Mx1 -labeled pulp progenitor cells are main contributors to postnatal odontoblasts and pulp cells in murine molars.

Regeneration of dentin and odontoblasts from dental pulp stem cells (DPSCs) is essential for permanent tooth maintenance. However, the in vivo identity and role of endogenous DPSCs in reparative dentinogenesis are elusive. Here, using pulp single-cell analysis before and after molar eruption, we revealed that endogenous DPSCs are enriched in Cxcl12- GFP + coronal papilla-like cells with Mx1- Cre labeling. These Mx1 + Cxcl12- GFP + cells are long-term repopulating cells that contribute to the majority of pulp cells and new odontoblasts after eruption. Upon molar injury, Mx1 + DPSCs localize into the injury site and differentiate into new odontoblasts, forming scleraxis -GFP + and osteocalcin -GFP + dentinal tubules and reparative dentin. Single-cell and FACS analysis showed that Mx1 + Cxcl12- GFP + DPSCs are the most primitive cells with stem cell marker expression and odontoblast differentiation. Taken together, our findings demonstrate that Mx1 labels postnatal DSPCs, which are the main source of pulp cells and new odontoblasts with reparative dentinogenesis in vivo .

Dental Pulp Stem Cells and Current in vivo Approaches to Study Dental Pulp Stem Cells in Pulp Injury and Regeneration.

Dental pulp stem cells (DPSCs) have garnered significant interest in dental research for their unique characteristics and potential in tooth development and regeneration. While there were many studies to define their stem cell-like characteristics and osteogenic differentiation functions that are considered ideal candidates for regenerating damaged dental pulp tissue, how endogenous DPSCs respond to dental pulp injury and supply new dentin-forming cells has not been extensively investigated in vivo. Here, we review the recent progress in identity, function, and regulation of endogenous DPSCs and their clinical potential for pulp injury and regeneration. In addition, we discuss current advances in new mouse models, imaging techniques, and its practical uses and limitations in the analysis of DPSCs in pulp injury and regeneration in vivo.

Differential regulation of skeletal stem/progenitor cells in distinct skeletal compartments.

Skeletal stem/progenitor cells (SSPCs), characterized by self-renewal and multipotency, are essential for skeletal development, bone remodeling, and bone repair. These cells have traditionally been known to reside within the bone marrow, but recent studies have identified the presence of distinct SSPC populations in other skeletal compartments such as the growth plate, periosteum, and calvarial sutures. Differences in the cellular and matrix environment of distinct SSPC populations are believed to regulate their stemness and to direct their roles at different stages of development, homeostasis, and regeneration; differences in embryonic origin and adjacent tissue structures also affect SSPC regulation. As these SSPC niches are dynamic and highly specialized, changes under stress conditions and with aging can alter the cellular composition and molecular mechanisms in place, contributing to the dysregulation of local SSPCs and their activity in bone regeneration. Therefore, a better understanding of the different regulatory mechanisms for the distinct SSPCs in each skeletal compartment, and in different conditions, could provide answers to the existing knowledge gap and the impetus for realizing their potential in this biological and medical space. Here, we summarize the current scientific advances made in the study of the differential regulation pathways for distinct SSPCs in different bone compartments. We also discuss the physical, biological, and molecular factors that affect each skeletal compartment niche. Lastly, we look into how aging influences the regenerative capacity of SSPCs. Understanding these regulatory differences can open new avenues for the discovery of novel treatment approaches for calvarial or long bone repair.

Advances and challenges in intravital imaging of craniofacial and dental progenitor cells.

Craniofacial and appendicular bone homeostasis is dynamically regulated by a balance between bone formation and resorption by osteoblasts and osteoclasts, respectively. Despite the developments in multiple imaging techniques in bone biology, there are still technical challenges and limitations in the investigation of spatial/anatomical location of rare stem/progenitor cells and their molecular regulation in tooth and craniofacial bones of living animals. Recent advances in live animal imaging techniques for the craniofacial and dental apparatus can provide new insights in real time into bone stem/progenitor cell dynamics and function in vivo. Here, we review the current inventions and applications of the noninvasive intravital imaging technique and its practical uses and limitations in the analysis of stem/progenitor cells in craniofacial and dental apparatus in vivo. Furthermore, we also explore the potential applications of intravital microscopy in the dental field.

Constitutive loss of DNMT3A causes morbid obesity through misregulation of adipogenesis.

DNA Methyltransferase 3 A (DNMT3A) is an important facilitator of differentiation of both embryonic and hematopoietic stem cells. Heterozygous germline mutations in DNMT3A lead to Tatton-Brown-Rahman Syndrome (TBRS), characterized by obesity and excessive height. While DNMT3A is known to impact feeding behavior via the hypothalamus, here we investigated a role in adipocyte progenitors utilizing heterozygous knockout mice that recapitulate cardinal TBRS phenotypes. These mice become morbidly obese due to adipocyte enlargement and tissue expansion. Adipose tissue in these mice exhibited defects in preadipocyte maturation and precocious activation of inflammatory gene networks, including interleukin-6 signaling. Adipocyte progenitor cell lines lacking DNMT3A exhibited aberrant differentiation. Furthermore, mice in which Dnmt3a was specifically ablated in adipocyte progenitors showed enlarged fat depots and increased progenitor numbers, partly recapitulating the TBRS obesity phenotypes. Loss of DNMT3A led to constitutive DNA hypomethylation, such that the DNA methylation landscape of young adipocyte progenitors resemble that of older wild-type mice. Together, our results demonstrate that DNMT3A coordinates both the central and local control of energy storage required to maintain normal weight and prevent inflammatory obesity.

Localized chondro-ossification underlies joint dysfunction and motor deficits in the Fkbp10 mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder that features wide-ranging defects in both skeletal and nonskeletal tissues. Previously, we and others reported that loss-of-function mutations in FK506 Binding Protein 10 (FKBP10) lead to skeletal deformities in conjunction with joint contractures. However, the pathogenic mechanisms underlying joint dysfunction in OI are poorly understood. In this study, we have generated a mouse model in which Fkbp10 is conditionally deleted in tendons and ligaments. Fkbp10 removal substantially reduced telopeptide lysyl hydroxylation of type I procollagen and collagen cross-linking in tendons. These biochemical alterations resulting from Fkbp10 ablation were associated with a site-specific induction of fibrosis, inflammation, and ectopic chondrogenesis followed by joint deformities in postnatal mice. We found that the ectopic chondrogenesis coincided with enhanced Gli1 expression, indicating dysregulated Hedgehog (Hh) signaling. Importantly, genetic inhibition of the Hh pathway attenuated ectopic chondrogenesis and joint deformities in Fkbp10 mutants. Furthermore, Hh inhibition restored alterations in gait parameters caused by Fkbp10 loss. Taken together, we identified a previously unappreciated role of Fkbp10 in tendons and ligaments and pathogenic mechanisms driving OI joint dysfunction.

Tendon and motor phenotypes in the Crtap-/- mouse model of recessive osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is characterized by short stature, skeletal deformities, low bone mass, and motor deficits. A subset of OI patients also present with joint hypermobility; however, the role of tendon dysfunction in OI pathogenesis is largely unknown. Using the Crtap-/- mouse model of severe, recessive OI, we found that mutant Achilles and patellar tendons were thinner and weaker with increased collagen cross-links and reduced collagen fibril size at 1- and 4-months compared to wildtype. Patellar tendons from Crtap-/- mice also had altered numbers of CD146+CD200+ and CD146-CD200+ progenitor-like cells at skeletal maturity. RNA-seq analysis of Achilles and patellar tendons from 1-month Crtap-/- mice revealed dysregulation in matrix and tendon marker gene expression concomitant with predicted alterations in TGF-ß, inflammatory, and metabolic signaling. At 4-months, Crtap-/- mice showed increased αSMA, MMP2, and phospho-NFκB staining in the patellar tendon consistent with excess matrix remodeling and tissue inflammation. Finally, a series of behavioral tests showed severe motor impairments and reduced grip strength in 4-month Crtap-/- mice - a phenotype that correlates with the tendon pathology.

A Framework (SOCRATex) for Hierarchical Annotation of Unstructured Electronic Health Records and Integration Into a Standardized Medical Database: Development and Usability Study.

BACKGROUND: Although electronic health records (EHRs) have been widely used in secondary assessments, clinical documents are relatively less utilized owing to the lack of standardized clinical text frameworks across different institutions. OBJECTIVE: This study aimed to develop a framework for processing unstructured clinical documents of EHRs and integration with standardized structured data. METHODS: We developed a framework known as Staged Optimization of Curation, Regularization, and Annotation of clinical text (SOCRATex). SOCRATex has the following four aspects: (1) extracting clinical notes for the target population and preprocessing the data, (2) defining the annotation schema with a hierarchical structure, (3) performing document-level hierarchical annotation using the annotation schema, and (4) indexing annotations for a search engine system. To test the usability of the proposed framework, proof-of-concept studies were performed on EHRs. We defined three distinctive patient groups and extracted their clinical documents (ie, pathology reports, radiology reports, and admission notes). The documents were annotated and integrated into the Observational Medical Outcomes Partnership (OMOP)-common data model (CDM) database. The annotations were used for creating Cox proportional hazard models with different settings of clinical analyses to measure (1) all-cause mortality, (2) thyroid cancer recurrence, and (3) 30-day hospital readmission. RESULTS: Overall, 1055 clinical documents of 953 patients were extracted and annotated using the defined annotation schemas. The generated annotations were indexed into an unstructured textual data repository. Using the annotations of pathology reports, we identified that node metastasis and lymphovascular tumor invasion were associated with all-cause mortality among colon and rectum cancer patients (both P=.02). The other analyses involving measuring thyroid cancer recurrence using radiology reports and 30-day hospital readmission using admission notes in depressive disorder patients also showed results consistent with previous findings. CONCLUSIONS: We propose a framework for hierarchical annotation of textual data and integration into a standardized OMOP-CDM medical database. The proof-of-concept studies demonstrated that our framework can effectively process and integrate diverse clinical documents with standardized structured data for clinical research.

Enhanced Electrical Properties of AZO/IZO Multilayered Thin Film with Post Laser Annealing Process.

In this research, alternative deposition process of ZnO-based thin films have been studied for transparent conducting oxide (TCO) application. To improve the electrical and optical properties of transparent oxide thin films, alternatively stacked Al-doped ZnO and In-doped ZnO thin films were investigated. Multilayer structure of alternative 6 layers of thin films were prepared in this research. Especially, Aluminum and Indium were chosen as dopant materials. Thin films of Al-doped ZnO (AZO) and In-doped ZnO (IZO) were alternatively deposited by spin coating with sol-gel method. After deposition of multilayered thin films, multi steps of furnace (F), rapid thermal annealing (R) and CO2 laser annealing (L) processes were carried out and investigated thin film properties by dependence of post-annealing sequence and thin film structures. The electrical and optical properties of thin films were investigated by 4-point probe and UV-vis spectroscopy and its shows the greatest sheet resistance value of 0.59 kΩ/sq. from AZO/IZO multilayered structure and upper 85% of transmittance. The structural property and surface morphology were measured by X-Ray Diffraction (XRD) and field emission scanning electron microscopy (FE-SEM). The Al- and In-doped ZnO thin film shows the highest intensity value at (002) peak of AZO/IZO multilayer structure which was performed FRL process.

Interferon Gamma Mediates Hematopoietic Stem Cell Activation and Niche Relocalization through BST2.

During chronic infection, the inflammatory cytokine interferon gamma (IFNγ) damages hematopoietic stem cells (HSCs) by disrupting quiescence and promoting excessive terminal differentiation. However, the mechanism by which IFNγ hinders HSC quiescence remains undefined. Using intravital 3-dimensional microscopy, we find that IFNγ disrupts the normally close interaction between HSCs and CXCL12-abundant reticular (CAR) cells in the HSC niche. IFNγ stimulation increases expression of the cell surface protein BST2, which we find is required for IFNγ-dependent HSC relocalization and activation. IFNγ stimulation of HSCs increases their E-selectin binding by BST2 and homing to the bone marrow, which depends on E-selectin binding. Upon chronic infection, HSCs from mice lacking BST2 are more quiescent and more resistant to depletion than HSCs from wild-type mice. Overall, this study defines a critical mechanism by which IFNγ promotes niche relocalization and activation in response to inflammatory stimulation and identifies BST2 as a key regulator of HSC quiescence. VIDEO ABSTRACT.

Application of Epidemiological Geographic Information System: An Open-Source Spatial Analysis Tool Based on the OMOP Common Data Model.

BACKGROUND: Spatial epidemiology is used to evaluate geographical variations and disparities in health outcomes; however, constructing geographic statistical models requires a labor-intensive process that limits the overall utility. We developed an open-source software for spatial epidemiological analysis and demonstrated its applicability and quality. METHODS: Based on standardized geocode and observational health data, the Application of Epidemiological Geographic Information System (AEGIS) provides two spatial analysis methods: disease mapping and detecting clustered medical conditions and outcomes. The AEGIS assesses the geographical distribution of incidences and health outcomes in Korea and the United States, specifically incidence of cancers and their mortality rates, endemic malarial areas, and heart diseases (only the United States). RESULTS: The AEGIS-generated spatial distribution of incident cancer in Korea was consistent with previous reports. The incidence of liver cancer in women with the highest Moran's I (0.44; p < 0.001) was 17.4 (10.3-26.9). The malarial endemic cluster was identified in Paju-si, Korea (p < 0.001). When the AEGIS was applied to the database of the United States, a heart disease cluster was appropriately identified (p < 0.001). CONCLUSIONS: As an open-source, cross-country, spatial analytics solution, AEGIS may globally assess the differences in geographical distribution of health outcomes through the use of standardized geocode and observational health databases.

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.  Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

Protein quality control through endoplasmic reticulum-associated degradation maintains haematopoietic stem cell identity and niche interactions.

Stem cells need to be protected from genotoxic and proteotoxic stress to maintain a healthy pool throughout life1-3. Little is known about the proteostasis mechanism that safeguards stem cells. Here we report endoplasmic reticulum-associated degradation (ERAD) as a protein quality checkpoint that controls the haematopoietic stem cell (HSC)-niche interaction and determines the fate of HSCs. The SEL1L-HRD1 complex, the most conserved branch of ERAD4, is highly expressed in HSCs. Deletion of Sel1l led to niche displacement of HSCs and a complete loss of HSC identity, and allowed highly efficient donor-HSC engraftment without irradiation. Mechanistic studies identified MPL, the master regulator of HSC identity5, as a bona fide ERAD substrate that became aggregated in the endoplasmic reticulum following ERAD deficiency. Restoration of MPL signalling with an agonist partially rescued the number and reconstitution capacity of Sel1l-deficient HSCs. Our study defines ERAD as an essential proteostasis mechanism to safeguard a healthy stem cell pool by regulating the stem cell-niche interaction.

Theoretical Design of Lithium Chloride Superionic Conductors for All-Solid-State High-Voltage Lithium-Ion Batteries.

The development of solid electrolytes (SEs) is a promising pathway to improve the energy density and safety of conventional Li-ion batteries. Several lithium chloride SEs, Li3MCl6 (M = Y, Er, In, and Sc), have gained popularity due to their high ionic conductivity, wide electrochemical window, and good chemical stability. This study systematically investigated 17 Li3MCl6 SEs to identify novel and promising lithium chloride SEs. Calculation results revealed that 12 Li3MCl6 (M = Bi, Dy, Er, Ho, In, Lu, Sc, Sm, Tb, Tl, Tm, and Y) were stable phase with a wide electrochemical stability window and excellent chemical stability against cathode materials and moisture. Li-ion transport properties were examined using bond valence site energy (BVSE) and ab initio molecular dynamics (AIMD) calculation. Li3MCl6 showed the lower migration energy barrier in monoclinic structures, while orthorhombic and trigonal structures exhibited higher energy barriers due to the sluggish diffusion along the two-dimensional path based on the BVSE model. AIMD results confirmed the slower ion migration along the 2D path, exhibiting lower ionic diffusivity and higher activation energy in orthorhombic and trigonal structures. For the further increase of ionic conductivity in monoclinic structures, Li-ion vacancy was formed by the substitution of M3+ with Zr4+. Zr-substituted phase (Li2.5M0.5Zr0.5Cl6, M = In, Sc) exhibited up to a fourfold increase in ionic conductivity. This finding suggested that the optimization of Li vacancy in the Li3MCl6 SEs could lead to superionic Li3MCl6 SEs.

GABRQ expression is a potential prognostic marker for patients with clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer. Novel biomarkers of ccRCC may provide crucial information on tumor features and prognosis. The present study aimed to determine whether the expression of γ-aminobutyric acid (GABA) A receptor subunit θ (GABRQ) could serve as a novel prognostic marker of ccRCC. GABA is the main inhibitory neurotransmitter in the brain that activates the receptor GABAA, which is comprised of three subunit isoforms: GABRA3, GABRB3 and GABRQ. A recent study reported that GABRQ is involved in the initiation and progression of hepatocellular carcinoma; however, the role of GABRQ in ccRCC remains unknown. In the present study, clinical and transcriptomic data were obtained from cohorts of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). Differential GABRQ expression levels among early (TI and II), late (TIII and IV), nonmetastatic (M0) and metastatic (M1, primary tumor) stages of ccRCC samples were then identified. Furthermore, the use of GABRQ as a prognostic gene was analyzed using Uno's C-index based on the time-dependent area under the curve (AUC), the AUC of the receiver operating characteristic curve at 5 years, the Kaplan-Meier survival curve and multivariate analysis. The survival curve analysis revealed that low GABRQ mRNA expression was significantly associated with a poor prognosis of ccRCC (P<0.001 and P=0.0012 for TCGA and ICGC data, respectively). In addition, analyses of the C-index and AUC values further supported this discriminatory power. Furthermore, the prognostic value of GABRQ mRNA expression was confirmed by multivariate Cox regression analysis. Taken together, these results suggested that GABRQ mRNA expression may be considered as a novel prognostic biomarker of ccRCC.

Identification of Functionally Distinct Mx1+αSMA+ Periosteal Skeletal Stem Cells.

The periosteum is critical for bone maintenance and healing. However, the in vivo identity and specific regulatory mechanisms of adult periosteum-resident skeletal stem cells are unknown. Here, we report animal models that selectively and durably label postnatal Mx1+αSMA+ periosteal stem cells (P-SSCs) and establish that P-SSCs are a long-term repopulating, functionally distinct SSC subset responsible for lifelong generation of periosteal osteoblasts. P-SSCs rapidly migrate toward an injury site, supply osteoblasts and chondrocytes, and recover new periosteum. Notably, P-SSCs specifically express CCL5 receptors, CCR3 and CCR5. Real-time intravital imaging revealed that the treatment with CCL5 induces P-SSC migration in vivo and bone healing, while CCL5/CCR5 deletion, CCR5 inhibition, or local P-SSC ablation reduces osteoblast number and delays bone healing. Human periosteal cells express CCR5 and undergo CCL5-mediated migration. Thus, the adult periosteum maintains genetically distinct SSC subsets with a CCL5-dependent migratory mechanism required for bone maintenance and injury repair.

Democratized image analytics by visual programming through integration of deep models and small-scale machine learning.

Analysis of biomedical images requires computational expertize that are uncommon among biomedical scientists. Deep learning approaches for image analysis provide an opportunity to develop user-friendly tools for exploratory data analysis. Here, we use the visual programming toolbox Orange ( http://orange.biolab.si ) to simplify image analysis by integrating deep-learning embedding, machine learning procedures, and data visualization. Orange supports the construction of data analysis workflows by assembling components for data preprocessing, visualization, and modeling. We equipped Orange with components that use pre-trained deep convolutional networks to profile images with vectors of features. These vectors are used in image clustering and classification in a framework that enables mining of image sets for both novel and experienced users. We demonstrate the utility of the tool in image analysis of progenitor cells in mouse bone healing, identification of developmental competence in mouse oocytes, subcellular protein localization in yeast, and developmental morphology of social amoebae.

Prognostic Role of TMED3 in Clear Cell Renal Cell Carcinoma: A Retrospective Multi-Cohort Analysis.

Transmembrane p24 trafficking protein 3 (TMED3) is a metastatic suppressor in colon cancer and hepatocellular carcinoma. However, its function in the progression of clear cell renal cell carcinoma (ccRCC) is unknown. Here, we report that TMED3 could be a new prognostic marker for ccRCC. Patient data were extracted from cohorts in the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium (ICGC). Differential expression of TMED3 was observed between the low stage (Stage I and II) and high stage (Stage III and IV) patients in the TCGA and ICGC cohorts and between the low grade (Grade I and II) and high grade (Grade III and IV) patients in the TCGA cohort. Further, we evaluated TMED3 expression as a prognostic gene using Kaplan-Meier survival analysis, multivariate analysis, the time-dependent area under the curve (AUC) of Uno's C-index, and the AUC of the receiver operating characteristics at 5 years. The Kaplan-Meier analysis revealed that TMED3 overexpression was associated with poor prognosis for ccRCC patients. Analysis of the C-indices and area under the receiver operating characteristic curve further supported this. Multivariate analysis confirmed the prognostic significance of TMED3 expression levels (P = 0.005 and 0.006 for TCGA and ICGC, respectively). Taken together, these findings demonstrate that TMED3 is a potential prognostic factor for ccRCC.