Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7-9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

Transcriptome analysis reveals the potential lncRNA-mRNA modules involved in genetic male sterility and fertility of Chinese cabbage (brassica rapa L. ssp. pekinensis).

BACKGROUND: Long non-coding RNAs (lncRNAs) play a crucial role in regulating gene expression vital for the growth and development of plants. Despite this, the role of lncRNAs in Chinese cabbage (Brassica rapa L. ssp. pekinensis) pollen development and male fertility remains poorly understood. RESULTS: In this study, we characterized a recessive genic male sterile mutant (366-2 S), where the delayed degradation of tapetum and the failure of tetrad separation primarily led to the inability to form single microspores, resulting in male sterility. To analyze the role of lncRNAs in pollen development, we conducted a comparative lncRNA sequencing using anthers from the male sterile mutant line (366-2 S) and the wild-type male fertile line (366-2 F). We identified 385 differentially expressed lncRNAs between the 366-2 F and 366-2 S lines, with 172 of them potentially associated with target genes. To further understand the alterations in mRNA expression and explore potential lncRNA-target genes (mRNAs), we performed comparative mRNA transcriptome analysis in the anthers of 366-2 S and 366-2 F at two stages. We identified 1,176 differentially expressed mRNAs. Remarkably, GO analysis revealed significant enrichment in five GO terms, most notably involving mRNAs annotated as pectinesterase and polygalacturonase, which play roles in cell wall degradation. The considerable downregulation of these genes might contribute to the delayed degradation of tapetum in 366-2 S. Furthermore, we identified 15 lncRNA-mRNA modules through Venn diagram analysis. Among them, MSTRG.9997-BraA04g004630.3 C (ß-1,3-glucanase) is associated with callose degradation and tetrad separation. Additionally, MSTRG.5212-BraA02g040020.3 C (pectinesterase) and MSTRG.13,532-BraA05g030320.3 C (pectinesterase) are associated with cell wall degradation of the tapetum, indicating that these three candidate lncRNA-mRNA modules potentially regulate pollen development. CONCLUSION: This study lays the foundation for understanding the roles of lncRNAs in pollen development and for elucidating their molecular mechanisms in regulating male sterility in Chinese cabbage.

HDAC4 represses ER stress induced chondrocyte apoptosis by inhibiting ATF4 and attenuates cartilage degeneration in an osteoarthritis rat model.

BACKGROUND: The present study evaluated whether the lack of histone deacetylase 4 (HDAC4) increases endoplasmic reticulum stress-induced chondrocyte apoptosis by releasing activating transcription factor 4 (ATF4) in human osteoarthritis (OA) cartilage degeneration. METHODS: Articular cartilage from the tibial plateau was obtained from patients with OA during total knee replacement. Cartilage extracted from severely damaged regions was classified as degraded cartilage, and cartilage extracted from a relatively smooth region was classified as preserved cartilage. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining was used to detect chondrocyte apoptosis. HDAC4, ATF4, and C/EBP homologous protein (CHOP) expression levels were measured using immunohistochemistry staining and real-time quantitative PCR. Chondrocytes were transfected with HDAC4 or HDAC4 siRNA for 24 h and stimulated with 300 µM H2O2 for 12 h. The chondrocyte apoptosis was measured using flow cytometry. ATF4, CHOP, and caspase 12 expression levels were measured using real-time quantitative PCR and western blotting. Male Sprague-Dawley rats (n = 15) were randomly divided into three groups and transduced with different vectors: ACLT + Ad-GFP, ACLT + Ad-HDAC4-GFP, and sham + Ad-GFP. All rats received intra-articular injections 48 h after the operation and every three weeks thereafter. Cartilage damage was assessed using Safranin O staining and quantified using the Osteoarthritis Research Society International score. ATF4, CHOP, and collagen II expression were detected using immunohistochemistry, and chondrocyte apoptosis was detected using terminal deoxynucleotidyl transferase dUTP nick end labeling staining. RESULTS: The chondrocyte apoptosis was higher in degraded cartilage than in preserved cartilage. HDAC4 expression was lower in degraded cartilage than in preserved cartilage. ATF4 and CHOP expression was increased in degraded cartilage. Upregulation of HDAC4 in chondrocytes decreased the expression of ATF4, while the expression of ATF4 was increased after downregulation of HDAC4. Upregulation of HDAC4 decreased the chondrocyte apoptosis under endoplasmic reticulum stress, and chondrocyte apoptosis was increased after downregulation of HDAC4. In a rat anterior cruciate ligament transection OA model, adenovirus-mediated transduction of HDAC4 was administered by intra-articular injection. We detected a stronger Safranin O staining with lower Osteoarthritis Research Society International scores, lower ATF4 and CHOP production, stronger collagen II expression, and lower chondrocyte apoptosis in rats treated with Ad-HDAC4. CONCLUSION: The lack of HDAC4 expression partially contributes to increased ATF4, CHOP, and endoplasmic reticulum stress-induced chondrocyte apoptosis in OA pathogenesis. HDAC4 attenuates cartilage damage by repressing ATF4-CHOP signaling-induced chondrocyte apoptosis in a rat model of OA.

Deficient gait function despite effect index of the Western Ontario and McMaster university osteoarthritis index score considered cured one year after bilateral total knee arthroplasty.

BACKGROUND: To clarify the value of gait analysis and its consistency with traditional scoring scales for the evaluation of knee joint function after total knee arthroplasty (TKA). METHODS: This study included 25 patients with knee osteoarthritis (KOA) who underwent bilateral TKA, and 25 conditionally matched healthy individuals, categorised into the experimental and control groups, respectively. Patients in the experimental group underwent gait analysis and Western Ontario and McMaster University Osteoarthritis Index (WOMAC) evaluation before and 1 year after TKA. Weight-bearing balance and walking stability were assessed using discrete trends of relevant gait indicators. Pearson's correlation analysis was performed on the gait and WOMAC score data of the experimental group before and after TKA. RESULTS: One year after TKA, patients' gait indices (except gait cycle) were significantly better than before surgery, but significantly worse than that of the control group (P < 0.01). The shape of patients' plantar pressure curves did not return to normal. Additionally, the discrete trend of related gait indicators reflecting weight-bearing balance and walking stability were smaller than before TKA, but still greater than that of the control group. The WOMAC scores of patients 1 year after TKA were significantly lower than those before TKA (P < 0.001), and the efficacy index was > 80%. The WOMAC scores and gait analysis results were significantly correlated before TKA (P < 0.05). CONCLUSIONS: Gait analysis should be used in conjunction with scoring scales to assess joint functions.

Biomechanical properties of articular cartilage in different regions and sites of the knee joint: acquisition of osteochondral allografts.

Osteochondral allograft (OCA) transplantation involves grafting of natural hyaline cartilage and supporting subchondral bone into the cartilage defect area to restore its biomechanical and tissue structure. However, differences in biomechanical properties and donor-host matching may impair the integration of articular cartilage (AC). This study analyzed the biomechanical properties of the AC in different regions of different sites of the knee joint and provided a novel approach to OCA transplantation. Intact stifle joints from skeletally mature pigs were collected from a local abattoir less than 8 h after slaughter. OCAs were collected from different regions of the joints. The patella and the tibial plateau were divided into medial and lateral regions, while the trochlea and femoral condyle were divided into six regions. The OCAs were analyzed and compared for Young's modulus, the compressive modulus, and cartilage thickness. Young's modulus, cartilage thickness, and compressive modulus of OCA were significantly different in different regions of the joints. A negative correlation was observed between Young's modulus and the proportion of the subchondral bone (r = - 0.4241, P < 0.0001). Cartilage thickness was positively correlated with Young's modulus (r = 0.4473, P < 0.0001) and the compressive modulus (r = 0.3678, P < 0.0001). During OCA transplantation, OCAs should be transplanted in the same regions, or at the closest possible regions to maintain consistency of the biomechanical properties and cartilage thickness of the donor and recipient, to ensure smooth integration with the surrounding tissue. A 7 mm depth achieved a higher Young's modulus, and may represent the ideal length.

Polyploidization: A Biological Force That Enhances Stress Resistance.

Organisms with three or more complete sets of chromosomes are designated as polyploids. Polyploidy serves as a crucial pathway in biological evolution and enriches species diversity, which is demonstrated to have significant advantages in coping with both biotic stressors (such as diseases and pests) and abiotic stressors (like extreme temperatures, drought, and salinity), particularly in the context of ongoing global climate deterioration, increased agrochemical use, and industrialization. Polyploid cultivars have been developed to achieve higher yields and improved product quality. Numerous studies have shown that polyploids exhibit substantial enhancements in cell size and structure, physiological and biochemical traits, gene expression, and epigenetic modifications compared to their diploid counterparts. However, some research also suggested that increased stress tolerance might not always be associated with polyploidy. Therefore, a more comprehensive and detailed investigation is essential to complete the underlying stress tolerance mechanisms of polyploids. Thus, this review summarizes the mechanism of polyploid formation, the polyploid biochemical tolerance mechanism of abiotic and biotic stressors, and molecular regulatory networks that confer polyploidy stress tolerance, which can shed light on the theoretical foundation for future research.

TMT quantitative proteomics reveals key proteins relevant to microRNA-1-mediated regulation in osteoarthritis.

Osteoarthritis (OA) is the second-commonest arthritis, but pathogenic and regulatory mechanisms underlying OA remain incompletely understood. Here, we aimed to identify the mechanisms associated with microRNA-1 (miR-1) treatment of OA in rodent OA models using a proteomic approach. First, N = 18 Sprague Dawley (SD) rats underwent sham surgery (n = 6) or ACL transection (n = 12), followed at an interval of one week by randomization of the ACL transection group to intra-articular administration of either 50 µL placebo (control group) or miR-1 agomir, a mimic of endogenous miR-1 (experimental group). After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and immunohistochemically stained for the presence of MMP-13. Second, N = 30 Col2a1-cre-ERT2 /GFPf1/fl -RFP-miR-1 transgenic mice were randomized to intra-articular administration of either placebo (control group, N = 15) or tamoxifen, an inducer of miR-1 expression (experimental group, N = 15), before undergoing surgical disruption of the medial meniscus (DMM) after an interval of five days. After allowing for eight weeks of remodeling, articular cartilage tissue was harvested and underwent differential proteomic analysis. Specifically, tandem mass tagging (TMT) quantitative proteomic analysis was employed to identify inter-group differentially-expressed proteins (DEP), and selected DEPs were validated using real-time quantitative polymerase chain reaction (RT-qPCR) technology. Immunohistochemically-detected MMP-13 expression was significantly lower in the experimental rat group, and proteomic analyses of mouse tissue homogenate demonstrated that of 3526 identified proteins, 345 were differentially expressed (relative up- and down-regulation) in the experimental group. Proteins Fn1, P4ha1, P4ha2, Acan, F2, Col3a1, Fga, Rps29, Rpl34, and Fgg were the *top ten most-connected proteins, implying that miR-1 may regulate an expression network involving these proteins. Of these ten proteins, three were selected for further validation by RT-qPCR: the transcript of Fn1, known to be associated with OA, exhibited relative upregulation in the experimental group, whereas the transcripts of P4ha1 and Acan exhibited relative downregulation. These proteins may thus represent key miR-1 targets during OA-regulatory mechanisms, and may provide additional insights regarding therapeutic mechanisms of miR-1 in context of OA.

Cytological and transcriptomic analyses provide insights into the pollen fertility of synthetic allodiploid Brassica juncea hybrids.

KEY MESSAGE: Imbalanced chromosomes and cell cycle arrest, along with down-regulated genes in DNA damage repair and sperm cell differentiation, caused pollen abortion in synthetic allodiploid Brassica juncea hybrids. Interspecific hybridization is considered to be a major pathway for species formation and evolution in angiosperms, but the occurrence of pollen abortion in the hybrids is common, prompting us to recheck male gamete development in allodiploid hybrids after the initial combination of different genomes. Here, we investigated the several key meiotic and mitotic events during pollen development using the newly synthesised allodiploid B. juncea hybrids (AB, 2n = 2× = 18) as a model system. Our results demonstrated the partial synapsis and pairing of non-homologous chromosomes concurrent with chaotic spindle assembly, affected chromosome assortment and distribution during meiosis, which finally caused difference in genetic constitution amongst the final tetrads. The mitotic cell cycle arrest during microspore development resulted in the production of anucleate pollen cells. Transcription analysis showed that sets of key genes regulating cyclin (CYCA1;2 and CYCA2;3), DNA damage repair (DMC1, NBS1 and MMD1), and ubiquitin-proteasome pathway (SINAT4 and UBC) were largely downregulated at the early pollen meiosis stages, and those genes involved in sperm cell differentiation (DUO1, PIRL1, PIRL9 and LBD27) and pollen wall synthesis (PME48, VGDH11 and COBL10) were mostly repressed at the late pollen mitosis stages in the synthetic allodiploid B. juncea hybrids (AB). In conclusion, this study elucidated the related mechanisms affecting pollen fertility during male gametophyte development at the cytological and transcriptomic levels in the synthetic allodiploid B. juncea hybrids.

HDAC6 inhibition regulates substrate stiffness-mediated inflammation signaling in chondrocytes.

Osteoarthritis (OA) is a chronic disease and is difficult to cure. Chondrocytes are highly mechanosensitive. Therefore, mechanical therapies have received attention as a therapeutic direction for OA. The stiffness, as a critical cue of the extracellular matrix (ECM), affects cell growth, development, and death. In this study, we use polydimethylsiloxane (PDMS) to create substrates with varying stiffness for chondrocyte growth, interleukin-1ß (IL-1ß) treatment to mimic the inflammatory environment, and Tubastatin A (Tub A) to inhibit histone deacetylase 6 (HDAC6). Our results show that stiff substrates can be anti-inflammatory and provide a better matrix environment than soft substrates. Inhibition of HDAC6 improves the inflammatory environment caused by IL-1ß and coordinates with inflammation to spread the chondrocyte area and primary cilia elongation. Without IL-1ß and Tub A treatments, the length of the primary cilia rather than frequency is stiffness-dependent, and their length on stiff substrates are greater than that on soft substrates. In conclusion, we demonstrate that stiff substrates, inflammation, and inhibition of HDAC6 enhance the mechanosensitivity of primary cilia and mediate substrate stiffness to suppress inflammation and protect the matrix.

Prevention of sclerosis around cannulated screw after treatment of femoral neck fractures with bioceramic nails: a finite element analysis.

PURPOSE: Conventional cannulated screws (CS) are the main treatment method for femoral neck fractures (FNF). However, the rate of femoral head necrosis remains high after FNF treatment. The study aimed to compare the biomechanical features of different internal fixation materials for the treatment of Pauwel type III FNF to explore new strategies for clinical management. METHODS: A new material was prepared by applying casting, freeze drying and sintering process. The independently developed calcium magnesium silicate ceramic powder and hydrogel solution were evenly mixed to obtain a high-viscosity bio-ink, and a bioceramic nail (BN) with high mechanical strength and high fracture toughness was successfully prepared. Four internal fixations were developed to establish the Pauwel type III FNF and healed fracture finite element models: A, three CSs; B, three BNs; C, two BNs and one CS; D, one BN and two CSs. Von Mises stress and displacement of the implants and femur were observed. RESULTS: The measured Mg content in ceramic powder was 2.08 wt%. The spectral data confirmed that the ceramic powder has high crystallinity, which coincides with the wollastonite-2 M (PDF# 27-0088). The maximum von Mises stresses for the four models were concentrated in the lower part of the fracture surface, at 318.42 Mpa, 103.52 MPa, 121.16 MPa, and 144.06 MPa in models A, B, C, and D, respectively. Moreover, the maximum Von-mises stresses of the implants of the four models were concentrated near the fracture end at 243.65 MPa (A) and 58.02 MPa (B), 102.18 MPa (C), and 144.06 MPa (D). The maximum displacements of the four models were 5.36 mm (A), 3.41 mm (B), 3.60 mm (C), and 3.71 mm (D). The displacements of the three models with BNs were similar and smaller than that of the triple CS fracture model. In the fracture healing models with and without three CSs, the greatest stress concentration was scattered among the lowest screw tail, femoral calcar region, and lateral femur shaft. The displacement and stress distributions in both models are generally consistent. The stress distribution and displacement of the three healed femoral models with BNs were essentially identical to the healing models with three CSs. The maximum von Mises stresses were 65.94 MPa (B), 64.61 MPa (C), and 66.99 MPa (D) while the maximum displacements of the three healed femoral models were 2.49 mm (B), 2.56 mm (C), and 2.49 mm (D), respectively. CONCLUSIONS: Bioceramic nails offer greater advantages than conventional canulated screws after femoral neck fractures. However, the combination of bioceramic nails and CSs is more clinically realistic; replacing all internal fixations with bioceramic nails after the healing of femoral neck fractures can solve the problem of sclerosis formation around CSs and improve bone reconstruction by their bioactivity.

Integrative Transcriptome, miRNAs, Degradome, and Phytohormone Analysis of Brassica rapa L. in Response to Plasmodiophora brassicae.

Clubroot is an infectious root disease caused by Plasmodiophora brassicae in Brassica crops, which can cause immeasurable losses. We analyzed integrative transcriptome, small RNAs, degradome, and phytohormone comprehensively to explore the infection mechanism of P. brassicae. In this study, root samples of Brassica rapa resistant line material BrT24 (R-line) and susceptible line material Y510-9 (S-line) were collected at four different time points for cytological, transcriptome, miRNA, and degradome analyses. We found the critical period of disease resistance and infection were at 0-3 DAI (days after inoculation) and 9-20 DAI, respectively. Based on our finding, we further analyzed the data of 9 DAI vs. 20 DAI of S-line and predicted the key genes ARF8, NAC1, NAC4, TCP10, SPL14, REV, and AtHB, which were related to clubroot disease development and regulating disease resistance mechanisms. These genes are mainly related to auxin, cytokinin, jasmonic acid, and ethylene cycles. We proposed a regulatory model of plant hormones under the mRNA-miRNA regulation in the critical period of P. brassicae infection by using the present data of the integrative transcriptome, small RNAs, degradome, and phytohormone with our previously published results. Our integrative analysis provided new insights into the regulation relationship of miRNAs and plant hormones during the process of disease infection with P. brassicae.

bra-miR167a Targets ARF8 and Negatively Regulates Arabidopsis thaliana Immunity against Plasmodiophora brassicae.

Clubroot is a soil-borne disease caused by Plasmodiophora brassicae, which can seriously affect the growth and production of cruciferous crops, especially Chinese cabbage crops, worldwide. At present, few studies have been conducted on the molecular mechanism of this disease's resistance response. In this experiment, we analyzed the bioinformation of bra-miR167a, constructed a silencing vector (STTM167a) and an overexpression vector (OE-miR167a), and transformed them to Arabidopsis to confirm the role of miR167a in the clubroot resistance mechanism of Arabidopsis. Afterwards, phenotype analysis and expression level analysis of key genes were conducted on transgenic plants. From the result, we found that the length and number of lateral roots of silence transgenic Arabidopsis STTM167a was higher than that of WT and OE-miR167a. In addition, the STTM167a transgenic Arabidopsis induced up-regulation of disease resistance-related genes (PR1, PR5, MPK3, and MPK6) at 3 days after inoculation. On the other hand, the auxin pathway genes (TIR1, AFB2, and AFB3), which are involved in maintaining the balance of auxin/IAA and auxin response factor (ARF), were down-regulated. These results indicate that bra-miR167a is negative to the development of lateral roots and auxins, but positive to the expression of resistance-related genes. This also means that the STTM167a can improve the resistance of clubroot by promoting lateral root development and the level of auxin, and can induce resistance-related genes by regulating its target genes. We found a positive correlation between miR167a and clubroot disease, which is a new clue for the prevention and treatment of clubroot disease.

[Research progress of chondrocyte mechanotransduction mediated by TRPV4 and PIEZOs].

Mechanical signal transduction are crucial for chondrocyte in response to mechanical cues during the growth, development and osteoarthritis (OA) of articular cartilage. Extracellular matrix (ECM) turnover regulates the matrix mechanical microenvironment of chondrocytes. Thus, understanding the mechanotransduction mechanisms during chondrocyte sensing the matrix mechanical microenvironment can develop effective targeted therapy for OA. In recent decades, growing evidences are rapidly advancing our understanding of the mechanical force-dependent cartilage remodeling and injury responses mediated by TRPV4 and PIEZOs. In this review, we highlighted the mechanosensing mechanism mediated by TRPV4 and PIEZOs during chondrocytes sensing mechanical microenvironment of the ECM. Additionally, the latest progress in the regulation of OA by inflammatory signals mediated by TRPV4 and PIEZOs was also introduced. These recent insights provide the potential mechanotheraputic strategies to target these channels and prevent cartilage degeneration associated with OA. This review will shed light on the pathogenesis of articular cartilage, searching clinical targeted therapies, and designing cell-induced biomaterials.

Ti3 C2 TX MXene Modified with ZnTCPP with Bacteria Capturing Capability and Enhanced Visible Light Photocatalytic Antibacterial Activity.

Light-assisted antibacterial therapy is a promising alternative to antibiotic therapy due to the high antibacterial efficacy without bacterial resistance. Recent research has mainly focused on the use of near-infrared light irradiation to kill bacteria by taking advantage of the synergistic effects rendered by hyperthermia and radical oxygen species. However, photocatalytic antibacterial therapy excited by visible light is more convenient and practical, especially for wounds. Herein, a visible light responsive organic-inorganic hybrid of ZnTCPP/Ti3 C2 TX is designed and fabricated to treat bacterial infection with antibacterial efficiency of 99.86% and 99.92% within 10 min against Staphylococcus aureus and Escherichia coli, respectively. The porphyrin-metal complex, ZnTCPP, is assembled on the surface of Ti3 C2 TX MXene to capture bacteria electrostatically and the Schottky junction formed between Ti3 C2 TX and ZnTCPP promotes visible light utilization, accelerates charge separation, and enhances the mobility of photogenerated charges, and finally increases the photocatalytic activity. As a result of the excellent bacteria capturing ability and photocatalytic antibacterial effects, ZnTCPP/Ti3 C2 TX exposed to visible light has excellent antibacterial properties in vitro and in vivo. Therefore, organic-inorganic materials that have been demonstrated to possess good biocompatibility and enhance wound healing have large potential in bio-photocatalysis, antibacterial therapy, as well as antibiotics-free treatment of wounds.

Inactivation of Ihh in Sp7-Expressing Cells Inhibits Osteoblast Proliferation, Differentiation, and Bone Formation, Resulting in a Dwarfism Phenotype with Severe Skeletal Dysplasia in Mice.

Indian hedgehog (Ihh) is an indispensable paracrine factor for proper tissue patterning, skeletogenesis, and cellular proliferation. Recent genetic studies have revealed critical roles of chondrocyte-derived Ihh in regulating chondrocyte proliferation, hypertrophy and cartilage ossification. However, the functions of Sp7-expressing cell-derived Ihh in osteoblast differentiation and bone formation remain unclear. Sp7 is an essential transcription factor for osteoblast differentiation. In the current study, we generated Sp7-iCre; Ihhfl/fl mice, in which the Ihh gene was specifically deleted in Sp7-expressing cells to investigate the roles of Ihh. Ihh ablation in Sp7-expressing cells resulted in a dwarfism phenotype with severe skeletal dysplasia and lethality at birth, but with normal joint segmentation. Sp7-iCre; Ihhfl/fl mice had fewer osteoblasts, almost no cortical and trabecular bones, smaller skulls, and wider cranial sutures. Additionally, the levels of osteogenesis- and angiogenesis-related genes, and of major bone matrix protein genes were significantly reduced. These results demonstrated that Ihh regulates bone formation in Sp7-expressing cells. Ihh deficiency in primary osteoblasts cultured in vitro inhibited their proliferation, differentiation, and mineralization ability, and reduced the expression of osteogenesis-related genes. Moreover, the deletion of Ihh also attenuated the Bmp2/Smad/Runx2 pathway in E18.5 tibial and primary osteoblasts. The activity of primary osteoblasts in mutant mice was rescued after treatment with rhBMP2. In summary, our data revealed that Ihh in Sp7-expressing cells plays an indispensable role in osteoblast differentiation, mineralization, and embryonic osteogenesis, further implicated that its pro-osteogenic role may be mediated through the canonical Bmp2/Smad/Runx2 pathway.

BrWAX2 plays an essential role in cuticular wax biosynthesis in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

KEY MESSAGE: Map-based cloning was used to identify the BrWAX2 gene, which was involved in the cuticular wax biosynthesis. The malfunction of BrWAX2 together with other reduced expression of genes in alkane-forming pathway caused the glossy phenotype. Cuticular wax covering the outer plant surface plays various roles in protecting against biotic and abiotic stresses. Wax-less mutant shows glossy in stem and leaf surface and plays important roles in enriching Chinese cabbage germplasm resources for breeding brilliant green varieties. However, genes responsible for the glossy trait in Chinese cabbage are rarely reported. In this study, we identified a glossy Chinese cabbage line Y1211-1. Genetic analysis indicated that the glossy trait in Y1211-1 was controlled by a single recessive locus, BrWAX2 (Brassica rapa WAX 2). Using bulked segregant sequencing (BSA-Seq) and kompetitive allele-specific PCR (KASP) assays, BrWAX2 was fine-mapped to an interval of 100.78 kb. Functional annotation analysis, expression analysis, and sequence variation analysis revealed that Bra032670, homologous to CER1 in Arabidopsis, was the most likely candidate gene for BrWAX2. The gene Bra032670 was absent in glossy mutant. Cuticular wax composition analysis and RNA-Seq analysis suggested that the absence of BrWAX2 together with the decreased expression of other genes in alkane-forming pathway reduced the wax amount and caused the glossy phenotype. Furthermore, we developed and validated the functional marker BrWAX2-sp for BrWAX2. Overall, these results provide insight into the molecular mechanism underlying cuticular wax biosynthesis and reveal valuable information for marker-assisted selection (MAS) breeding in Chinese cabbage.

Improving the symptoms of post-traumatic osteoarthritis by α2-macroglobulin-rich serum.

PURPOSE: Altered joint loading by trauma induces joint degeneration, eventually leading to the generation of post-traumatic osteoarthritis (PTOA). Recent studies have shown that α2-macroglobulin (A2M) inhibits PTOA, induced by anterior cruciate ligament transection (ACLT), pathogenesis by regulating proinflammatory cytokines and matrix metalloproteinases. However, the application of A2M is limited due to high prices. Therefore, the aim of this study is to explore the novel preparation of A2M. MATERIALS AND METHODS: The early change of A2M in synovial fluid and serum was measured by ELISA. Ultra-filtered centrifugation was performed to prepare α2-macroglobulin-rich serum (A2MRS). The bioactivity of A2M in A2MRS was detected by improved Ellis and Gollas-Galvan method. The effects of A2MRS on PTOA were observed using immunohistochemistry, safranine O staining, micro X-ray, fluorescence molecular tomography etc. RESULTS: The concentration of A2M in PTOA group was significantly higher than that in Sham group in synovial fluid on the third day after ACLT in rat PTOA model. On the contrary, a significant downregulation of A2M levels in PTOA group was observed compared to the Sham group in serum at the seventh day after ACLT. Secondly, A2MRS was prepared successfully, and the concentration and bioactivity of A2M in A2MRS was significantly higher than that in serum. Lastly, A2MRS not only reduced notably the production of secondary cartilage ossification, type 10 collagen and matrix metalloproteinase 13, but also increased profoundly the generation of type 2 collagen, aggrecan, and chondrocytes' number. CONCLUSION: Our results indicate that A2MRS has protective effects on PTOA.

Comparison of femoral mechanics before and after internal fixation removal and the effect of sclerosis on femoral stress: a finite element analysis.

BACKGROUND: Femoral neck fractures are a common traumatic injury. The removal of the internal fixation remains controversial, especially in terms of mechanical stability. Moreover, collapsed necrosis of the femoral head continues to occur after fracture healing. We believe that sclerotic cancellous bone (SCB) formation around the screw is associated with femoral head necrosis. We aimed to compare mechanical features before and after implant removal and determine the effect of SCB formation on stress distribution. METHODS: Cylindrical cancellous bone sections were collected from a relatively normal region and an SCB region of a necrotic femoral head, and their elastic moduli were measured. Four femoral finite element models were developed: a) femoral neck fracture healing with implants, b) fracture healing without implants, c) sclerosis around the screw with implants, and d) sclerosis around the screw without implants. RESULTS: The maximum von Mises peak stresses of models a and b were 66.643 MPa and 63.76 MPa, respectively, and were concentrated in the upper lateral femur. The main stress was scattered at the lowest screw tail, femoral calcar region, and lateral femur shaft. Moreover, coronal plane strain throughout the screw paths near the femoral head in models a and b was mostly in the range of 1000-3000 µÎµ. The maximum stress concentrations in models c and d were located at the lower femoral head and reached 91.199 MPa and 78.019 MPa, respectively. CONCLUSIONS: The stresses in the sclerotic model around the cannulated screws are more concentrated on the femoral head than in the healing model without sclerotic bone. The overall stresses in the healing femoral neck fracture model were essentially unchanged before and after removal of the internal fixation.

GDF15 enhances proliferation of aged chondrocytes by phosphorylating SMAD2.

BACKGROUND: Aging is one of the primary factors influencing development of osteoarthritis, and the TGF-ß pathway plays an important role in age-related osteoarthritis. Specifically, GDF15 phosphorylates SMAD2/3 in the TGF-ß pathway to inhibit cardiomyocyte hypertrophy, and promote proliferation of chondrocytes. However, age-dependent changes in the level of GDF15 are unclear, as is whether GDF15 phosphorylates SMAD2/3 in the TGF-ß pathway to promote proliferation of old chondrocytes. This study, therefore, sought to examine the effect of various GDF15 concentrations on old chondrocyte proliferation. METHODS: Serum and cartilage specimens of young adults and older adults were collected, and GDF15 expression was quantified. Human chondrocytes were then cultured following routine protocols, and different concentrations of recombinant human GDF15 or pSMAD2 inhibitor were added into the culture medium. After 48 h of culturing, the proliferation of chondrocytes was detected by EdU, and the expression MMP13, SMAD2, and pSMAD2 was detected in chondrocytes via western blot and qRT-PCR analysis. RESULTS: The GDF15 content in serum and cartilage of young adults was higher than that of older adults (p < 0.05). The number of EdU-positive cells in the experimental group (containing recombinant human GDF15) was higher than that in the control group (medium only) (p < 0.05). Compared with the control group, chondrocytes in the experimental group showed increased pSMAD2 and type II collagen content (p < 0.05) and decreased MMP13 (p < 0.05), with no significant difference in SMAD2 content (p > 0.05). Moreover, no significant differences were observed between the control group and the TGF-ß signaling inhibitor group. The gene expression level of each index was consistent with the protein expression level. CONCLUSIONS: The GDF15 content of serum and cartilage in young adults is higher than in older adults, and GDF15 functions to promote the proliferation of chondrocytes by phosphorylating SMAD2 in older individuals.