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.

A Novel Algorithm for Thickness Prediction in Incremental Sheet Metal Forming.

Incremental sheet metal forming characterized as increased flexibility and local plastic deformation is well suitable for low-production-run manufacturing and a new sample trial production of complex shapes. Thickness thinning is still an obstacle to the application of incremental forming. In this study, a novel mathematical algorithm based on a non-uniform rational B-spline (NURBS) surface was proposed and implemented which focuses on predicting and calculating the final thickness for arbitrary parts in incremental forming. In order to evaluate the validity of the proposed model, the finite element simulation and forming experiments of three kinds of parts, such as truncated cones, truncated pyramids and ellipsoid parts, were conducted. The thickness of theoretical prediction was compared with that of finite element simulation and experiment, and good agreements were obtained. The results show that the proposed model and the method are effective and robust for predicting the thickness of the formed parts in incremental sheet metal forming.

Isolation and Evaluation of Rhizosphere Actinomycetes With Potential Application for Biocontrolling Fusarium Wilt of Banana Caused by Fusarium oxysporum f. sp. cubense Tropical Race 4.

Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) is globally one of the most destructive soil-borne fungal diseases. Biological control using environmental microorganisms is considered as an alternative and sustainable strategy. Actinomycetes have the potential to explore biocontrol agents due to their production of diverse metabolites. The isolation and identification of high-efficiency and broad-spectrum antagonistic actinomycetes are the key for the application of biocontrol agents. In the present study, 60 actinomycetes were obtained from the rhizosphere soil of Machilus pingii in the primitive ecological natural reserve of Hainan province, China. Seventeen isolates and their extracts exhibited significant antifungal activity against F. oxysporum TR4. Particularly, strain BITDG-11 with the strongest inhibition ability had a broad-spectrum antifungal activity. The assay of its physiological and biochemical profiles showed that strain BITDG-11 had the ability to produce IAA and siderophores and had a positive response to gelatin liquefaction and nitrate reduction. Enzyme activities of chitinase, ß-1,3-glucanase, lipase, and urease were also detected. Average nucleotide identity calculated by comparison with the standard strain genome of Streptomyces albospinus JCM3399 was 86.55% below the novel species threshold, suggesting that the strain could be a novel species. In addition, Streptomyces BITDG-11 obviously reduced the disease index of banana plantlets and promoted plant growth at 45 days post inoculation. The higher and lasting expression levels of defense genes and activities of antioxidant enzymes were induced in the roots of banana. Genome sequencing revealed that the Streptomyces BITDG-11 chromosome contained large numbers of conserved biosynthesis gene clusters encoding terpenes, non-ribosomal peptides, polyketides, siderophores, and ectoines. Fifteen bioactive secondary metabolites were further identified from Streptomyces BITDG-11 extract by gas chromatography-mass spectrometry. Dibutyl phthalate demonstrating a strong antifungal activity was the major compound with the highest peak area. Hence, Streptomyces sp. BITDG-11 has a great potential to become an essential constituent of modern agricultural practice as biofertilizers and biocontrol agents.

Single-cell RNA sequencing deconvolutes the in vivo heterogeneity of human bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of the transcriptomes suggest that osteoblast precursors induce angiogenesis coupled with osteogenesis, and chondrocyte precursors have the potential to differentiate into myocytes. We also discovered transcripts for several clusters of differentiation (CD) markers that were either highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs, representing potential novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing an insight into the extent of their cellular heterogeneity and roles in maintaining bone homeostasis.

A systematic dissection of human primary osteoblasts in vivo at single-cell resolution.

Human osteoblasts are multifunctional bone cells, which play essential roles in bone formation, angiogenesis regulation, as well as maintenance of hematopoiesis. However, the categorization of primary osteoblast subtypes in vivo in humans has not yet been achieved. Here, we used single-cell RNA sequencing (scRNA-seq) to perform a systematic cellular taxonomy dissection of freshly isolated human osteoblasts from one 31-year-old male with osteoarthritis and osteopenia after hip replacement. Based on the gene expression patterns and cell lineage reconstruction, we identified three distinct cell clusters including preosteoblasts, mature osteoblasts, and an undetermined rare osteoblast subpopulation. This novel subtype was found to be the major source of the nuclear receptor subfamily 4 group A member 1 and 2 (NR4A1 and NR4A2) in primary osteoblasts, and the expression of NR4A1 was confirmed by immunofluorescence staining on mouse osteoblasts in vivo. Trajectory inference analysis suggested that the undetermined cluster, together with the preosteoblasts, are involved in the regulation of osteoblastogenesis and also give rise to mature osteoblasts. Investigation of the biological processes and signaling pathways enriched in each subpopulation revealed that in addition to bone formation, preosteoblasts and undetermined osteoblasts may also regulate both angiogenesis and hemopoiesis. Finally, we demonstrated that there are systematic differences between the transcriptional profiles of human and mouse osteoblasts, highlighting the necessity for studying bone physiological processes in humans rather than solely relying on mouse models. Our findings provide novel insights into the cellular heterogeneity and potential biological functions of human primary osteoblasts at the single-cell level.

Single-cell RNA sequencing of human femoral head in vivo.

The homeostasis of bone metabolism depends on the coupling and precise regulation of various types of cells in bone tissue. However, the communication and interaction between bone tissue cells at the single-cell level remains poorly understood. Thus, we performed single-cell RNA sequencing (scRNA-seq) on the primary human femoral head tissue cells (FHTCs). Nine cell types were identified in 26,574 primary human FHTCs, including granulocytes, T cells, monocytes, B cells, red blood cells, osteoblastic lineage cells, endothelial cells, endothelial progenitor cells (EPCs) and plasmacytoid dendritic cells. We identified serine protease 23 (PRSS23) and matrix remodeling associated protein 8 (MXRA8) as novel bone metabolism-related genes. Additionally, we found that several subtypes of monocytes, T cells and B cells were related to bone metabolism. Cell-cell communication analysis showed that collagen, chemokine, transforming growth factor and their ligands have significant roles in the crosstalks between FHTCs. In particular, EPCs communicated with osteoblastic lineage cells closely via the "COL2A1-ITGB1" interaction pair. Collectively, this study provided an initial characterization of the cellular composition of the human FHTCs and the complex crosstalks between them at the single-cell level. It is a unique starting resource for in-depth insights into bone metabolism.

Alkaline amendments improve the health of soils degraded by metal contamination and acidification: Crop performance and soil bacterial community responses.

Soil degradation due to heavy metal contamination and acidification has negative effects on soil health and crop growth. Many previous studies have tried to improve the growth of crops and decrease their metal uptake. The recovery of soil health, however, has rarely been focused in soil remediation. In this study, a pot trial was conducted with lettuce (Lactuca sativa L.) growing in heavy metal contaminated and acidic soils, to examine the effects of alkaline amendments (limestone, LS; calcium magnesium phosphate fertilizer, Pcm) and organic amendments (cow manure compost, CMC; biochar, BC) on the growth of lettuce and on the availability of heavy metals, enzyme activities, and bacterial community structures in the soils. The results showed that, in comparison with the CMC and BC treatments, LS and Pcm were more effective at improving lettuce growth and reducing metal concentrations in shoots. Urease and catalase activities in LS and Pcm amended soils were consistently higher than in those with CMC and BC. Additionally, the alkaline amendments dramatically improved the bacterial diversity and shaped more favorable bacterial community structures. Proteobacteria and Gemmatimonadetes were predominant in soils amended with alkaline treatments. The beneficial bacterial genera Gemmatimonas and f_Gemmatimonadaceae, which are vital for phosphate dissolution, microbial nitrogen metabolism, and soil respiration, were also enriched. The results suggest that alkaline amendments were superior to organic amendments, and thus may be useful for the future recovery of soil functions and health under heavy metal contamination and low pH.

Polymers with controlled assembly and rigidity made with click-functional peptide bundles.

The engineering of biological molecules is a key concept in the design of highly functional, sophisticated soft materials. Biomolecules exhibit a wide range of functions and structures, including chemical recognition (of enzyme substrates or adhesive ligands1, for instance), exquisite nanostructures (composed of peptides2, proteins3 or nucleic acids4), and unusual mechanical properties (such as silk-like strength3, stiffness5, viscoelasticity6 and resiliency7). Here we combine the computational design of physical (noncovalent) interactions with pathway-dependent, hierarchical 'click' covalent assembly to produce hybrid synthetic peptide-based polymers. The nanometre-scale monomeric units of these polymers are homotetrameric, α-helical bundles of low-molecular-weight peptides. These bundled monomers, or 'bundlemers', can be designed to provide complete control of the stability, size and spatial display of chemical functionalities. The protein-like structure of the bundle allows precise positioning of covalent linkages between the ends of distinct bundlemers, resulting in polymers with interesting and controllable physical characteristics, such as rigid rods, semiflexible or kinked chains, and thermally responsive hydrogel networks. Chain stiffness can be controlled by varying only the linkage. Furthermore, by controlling the amino acid sequence along the bundlemer periphery, we use specific amino acid side chains, including non-natural 'click' chemistry functionalities, to conjugate moieties into a desired pattern, enabling the creation of a wide variety of hybrid nanomaterials.

Nanotubes, Plates, and Needles: Pathway-Dependent Self-Assembly of Computationally Designed Peptides.

Computationally designed peptides form desired antiparallel, tetrameric coiled-coil bundles that hierarchically assemble into a variety of well-controlled nanostructures depending on aqueous solution conditions. The bundles selectively self-assemble into different structures: nanotubes, platelets, or needle-like structures at solution pH values of 4.5, 7, and 10, respectively. The self-assembly produces hollow tubes or elongated needle-like structures at pH conditions associated with charged bundles (pH 4.5 or 10); at neutral pH, near the pI of the bundle, a plate-like self-assembled structure forms. Transmission electron microscopy and small-angle X-ray scattering show the nanotubes to be uniform with a tube diameter of ∼13 nm and lengths of up to several µm, yielding aspect ratios >1000. Combining the measured nanostructure geometry with the apparent charged states of the constituent amino acids, a tilted-bundle packing model is proposed for the formation of the homogeneous nanotubes. This work demonstrates the successful use of assembly pathway control for the construction of nanostructures with diverse, well-structured morphologies associated with the folding and self-association of a single type of molecule.

Self-assembly and soluble aggregate behavior of computationally designed coiled-coil peptide bundles.

Coiled-coil peptides have proven useful in a range of materials applications ranging from the formation of well-defined fibrils to responsive hydrogels. The ability to design from first principles their oligomerization and subsequent higher order assembly offers their expanded use in producing new materials. Toward these ends, homo-tetrameric, antiparallel, coiled-coil, peptide bundles have been designed computationally, synthesized via solid-phase methods, and their solution behavior characterized. Two different bundle-forming peptides were designed and examined. Within the targeted coiled coil structure, both bundles contained the same hydrophobic core residues. However, different exterior residues on the two different designs yielded sequences with different distributions of charged residues and two different expected isoelectric points of pI 4.4 and pI 10.5. Both coiled-coil bundles were extremely stable with respect to temperature (Tm > 80 C) and remained soluble in solution even at high (millimolar) peptide concentrations. The coiled-coil tetramer was confirmed to be the dominant species in solution by analytical sedimentation studies and by small-angle neutron scattering, where the scattering form factor is well represented by a cylinder model with the dimensions of the targeted coiled coil. At high concentrations (5-15 mM), evidence of interbundle structure was observed via neutron scattering. At these concentrations, the synthetic bundles form soluble aggregates, and interbundle distances can be determined via a structure factor fit to scattering data. The data support the successful design of robust coiled-coil bundles. Despite their different sequences, each sequence forms loosely associated but soluble aggregates of the bundles, suggesting similar dissociated states for each. The behavior of the dispersed bundles is similar to that observed for natural proteins.

Transition from disordered aggregates to ordered lattices: kinetic control of the assembly of a computationally designed peptide.

Natural biomolecular self-assembly typically occurs under a narrow range of solution conditions, and the design of sequences that can form prescribed structures under a range of such conditions would be valuable in the bottom-up assembly of predetermined nanostructures. We present a computationally designed peptide that robustly self-assembles into regular arrays under a wide range of solution pH and temperature conditions. Controling the solution conditions provides the opportunity to exploit a simple and reproducible approach for altering the pathway of peptide solution self-assembly. The computationally designed peptide forms a homotetrameric coiled-coil bundle that further self-assembles into 2-D plate structures with well-defined inter-bundle symmetry. Herein, we present how modulation of solution conditions, such as pH and temperature, can be used to control the kinetics of the inter-bundle assembly and manipulate the final morphology. Changes in solution pH primarily influence the inter-bundle assembly by affecting the charged state of ionizable residues on the bundle exterior while leaving the homotetrameric coiled-coil structure intact. At low pH, repulsive interactions prevent 2-D lattice nanostructure formation. Near the estimated isoelectric point of the peptide, bundle aggregation is rapid and yields disordered products, which subsequently transform into ordered nanostructures over days to weeks. At elevated temperatures (T = 40 °C or 50 °C), the formation of disordered, kinetically-trapped products largely can be eliminated, allowing the system to quickly assemble into plate-like nanostructured lattices. Moreover, subtle changes in pH and in the peptide charge state have a significant influence on the thickness of formed plates and on the hierarchical manner in which plates fuse into larger material structures with observable grain boundaries. These findings confirm the ability to finely tune the peptide assembly process to achieve a range of engineered structures with one simple 29-residue peptide building block.

Computationally designed peptides for self-assembly of nanostructured lattices.

Folded peptides present complex exterior surfaces specified by their amino acid sequences, and the control of these surfaces offers high-precision routes to self-assembling materials. The complexity of peptide structure and the subtlety of noncovalent interactions make the design of predetermined nanostructures difficult. Computational methods can facilitate this design and are used here to determine 29-residue peptides that form tetrahelical bundles that, in turn, serve as building blocks for lattice-forming materials. Four distinct assemblies were engineered. Peptide bundle exterior amino acids were designed in the context of three different interbundle lattices in addition to one design to produce bundles isolated in solution. Solution assembly produced three different types of lattice-forming materials that exhibited varying degrees of agreement with the chosen lattices used in the design of each sequence. Transmission electron microscopy revealed the nanostructure of the sheetlike nanomaterials. In contrast, the peptide sequence designed to form isolated, soluble, tetrameric bundles remained dispersed and did not form any higher-order assembled nanostructure. Small-angle neutron scattering confirmed the formation of soluble bundles with the designed size. In the lattice-forming nanostructures, the solution assembly process is robust with respect to variation of solution conditions (pH and temperature) and covalent modification of the computationally designed peptides. Solution conditions can be used to control micrometer-scale morphology of the assemblies. The findings illustrate that, with careful control of molecular structure and solution conditions, a single peptide motif can be versatile enough to yield a wide range of self-assembled lattice morphologies across many length scales (1 to 1000 nm).

Engineering complementary hydrophobic interactions to control ß-hairpin peptide self-assembly, network branching, and hydrogel properties.

The MAX1 ß-hairpin peptide (VKVKVKVK-V(D)PPT-KVKVKVKV-NH2) has been shown to form nanofibrils having a cross-section of two folded peptides forming a hydrophobic, valine-rich core, and the polymerized fibril exhibits primarily ß-sheet hydrogen bonding.1-7 These nanofibrils form hydrogel networks through fibril entanglements as well as fibril branching.8 Fibrillar branching in MAX1 hydrogel networks provide the ability to flow under applied shear stress and immediately reform a hydrogel solid on cessation of shear. New ß-hairpins were designed to limit branching during nanofibril growth because of steric specificity in the assembled fibril hydrophobic core. The nonturn valines of MAX1 were substituted by 2-naphthylalanine (Nal) and alanine (A) residues, with much larger and smaller side chain volumes, respectively, to obtain LNK1 (Nal)K(Nal)KAKAK-V(D)PPT-KAKAK(Nal)K(Nal)-NH2. LNK1 was targeted to self-associate with a specific "lock and key" complementary packing in the hydrophobic core in order to accommodate the Nal and Ala residue side chains. The experimentally observable manifestation of reduced fibrillar branching in the LNK1 peptide is the lack of solid hydrogel formation after shear in stark contrast to the MAX1 branched fibril system. Molecular dynamics simulations provide a molecular picture of interpeptide interactions within the assembly that is consistent with the branching propensity of MAX1 vs LNK1 and in agreement with experimental observations.