Dissection of Cellular Communication between Human Primary Osteoblasts and Bone Marrow Mesenchymal Stem Cells in Osteoarthritis at Single-Cell Resolution.

Background and Objectives: Osteoblasts are derived from bone marrow mesenchymal stem cells (BMMSCs) and play important role in bone remodeling. While our previous studies have investigated the cell subtypes and heterogeneity in osteoblasts and BMMSCs separately, cell-to-cell communications between osteoblasts and BMMSCs in vivo in humans have not been characterized. The aim of this study was to investigate the cellular communication between human primary osteoblasts and bone marrow mesenchymal stem cells. Methods and Results: To investigate the cell-to-cell communications between osteoblasts and BMMSCs and identify new cell subtypes, we performed a systematic integration analysis with our single-cell RNA sequencing (scRNA-seq) transcriptomes data from BMMSCs and osteoblasts. We successfully identified a novel preosteoblasts subtype which highly expressed ATF3, CCL2, CXCL2 and IRF1. Biological functional annotations of the transcriptomes suggested that the novel preosteoblasts subtype may inhibit osteoblasts differentiation, maintain cells to a less differentiated status and recruit osteoclasts. Ligand-receptor interaction analysis showed strong interaction between mature osteoblasts and BMMSCs. Meanwhile, we found FZD1 was highly expressed in BMMSCs of osteogenic differentiation direction. WIF1 and SFRP4, which were highly expressed in mature osteoblasts were reported to inhibit osteogenic differentiation. We speculated that WIF1 and sFRP4 expressed in mature osteoblasts inhibited the binding of FZD1 to Wnt ligand in BMMSCs, thereby further inhibiting osteogenic differentiation of BMMSCs. Conclusions: Our study provided a more systematic and comprehensive understanding of the heterogeneity of osteogenic cells. At the single cell level, this study provided insights into the cell-to-cell communications between BMMSCs and osteoblasts and mature osteoblasts may mediate negative feedback regulation of osteogenesis process.

Imputation of Human Primary Osteoblast Single Cell RNA-Seq Data Identified Three Novel Osteoblastic Subtypes.

BACKGROUND: Recently, single-cell RNA sequencing (scRNA-seq) technology was increasingly used to study transcriptomics at a single-cell resolution, scRNA-seq analysis was complicated by the "dropout", where the data only captures a small fraction of the transcriptome. This phenomenon can lead to the fact that the actual expressed transcript may not be detected. We previously performed osteoblast subtypes classification and dissection on freshly isolated human osteoblasts. MATERIALS AND METHODS: Here, we used the scImpute method to impute the missing values of dropout genes from a scRNA-seq dataset generated on freshly isolated human osteoblasts. RESULTS: Based on the imputed gene expression patterns, we discovered three new osteoblast subtypes. Specifically, these newfound osteoblast subtypes are osteoblast progenitors, and two undetermined osteoblasts. Osteoblast progenitors showed significantly high expression of proliferation related genes (FOS, JUN, JUNB and JUND). Analysis of each subtype showed that in addition to bone formation, these undetermined osteoblasts may involve osteoclast and adipocyte differentiation and have the potential function of regulate immune activation. CONCLUSIONS: Our findings provided a new perspective for studying the osteoblast heterogeneity and potential biological functions of these freshly isolated human osteoblasts at the single-cell level, which provides further insight into osteoblasts subtypes under various (pathological) physiological conditions.

High performance low dielectric polybenzocyclobutene nanocomposites with organic-inorganic hybrid silicon nanoparticles.

In this study, benzocyclobutene-functionalized organic-inorganic hybrid spherical silicon nanoparticles (BCBNPs) with controllable size (200-600 nm) and good dispersion were synthesized by a one-step sol-gel method in aqueous solution. The effect of the reaction conditions (time, precursor concentration, pH value and temperature) on the particle size of the BCBNPs and the formation mechanism of the BCBNPs were studied. What is more, homogeneously dispersed BCBNPs/divinyltetramethyldisiloxane-bis(benzocyclobutene) (BCBNPs/PDVSBCB) nanocomposites were prepared and the influence of the incorporation of BCBNPs on the properties of the nanocomposites was investigated. The dielectric constants of the BCBNPs/PDVSBCB nanocomposites were drastically reduced relative to neat PDVSBCB, and were as low as 2.25 (30 MHz). Besides, the thermal and mechanical properties of the BCBNPs/PDVSBCB nanocomposites were significantly improved, evidencing their potential applications in the field of high-performance dielectric materials.

Extracellular Vesicles from Human Urine-Derived Stem Cells Ameliorate Particulate Polyethylene-Induced Osteolysis.

PURPOSE: Wear debris particle-induced periprosthetic osteolysis is a severe complication of total joint replacement that results in aseptic loosening and subsequent arthroplasty failure. No effective therapeutic agents or drugs have been approved to prevent or treat osteolysis; thus, revision surgery is often needed. Extracellular vesicles (EVs) are vital nanosized regulators of intercellular communication that can be directly applied to promote tissue repair and regeneration. In this study, we assessed the therapeutic potential of EVs from human urine-derived stem cells (USCs) (USC-EVs) in preventing ultrahigh-molecular-weight polyethylene (UHMWPE) particle-induced osteolysis. METHODS: USCs were characterized by measuring induced multipotent differentiation and flow cytometry. USC-EVs were isolated and characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS) and Western blotting. RAW264.7 cells and bone marrow mesenchymal stem cells (BMSCs) were cultured with USC-EVs to verify osteoclast differentiation and osteoblast formation, respectively, in vitro. The effects of USC-EVs were investigated on a UHMWPE particle-induced murine calvarial osteolysis model by assessing bone mass, the inflammatory reaction, and osteoblast and osteoclast formation. RESULTS: USCs differentiated into osteogenic, adipogenic and chondrogenic cells in vitro and were positive for CD44, CD73, CD29 and CD90 but negative for CD34 and CD45. USC-EVs exhibited a cup-like morphology with a double-layered membrane structure and were positive for CD63 and TSG101 and negative for calnexin. In vitro, USC-EVs promoted the osteogenic differentiation of BMSCs and reduced proinflammatory factor production and osteoclastic activity in RAW264.7 cells. In vivo, local injection of USC-EVs around the central sites of the calvaria decreased inflammatory cytokine generation and osteolysis compared with the control groups and significantly increased bone formation. CONCLUSION: Based on our findings, USC-EVs prevent UHMWPE particle-induced osteolysis by decreasing inflammation, suppressing bone resorption and promoting bone formation.

Activation of N-methyl-D-aspartate receptor regulates insulin sensitivity and lipid metabolism.

RATIONALE: Although significant progress has been made in understanding the mechanisms of steatosis and insulin resistance, the physiological functions of regulators in these processes remain largely elusive. Evidence has suggested that the glutamate/N-methyl-D-aspartic acid receptor (NMDAR) axis contributes to acute lung injury, pulmonary arterial hypertension, and diabetes, but the specific metabolic contribution of the glutamate/NMDAR axis is not clear. Here we provide data at the animal, cellular, and molecular levels to support the role of the glutamate/NMDAR axis as a therapeutic target for metabolic syndrome in obesity. Methods: We examined the glutamate level in the obese mouse induced by a high-fat diet (HFD) for 12 weeks. To assess the role of NMDAR in insulin sensitivity and lipid metabolism, we tested the effects of Memantine (an NMDAR antagonist) and NMDA (an NMDAR agonist) on mice fed with HFD or standard chow diet. The in vitros NMDAR roles were analyzed in hepatocytes and potential mechanisms involved in regulating lipid metabolism were investigated. Results: Glutamate was increased in the serum of HFD-treated mice. The NMDAR blockade by Memantine decreased the susceptibility to insulin resistance and hepatic steatosis in obese mice. NMDA treatment for 6 months induced obesity in mice, characterized by hyperglycemia, hyperlipidemia, insulin resistance, and pathological changes in the liver. We provided in vitro evidence demonstrating that NMDAR activation facilitated metabolic syndrome in obesity through promoting lipid accumulation. NMDAR inhibition attenuated lipid accumulation induced by palmitic acid. Mechanistically, NMDAR activation impaired fatty acid oxidation by reducing PPARα phosphorylation and activity. The PPARα activity reduction induced by NMDAR activation was reversibly mediated by ERK1/2 signaling. Conclusion: These findings revealed that targeting NMDAR might be a promising therapeutic strategy for metabolic syndrome in obesity.

Extracellular vesicles from human urine-derived stem cells inhibit glucocorticoid-induced osteonecrosis of the femoral head by transporting and releasing pro-angiogenic DMBT1 and anti-apoptotic TIMP1.

Osteonecrosis of the femoral head (ONFH) frequently occurs after glucocorticoid (GC) treatment. Extracellular vesicles (EVs) are important nano-sized paracrine mediators of intercellular crosstalk. This study aimed to determine whether EVs from human urine-derived stem cells (USC-EVs) could protect against GC-induced ONFH and focused on the impacts of USC-EVs on angiogenesis and apoptosis to explore the mechanism by which USC-EVs attenuated GC-induced ONFH. The results in vivo showed that the intravenous administration of USC-EVs at the early stage of GC exposure could rescue angiogenesis impairment, reduce apoptosis of trabecular bone and marrow cells, prevent trabecular bone destruction and improve bone microarchitecture in the femoral heads of rats. In vitro, USC-EVs reversed the GC-induced suppression of endothelial angiogenesis and activation of apoptosis. Deleted in malignant brain tumors 1 (DMBT1) and tissue inhibitor of metalloproteinases 1 (TIMP1) proteins were enriched in USC-EVs and essential for the USC-EVs-induced pro-angiogenic and anti-apoptotic effects in GC-treated cells, respectively. Knockdown of TIMP1 attenuated the protective effects of USC-EVs against GC-induced ONFH. Our study suggests that USC-EVs are a promising nano-sized agent for the prevention of GC-induced ONFH by delivering pro-angiogenic DMBT1 and anti-apoptotic TIMP1. STATEMENT OF SIGNIFICANCE: This study demonstrates that the intravenous injection of extracellular vesicles from human urine-derived stem cells (USC-EVs) at the early stage of glucocorticoid (GC) exposure efficiently protects the rats from the GC-induced osteonecrosis of the femoral head (ONFH). Moreover, this study identifies that the promotion of angiogenesis and inhibition of apoptosis by transferring pro-angiogenic DMBT1 and anti-apoptotic TIMP1 proteins contribute importantly to the USC-EVs-induced protective effects against GC-induced ONFH. This study suggests the promising prospect of USC-EVs as a new nano-sized agent for protecting against GC-induced ONFH, and the potential of DMBT1 and TIMP1 as the molecular targets for further augmenting the protective function of USC-EVs.

Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2.

BACKGROUND: Some microRNAs (miRNAs) are involved in osteogenic differentiation. In recent years, increasing evidences have revealed that exosomes contain specific miRNAs. However, the effect and mechanism of miR-23a-5p-containing exosomes in osteoblast remain largely unclear. METHODS: We extracted exosomes from RANKL-induced RAW 264.7 cells, and identified exosomes via transmission electron microscopy, western blot and flow cytometry analysis. In addition, exosome secretion was inhibited by GW4869 and Rab27a siRNAs. miR-23a-5p expression was analyzed by qRT-PCR, and the related protein levels were examined by western blot assay. Furthermore, the number and distribution of osteoclasts were detected by TRAP staining, and early osteogenesis was evaluated by ALP staining. Combination of YAP1 and Runx2 was verified by Co-IP assay, and the regulation of miR-23a-5p and Runx2 was measured by dual luciferase reporter assay. RESULTS: We successfully extracted exosomes from RANKL-induced RAW 264.7 cells, and successfully verified exosomes morphology. We also indicated that miR-23a-5p was highly expressed in exosomes from RANKL-induced RAW 264.7 cells, and osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity, while its inhibition weakened osteoclasts. In mechanism, we demonstrated that Runx2 was a target gene of miR-23a-5p, YAP interacted with Runx2, and YAP or Runx2 inhibited MT1DP expression. In addition, we proved that knockdown of MT1DP facilitated osteogenic differentiation by regulating FoxA1 and Runx2. CONCLUSIONS: We demonstrated that osteoclast-derived miR-23a-5p-containing exosomes could efficiently suppress osteogenic differentiation by inhibiting Runx2 and promoting YAP1-mediated MT1DP. Therefore, we suggested miR-23a-5p in exosomes might provide a novel mechanism for osteoblast function.

DNA barcodes and species distribution models evaluate threats of global climate changes to genetic diversity: a case study from Nanorana parkeri (Anura: Dicroglossidae).

Anthropogenic global climate changes are one of the greatest threats to biodiversity. Distribution modeling can predict the effects of climate changes and potentially their effects on genetic diversity. DNA barcoding quickly identifies patterns of genetic diversity. As a case study, we use DNA barcodes and distribution models to predict threats under climate changes in the frog Nanorana parkeri, which is endemic to the Qinghai-Tibetan Plateau. Barcoding identifies major lineages W and E. Lineage W has a single origin in a refugium and Lineage E derives from three refugia. All refugia locate in river valleys and each greatly contributes to the current level of intraspecific genetic diversity. Species distribution models suggest that global climate changes will greatly influence N. parkeri, especially in the level of genetic diversity, because two former refugia will fail to provide suitable habitat. Our pipeline provides a novel application of DNA barcoding and has important implications for the conservation of biodiversity in southern areas of the Qinghai-Tibetan Plateau.

DNA barcoding of Vietnamese bent-toed geckos (Squamata: Gekkonidae: Cyrtodactylus) and the description of a new species.

Species of bent-toed gecko (Cyrtodactylus) in Vietnam have been described at a rate of nearly four species per year since 2007 mostly based on morphological data. A tool that guides species delimitation will accelerate the rate of documentation, and at a time when the recognition of species greatly benefits conservation. We use DNA barcoding using COI (550 bp) to re-examine the levels of genetic divergence and taxonomic status of 21 described species of Vietnamese bent-toed geckos. Tree-based analyses resolve all sampled species and identify potential undescribed taxa. Kimura 2-parameter genetic distances between the described species average 21.0±4.2% and range from 4.3% to 28.7%. Further, our analyses discover two potentially new species from Vietnam, two from Laos and one from China. Herein we describe the new species Cyrtodactylus puhuensis sp. nov. from Vietnam on the basis of both genetics and morphology. Genetically, it differs from the remaining species by an average K2P distance of 24.0±1.8%. Morphologically, the new species is diagnosed by its medium-size (snout-vent length 79.24 mm and tail length 82.59 mm, for the single known individual), in having a series of moderately enlarged transverse subcaudals and a series of moderately enlarged femoral scales that extend from precloacal scales, in possessing femoral scales without pores, with males having five precloacal pores, and in exhibiting 8 supralabials, 10 infralabials, 23 narrow subdigital lamellae on its fourth toe, and 36 transverse ventrals.

Universal COI primers for DNA barcoding amphibians.

DNA barcoding is a proven tool for the rapid and unambiguous identification of species, which is essential for many activities including the vouchering tissue samples in the genome 10K initiative, genealogical reconstructions, forensics and biodiversity surveys, among many other applications. A large-scale effort is underway to barcode all amphibian species using the universally sequenced DNA region, a partial fragment of mitochondrial cytochrome oxidase subunit I COI. This fragment is desirable because it appears to be superior to 16S for barcoding, at least for some groups of salamanders. The barcoding of amphibians is essential in part because many species are now endangered. Unfortunately, existing primers for COI often fail to achieve this goal. Herein, we report two new pairs of primers (➀, ➁) that in combination serve to universally amplify and sequence all three orders of Chinese amphibians as represented by 36 genera. This taxonomic diversity, which includes caecilians, salamanders and frogs, suggests that the new primer pairs will universally amplify COI for the vast majority species of amphibians.

A phylogeny of the Tylototriton asperrimus group (Caudata: Salamandridae) based on a mitochondrial study: suggestions for a taxonomic revision.

A phylogenetic hypothesis for the Asian newts of the Tylototriton asperrimus group was generated using data from two mitochondrial fragments including COI and the ND1-ND2 regions. Four distinct clades (A, B, C, D) were resolved with high nodal support within this monophyletic group. Clade A included T. asperrimus, T. hainanensis, T. notialis, "T. vietnamensis", and two unnamed salamander populations from Vietnam. Clade A, constituted the sister group of clades B + C. Newly identified clade C likely represents a new cryptic species. Clade C was the sister group of T. wenxianensis. The true T. vietnamensis exclusively constituted clade D. Our results bring into question some previous taxonomic decisions, and a revision is required. This study illustrates the necessity to include samples from type localities in taxonomic studies, and highlights the importance of fine-grained geographical sampling.

Validity and systematic position of Rana altaica (Rana: ranidae): results of a phylogenetic analysis.

In order to evaluate the phylogenetic position and validity of Rana altaica, we investigated the phylogeny of brown frogs in Eurasia by Bayesian Inference and Maximum Parsimony analyses of a fragment from the mitochondrial DNA gene Cytochrome b. Both analyses resolved R. altaica as nesting deeply within R. arvalis. Most samples of the nominal R. altaica from the Altai region and specimens from Central Siberia shared a haplotype with R. arvalis based on the network analysis. The matrilineal relationships suggested that R. altaica should be considered as a junior synonym of R. arvalis. Furthermore, our study suggested that the species group division of Chinese brown frogs should be re-evaluated within a phylogenetic context.

1-[Bicyclo[4.2.0]octa-1(6),2,4-trien-3-yl]-3-[bicyclo[4.2.0]octa-1(6),2,4-trien-3-yl-methyl]imidazolium hexa-fluoro-phos-phate.

In the title compound, C(20)H(19)N(2) (+)·PF(6) (-), the two benzocyclo-butene units are essentially planar and they form dihedral angles of 38.0 (2) and 72.7 (2)°, with the central imidazolium ring. In the crystal structure, weak C-H⋯π and π--π stacking inter-actions [centroid-centroid distance = 3.742 (2) Å] contribute to the stability of the crystal structure. The PF(6) (-) ion is disordered over two positions with site occupancies of 0.869 (9) and 0.131 (9).

1-[Bicyclo-[4.2.0]octa-1(6),2,4-trien-3-yl]-3-(but-3-en-yl)imidazolium bromide.

In the title compound, C(15)H(17)N(2) (+)·Br(-), the cyclo-butene and benzene rings are coplanar. The dihedral angle between the benzene and imidazolium rings is 21.2 (3)°. In the crystal structure, the C(15)H(17)N(2) (+) and Br(-) ions are linked into a zigzag chain along the b axis by C-H⋯Br hydrogen bonds, and weak C-H⋯π inter-actions involving the benzene ring of a screw-related cation.