtual surgery system for physical model construction. Insertion model and cutting force for soft tissues are meaningful and applicable and can be utilized to promote the accuracy of response for haptic feedback sensations.

Development process of traumatic heterotopic ossification of the temporomandibular joint in mice.

PURPOSE: The exact development process underlying traumatic heterotopic ossification of the temporomandibular joint (THO-TMJ) is largely unclear. In this study, we try to explore the histological development process of THO-TMJ. MATERIALS AND METHODS: Condylar cartilage of one-month-old male mice was partially removed from the left joint with small scissors to induce THO-TMJ. The phenotypes were observed using gross observation, microcomputed tomography (micro-CT) scans and histological examination from one month to six months after surgery. RESULTS: The micro-CT examination results showed that the injured condyle integrated with ectopic bone tissue to form an osteophyte and that the volume and density of the osteophyte grew exponentially with time. Hematoxylin and eosin (H&E), safranin O and fast green staining of the THO-TMJ specimens revealed that the ectopic bone tissue was mainly nonmineralized fibrous tissue 1 month after surgery. This tissue gradually transformed into cartilage 3 months after surgery. Finally, the tissues transformed into mature bone tissue 6 months after surgery. Immunofluorescence staining showed VEGF-α expression in the heterotopic tissue 1 month after surgery, and the expression of Sox9 in the heterotopic tissue was obvious 3 months after surgery. Furthermore, OCN expression was evident in most of the heterotopic tissue 6 months after surgery. The results also showed clear hypoxia-inducible factor 1-alpha (Hif-1α) expression in the injured chondrocytes of the condyle, especially in the articular proliferative zone and fibrocartilaginous zone. CONCLUSIONS: The THO-TMJ imaging characteristics indicated an exponential change with time. Histologically, the development process of THO-TMJ is an endochondral ossification process and includes three stages, fibroproliferative, chondrogenic and osteogenic stage. In addition, Hif-1α, which was expressed in some of the injured chondrocytes, may play an essential role in the initial THO-TMJ.

Effects of a single condylar neck fracture without condylar cartilage injury on traumatic heterotopic ossification around the temporomandibular joint in mice.

OBJECTIVES: In this study, we tried to explore the effects of a single condylar neck fracture without condylar cartilage injury during the pathogenesis process of traumatic heterotopic ossification around the temporomandibular joint (THO-TMJ). STUDY DESIGN: One-month-old C57 BL/6 J male mice were divided into 2 groups. In group 1, condylar cartilage was partially removed in the right joint to induce THO. In group 2, a single fracture on the condylar neck was created using small scissors. The condylar head was repositioned to its original place if any displacement occurred. The phenotypes were observed using gross observation, micro-computed tomography, and histologic examination. RESULTS: The results showed obvious hyperplasia in the right condyle in group 1, with ectopic bones and cartilage in the periarticular region. In group 2, the surface of condyle was smooth, but the size of the right condylar head became smaller. CONCLUSIONS: Taking these findings together, we concluded that it is condylar cartilage injury, and not a single condylar neck fracture without condylar cartilage injury, that contributes to the development of THO-TMJ.

Postnatal Development of the Spheno-occipital Synchondrosis: A Histological Analysis.

The spheno-occipital synchondrosis (SOS) in cranial base is an important growth center for the craniofacial skeleton, and also is a guide rail for development of the maxilla, midface, and mandible. Previous studies showed that SOS may be a treatment target for youngsters with midfacial hypoplasia and small cranial vault secondary to craniosynostosis. However, most of studies about the SOS are based on imaging data. In this study, we try to explore the characteristics of postnatal development of the mouse SOS based on histological analysis. Our findings showed that the width of the SOS in mice were gradually decreased from newborn mice to adult mice, and the SOS cartilage was gradually became small, then almost completely ossificated in adult mice. The resting and proliferative layers in SOS cartilage were gradually decreased, and almost only hypertrophic chondrocytes while no resting and proliferative layer chondrocytes in adult mice. The proliferative ability of SOS chondrocytes also gradually decreased. These findings will be of benefit for the further clinical treatment for patients with midfacial hypoplasia or small cranial vault secondary to craniosynostosis. Further evidence-based research about the clinical implication is necessary in future.

A fiber-progressive-engagement model to evaluate the composition, microstructure, and nonlinear pseudoelastic behavior of porcine arteries and decellularized derivatives.

The theoretical fiber-progressive-engagement model was proposed to describe the pseudoelastic behavior of an artery pre- and post-decellularization treatments. Native porcine arteries were harvested and decellularized with 0.05% trypsin for 12 h. The uniaxial tensile test data were fitted to the fiber-progressive-engagement model proposed herein. The effects of decellularization on the morphology, structural characteristics, and composition of vessel walls were studied. The experimental stress-strain curve was fitted to the model in the longitudinal and circumferential direction, which demonstrated the adequacy of the proposed model (R2>0.99). The initial and turning strains were similar in the longitudinal and circumferential directions in the aorta, suggesting the occurrence of collagen conjugation in both directions. Discrepancies in the initial and turning strain and initial and stiff modulus in both directions in the coronary artery revealed the anisotropic features of this vessel. Decellularization induced a decrease in the initial and turning strains, a slight change in the initial modulus, and a substantial decrease in the stiffness modulus. The decrease in the initial and turning strain can be attributed to the loss of waviness of collagen bundles because of the considerable decrease in elastin and glycosaminoglycan contents. This simple non-linear model can be used to determine the fiber modulus and waviness degree of vascular tissue. Based on these results, this mechanical test can be used as a screening tool for the selection of an optimized decellularization protocol for arterial tissues. STATEMENT OF SIGNIFICANCE: Decellularized vascular graft has potential in clinical application, such as coronary artery bypass surgery, peripheral artery bypass surgery or microsurgery. An ideal decellularization protocol requires balance in cell removal efficiency and extracellular matrix preserving. Both biochemical and biomechanical properties are crucial to the success of scaffold in cell seeding and animal study. A comprehensive understanding of the composition, microstructure, and mechanical behavior of the arterial wall is the key to the development of decellularized vascular grafts. For this purpose, we proposed this "Fiber-Progressive-Engagement" model to evaluate the microstructure, composition and mechanical properties of porcine coronary artery. The model provides a new perspective regarding the non-linear behavior of arterial tissue and its decellularized derivatives. It can be widely applied to different types of tissues, as demonstrated in the aorta and coronary artery. This model has several advantages; it provides an improved fit of non-linear curves (R2>0.99), can be used to elucidate the pseudoelastic properties of porcine vascular tissues using the concept of fiber engagement, and can estimate an elastic modulus with greater accuracy (compared to the graphical estimation or calculation by simple linear fittings), as well as to plot typical stress-strain curves.

Type AII lantibiotic bovicin HJ50 with a rare disulfide bond: structure, structure-activity relationships and mode of action.

Lantibiotics are ribosomally synthesized antimicrobial peptides containing unusual amino acids. As promising alternatives to conventional antibiotics, they have a high potential for alleviating the problem of emergent antibiotic resistance, with possible applications in many industries that have antibacterial demand. Bovicin HJ50 is a type AII lantibiotic, the largest group of lantibiotics, comprising a linear N-terminal region and a globular C-terminal region. Interestingly, bovicin H50 has a disulfide bond that is rare in this group. Owing to limited information about the spatial structures of type AII lantibiotics, the functional regions of this type and the role of the disulfide bond are still unknown. In the present study, we resolved the solution structure of bovicin HJ50 using NMR spectroscopy. This is the first spatial structure of a type AII lantibiotic. Bovicin HJ50 exhibited high flexibility in aqueous solution, whereas varied rigidities were observed in the different rings with the conserved ring A being the most rigid. The charged residues Lys¹¹, Asp¹² and Lys³°, as well as the essential disulfide bond were critical for antimicrobial activity. Importantly, bovicin HJ50 showed not only peptidoglycan precursor lipid II-binding ability, but also pore-forming activity, which is significantly different from other bacteriostatic type AII lantibiotics, suggesting a novel antimicrobial mechanism.

Microbial production of antioxidant food ingredients via metabolic engineering.

Antioxidants are biological molecules with the ability to protect vital metabolites from harmful oxidation. Due to this fascinating role, their beneficial effects on human health are of paramount importance. Traditional approaches using solvent-based extraction from food/non-food sources and chemical synthesis are often expensive, exhaustive, and detrimental to the environment. With the advent of metabolic engineering tools, the successful reconstitution of heterologous pathways in Escherichia coli and other microorganisms provides a more exciting and amenable alternative to meet the increasing demand of natural antioxidants. In this review, we elucidate the recent progress in metabolic engineering efforts for the microbial production of antioxidant food ingredients - polyphenols, carotenoids, and antioxidant vitamins.

Combinatorial biosynthesis of plant-specific coumarins in bacteria.

Coumarins are plant secondary metabolites that have demonstrated a variety of important therapeutic properties, such as antibacterial, anti-inflammatory, and anti-coagulant effects, as well as anti-cancer and anti-AIDS activities. However, knowledge regarding their biosynthesis is relatively limited even for the simplest coumarin molecule, which serves as the gateway molecule to many pharmaceutically important coumarin derivatives. Here we reported the design and validation of artificial pathways leading to the biosynthesis of plant-specific simple coumarins in bacteria. First, Escherichia coli strains were engineered to convert inexpensive phenylpropanoid acid precursors, 4-coumarate and ferulate to simple coumarins, umbelliferone (4.3 mg/L) and scopoletin (27.8 mg/L), respectively. Furthermore, we assembled the complete artificial pathways in E. coli and achieved de novo biosynthesis of umbelliferone and scopoletin without addition of precursors. This study lays the foundation for microbial production of more diverse coumarin compounds.

The synthetic flavonoid WYC02-9 inhibits colorectal cancer cell growth through ROS-mediated activation of MAPK14 pathway.

AIM: Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. In this study, we explored the anti-cancer activity of WYC02-9, a synthetic protoapigenone, on human HCT116 CRC cells. MAIN METHODS: The anti-cancer activity of WYC02-9 and its underlying mechanisms were analyzed using XTT cell proliferation assays, colony formation assays, FACS analysis, annexin V staining, immunoblotting analysis, reactive oxygen species (ROS) generation assays, soft agar assays, a nude mice xenograft study and immunohistochemistry assays. KEY FINDINGS: Data showed that WYC02-9 suppressed CRC cell growth by arresting cells at G2/M and inducing cell death via apoptotic pathways. Further analysis demonstrated that WYC02-9-induced apoptosis was mediated by the activation of a ROS-mediated MAPK14 pathway. An in vivo xenograft study revealed that WYC02-9 enhanced MAP2K3/6 and MAPK14 phosphorylation, induced apoptosis, and suppressed CRC tumor growth. SIGNIFICANCE: WYC02-9 exerts its anti-tumor effect via ROS/MAPK14-induced apoptosis and has the potential to be developed as a chemotherapeutic agent for CRC.