Dissecting the genetic basis of heterosis in elite super-hybrid rice.

Y900 is one of the top hybrid rice (Oryza sativa) varieties, with its yield exceeding 15 t·hm-2. To dissect the mechanism of heterosis, we sequenced the male parent line R900 and female parent line Y58S using long-read and Hi-C technology. High-quality reference genomes of 396.41 Mb and 398.24 Mb were obtained for R900 and Y58S, respectively. Genome-wide variations between the parents were systematically identified, including 1,367,758 single-nucleotide polymorphisms, 299,149 insertions/deletions, and 4,757 structural variations. The level of variation between Y58S and R900 was the lowest among the comparisons of Y58S with other rice genomes. More than 75% of genes exhibited variation between the two parents. Compared with other two-line hybrids sharing the same female parent, the portion of Geng/japonica (GJ)-type genetic components from different male parents increased with yield increasing in their corresponding hybrids. Transcriptome analysis revealed that the partial dominance effect was the main genetic effect that constituted the heterosis of Y900. In the hybrid, both alleles from the two parents were expressed, and their expression patterns were dynamically regulated in different tissues. The cis-regulation was dominant for young panicle tissues, while trans-regulation was more common in leaf tissues. Overdominance was surprisingly prevalent in stems and more likely regulated by the trans-regulation mechanism. Additionally, R900 contained many excellent GJ haplotypes, such as NARROW LEAF1, Oryza sativa SQUAMOSA PROMOTER BINDING PROTEIN-LIKE13, and Grain number, plant height, and heading date8, making it a good complement to Y58S. The fine-tuned mechanism of heterosis involves genome-wide variation, GJ introgression, key functional genes, and dynamic gene/allele expression and regulation pattern changes in different tissues and growth stages.

ERK1 loss accelerates the progression of osteoarthritis in aged mice via NRF2/BACH1 signaling.

Osteoarthritis (OA) is now a common degenerative joint related disease. However, the clinical efficacy of drugs associated with cartilage regeneration remains limited. In our study, we firstly explored the role of ERK1 in the progression of OA. We clarified that ERK1-deficient mice were susceptible to age-related OA. The higher OARSI scores and more severe cartilage degeneration was observed in the ERK1-deficient mice. ERK1 deficiency decreased the nuclear transportation of Nrf2 in the chondrocytes and accelerated chondrocyte aging in vitro. Moreover, chondrocytes with ERK1 deficiency elevated the nuclear expression of BACH1, resulting in lowered expression of antioxidant enzymes in ERK1-deficient chondrocytes. The Nrf2 activator dimethyl fumarate (DMF) was used. Our experiments demonstrated the protective function of DMF against OA in ERK1 knockout mice. Above all, we confirmed the effects of ERK1 on the progression of OA and clarified the mechanisms underlying these effects. DMF might has significant use in the development of novel drugs for the therapy of OA in the future.

ML264 inhibits osteosarcoma growth and metastasis via inhibition of JAK2/STAT3 and WNT/ß-catenin signalling pathways.

Osteosarcoma, the most common bone malignancy, has a high morbidity rate and poor prognosis. Krüppel-like factor 5 (KLF5) is a key transcriptional regulator of cellular proliferation whose overexpression is observed in osteosarcoma cell lines (U2OS, 143B, MG63 and SAOS2). ML264, a small-molecule inhibitor of KLF5, exerts antiproliferative effects in colorectal cancer; however, its function in osteosarcoma remains unknown. Here, we explored the possible antitumour effects of ML264 on 143B and U2OS cell lines and murine tumour xenograft model. ML264 suppressed proliferation and clonogenic ability of osteosarcoma cells in a dose-dependent manner. Moreover, ML264 induced G0/G1 cell cycle arrest, with no influence on apoptosis, and inhibited the migratory and invasive abilities of osteosarcoma cells, as demonstrated by wound-healing and Transwell assays. Exposure to ML264 reduced the mRNA and protein levels of molecules associated with epithelial-mesenchymal transition phenotype, including N-cadherin, vimentin, Snail, matrix metalloproteinase (MMP) 9 and MMP13. Inhibition of signal transducer and activator of transcription (STAT) 3 phosphorylation and Wnt signalling was also observed. In the murine model of osteosarcoma, tumour growth was efficiently suppressed following a 10-day treatment with ML264. Collectively, our findings demonstrate the potential value of ML264 as a novel anticancer drug for osteosarcoma.

Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling.

The endogenous metabolite itaconate has emerged as a regulator of macrophage function that limits inflammation. However, its effect on cell differentiation and osteoclast-related diseases is unclear. Here, for the first time, we explored the effect of itaconate and its cell-permeable itaconate derivative, 4-octyl itaconate (OI) on osteoclast differentiation in vitro and in vivo. Firstly, we demonstrated that itaconate concentration was lower in estrogen-deficient mice. OI released itaconate and induced the expression of nuclear factor-erythroid 2-related factor 2 (Nrf2) in bone marrow-derived macrophages during osteoclastogenesis. Furthermore, OI significantly suppressed the early, middle, and late stages of osteoclastogenesis induced by receptor activator of NF-κB ligand in vitro, as confirmed by tartrate-resistant acid phosphatase staining. Moreover, it significantly inhibited fibrous actin ring formation and bone resorption in vitro. Mechanistically, we observed that OI enhanced Nrf2 expression by suppressing its association with ubiquitin via inhibition of the E3 ubiquitin ligase (Hrd1). OI also inhibited LPS-induced the reactive oxygen species and inflammatory responses via Hrd1. An estrogen deficiency (via ovariectomy)-induced osteoporosis model was also established. Here, on micro-computed tomography and histologic analysis showed that OI effectively suppressed ovariectomy-induced bone loss. In summary, OI, an itaconate derivative, can inhibit osteoclastogenesis in vitro and in vivo, indicating that OI could be a potential drug to treat osteoclast-related diseases; our results also link itaconate to the development of osteoporosis.-Sun, X., Zhang, B., Pan, X., Huang, H., Xie, Z., Ma, Y., Hu, B., Wang, J., Chen, Z., Shi, P. Octyl itaconate inhibits osteoclastogenesis by suppressing Hrd1 and activating Nrf2 signaling.

Tomatidine suppresses osteoclastogenesis and mitigates estrogen deficiency-induced bone mass loss by modulating TRAF6-mediated signaling.

Postmenopausal osteoporosis is initiated by estrogen withdrawal and is characterized mainly by overactivated osteoclastic bone resorption. Targeting TNF receptor-associated factor 6 (TRAF6) or its downstream signaling pathways to modulate osteoclast formation and function is an appealing strategy for osteoclast-related disorders. In the present study, we determined the effect of tomatidine, a steroidal alkaloid derived from Solanaceae, on the formation and function of receptor activator of NF-κB (RANK) ligand-induced osteoclasts and the underlying mechanism. Tomatidine inhibited osteoclast formation in a dose-dependent manner and decreased the expression of osteoclast marker genes. Actin ring formation and osteoclastic bone resorption were attenuated in the presence of tomatidine in vitro. Eight weeks after ovariectomy, tomatidine prevented estrogen deficiency-induced bone loss and restored the mechanical properties of the femur. At the molecular level, tomatidine abrogated phosphorylation of c-Jun N-terminal kinase (JNK)/p38, NF-κB, and protein kinase B (Akt) pathway proteins by suppressing RANK expression, inhibiting the binding of TRAF6 to RANK, and downregulating the osteoclastogenesis marker-related protein expression. In summary, these data demonstrated that tomatidine attenuated osteoclast formation and function by modulating multiple TRAF6-mediated pathways. Therefore, tomatidine could be a novel candidate for the treatment of osteoclast-related disorders, including osteoporosis.-Hu, B., Sun, X., Yang, Y., Ying, Z., Meng, J., Zhou, C., Jiang, G., Li, S., Wu, F., Zhao, X., Zhu, H., Wu, H., Cai, X., Shi, Z., Yan, S. Tomatidine suppresses osteoclastogenesis and mitigates estrogen deficiency-induced bone mass loss by modulating TRAF6-mediated signaling.

Glabridin inhibits osteosarcoma migration and invasion via blocking the p38- and JNK-mediated CREB-AP1 complexes formation.

Osteosarcoma is the most common bone malignancy, and it seriously affects the quality of life of affected children and adolescents. Glabridin (GLA), a major component of licorice root extract, has been reported to exert antitumor effects against a variety of tumor types; however, its effects on osteosarcoma have not been elucidated. In the current study, we investigate the effects and potential antimetastatic mechanisms of GLA on osteosarcoma in vitro and in vivo. Flow cytometry showed that GLA induced G2/M cell cycle phase arrest and promoted cell apoptosis. Transwell and wound-healing assays showed that GLA significantly decreased the migration and invasion of osteosarcoma cells. Further western blotting and quantitative real-time polymerase chain reaction showed that the expression of matrix metalloproteinase (MMP)-2 and MMP-9 in MG63 and HOS cells were reduced after GLA treatment. Moreover, western blotting demonstrated that GLA downregulated the phosphorylation of p38 mitogen-activated protein kinases and c-Jun N-terminal kinase. A coimmunoprecipitation assay illustrated that formation of cAMP response element-binding protein (CREB)-activating protein 1 (AP1) complexes and the DNA binding activities of CREB and AP1 in MG63 and HOS cells were impaired following treatment with GLA. Finally, GLA inhibited tumor growth and suppressed osteosarcoma cell metastasis in vivo. Overall, our findings highlight the potential of GLA as a therapeutic agent for the prevention and treatment of tumor metastasis.

TGF-ß inhibits osteogenesis by upregulating the expression of ubiquitin ligase SMURF1 via MAPK-ERK signaling.

High incidence of osteoporotic fractures emphasizes the necessity of developing effective measures to promote osteogenesis. In our study, we investigated a possible role of MAPK-ERK signaling in the TGF-ß-mediated osteoblastic differentiation. Our results indicated that TGF-ß activated the MAPK-ERK pathway and inhibited osteogenesis in mesenchymal pluripotent cell line, C3H10T1/2, and preosteoblastic cell line, MC3T3 cells. And the downregulation of MAPK-ERK signaling using pharmacological inhibitor U0126 and RNA interference rescued osteoblast differentiation suppressed by TGF-ß, which was confirmed by Alkaline phosphatase (ALP) staining and alizarrn red staining, and the enhanced expression of osteogenesic markers. Western blotting analysis indicated that TGF-ß induced protein expression of E3 ubiquitin-protein ligase SMURF1, which contributed to the degradation of RUNX2 and SMAD1 as evidenced by SMURF1 inhibition using RNA interference and proteasome inhibitor MG132. Moreover, we observed that the expression of SMURF1 was decreased, while that of SMAD1 and RUNX2 increased by MAPK-ERK inhibitor U0126 in TGF-ß-treated differentiating preosteoblasts, suggesting that MAPK-ERK regulated the transcription of osteogenesis-related genes. Furthermore, a synergistic effect between U0126 and bone morphogenic protein (BMP)-2 on osteoblast differentiation and bone formation was observed both in cell cultures and experimental animals. In conclusion, our results revealed that TGF-ß inhibited osteoblastic differentiation by inducing the MAPK-ERK pathway which upregulated the expression of ubiquitin ligase SMURF1 and resulted in reduced presence of osteogenic proteins. In addition, the potentiation of BMP-2 on osteogenic activity by ERK1/2 inhibitor U0126 suggests that it may have potential clinical utility for promoting osteogenesis in bone fracture repair.

TGF-ß synergizes with ML264 to block IL-1ß-induced matrix degradation mediated by Krüppel-like factor 5 in the nucleus pulposus.

Intervertebral disc degeneration causes low back pain.Interleukin-1ß (IL-1ß) is a well-known inflammatory mediator that is involved in disc degeneration but its molecular mechanisms on catabolic and anabolic events in nucleus pulposus (NP) cells remain unclear. Krüppel-like factor 5 (KLF5) is associated with inflammation and was previously shown to cause cartilage degradation. In this study, we revealed that KLF5 is involved in IL-1ß activated NF-kB cascade by enhancing both p65 phosphorylation and p65 acetylation. Moreover, the catabolic effect of KLF5 can be abolished by transforming growth factor-ß (TGF-ß) via promoting the proteasomal degradation of KLF5. Therefore, a KLF5 inhibitor ML264 was further proved to synergize with TGF-ß to attenuate IL-1ß-induced intervertebral disc degeneration. These results indicate the critical role of KLF5 in regulating intervertebral disc metabolism and suggest KLF5 inhibitor such as ML264 as potential compound for treatment of degenerative disc disease.

Role of the extracellular signal-regulated kinase 1/2 signaling pathway in the process of thrombin-promoting airway remodeling in ovalbumin-allergic rats.

CONTEXT: Although it is recognized that thrombin plays a key role in airway remodeling during chronic asthma. In a previous study, we have proved that thrombin promotes airway remodeling via PAR-1 in OVA-allergic rats, but little is known about intracellular signaling pathway involved in the event. OBJECTIVE: In this study, we intend to explore the impact of pERK1/2 signaling pathway on the process of thrombin-induced airway remodeling in OVA-allergic rats. MATERIALS AND METHODS: A rat model of chronic asthma was set up by systemic sensitization and repeated challenge to OVA. The doses of thrombin, recombinant hirudin, PAR-1 inhibitor ER-112780-06, and pERK1/2 inhibitor PD98059 varied for different groups. The expression of pERK1/2 was analyzed by western blot and RT-PCR. Secretion of TGF-ß1 and IL-6 was detected by ELISA. RESULTS: The expression of pERK1/2 was higher in the airway of asthmatic rats than those of normal rats, and was significantly increased by thrombin treatment but decreased by thrombin-inhibitor treatment. Airway remodeling was enhanced by thrombin but weakened by pERK1/2 inhibitor. Expression of growth factors and IL-6 in asthmatic rats was significantly increased by thrombin treatment and decreased by thrombin-inhibitor treatment and pERK1/2 inhibitor treatment. CONCLUSION: These results suggest that ERK1/2 signaling pathway may play an important role in the process of thrombin-promoting airway remodeling in OVA-allergic rats, and pERK1/2 inhibitor effectively inhibits the process.

Multi-year crop rotation and quicklime application promote stable peanut yield and high nutrient-use efficiency by regulating soil nutrient availability and bacterial/fungal community.

Diversifying cultivation management, including different crop rotation patterns and soil amendment, are effective strategies for alleviating the obstacles of continuous cropping in peanut (Arachis hypogaea L.). However, the peanut yield enhancement effect and temporal changes in soil chemical properties and microbial activities in response to differential multi-year crop rotation patterns and soil amendment remain unclear. In the present study, a multi-year localization experiment with the consecutive application of five different cultivation managements (including rotation with different crops under the presence or absence of external quicklime as soil amendment) was conducted to investigate the dynamic changes in peanut nutrient uptake and yield status, soil chemical property, microbial community composition and function. Peanut continuous cropping led to a reduction in peanut yield, while green manure-peanut rotation and wheat-maize-peanut rotation increased peanut yield by 40.59 and 81.95%, respectively. A combination of quicklime application increased yield by a further 28.76 and 24.34%. Alterations in cultivation management also strongly affected the soil pH, nutrient content, and composition and function of the microbial community. The fungal community was more sensitive than the bacterial community to cultivation pattern shift. Variation in bacterial community was mainly attributed to soil organic carbon, pH and calcium content, while variation in fungal community was more closely related to soil phosphorus content. Wheat-maize-peanut rotation combined with quicklime application effectively modifies the soil acidification environment, improves the soil fertility, reshapes the composition of beneficial and harmful microbial communities, thereby improving soil health, promoting peanut development, and alleviating peanut continuous cropping obstacles. We concluded that wheat-maize-peanut rotation in combination with quicklime application was the effective practice to improve the soil fertility and change the composition of potentially beneficial and pathogenic microbial communities in the soil, which is strongly beneficial for building a healthy soil micro-ecology, promoting the growth and development of peanut, and reducing the harm caused by continuous cropping obstacles to peanut.

Urolithin B reduces cartilage degeneration and alleviates osteoarthritis by inhibiting inflammation.

Osteoarthritis is the most prevalent degenerative joint disease reported worldwide. Conventional treatment strategies mainly focus on medication and involve surgical joint replacement. The use of these therapies is limited by gastrointestinal complications and the lifespan of joint prostheses. Hence, safe and efficacious drugs are urgently needed to impede the osteoarthritis progression. Urolithin B, a metabolite of ellagic acid in the gut, exhibits anti-inflammatory and antioxidant properties; however, its role in osteoarthritis remains unclear. In this study, we demonstrated that urolithin B efficiently inhibits the inflammatory factor-induced production of matrix metalloproteinases (MMP3 and MMP13) in vitro and upregulates the expression of type II collagen and aggrecan. Urolithin B alleviates cartilage erosion and osteophyte formation induced by anterior cruciate ligament transections. Moreover, urolithin B inhibits the activation of the NF-κB pathway by reducing the phosphorylation of Iκb-α and the nuclear translocation of P65. In summary, urolithin B significantly inhibits inflammation and alleviates osteoarthritis. Hence, urolithin B can be considered a potential agent suitable for the effective treatment of osteoarthritis in the future.

Restrained Mitf-associated autophagy by Mulberroside A ameliorates osteoclastogenesis and counteracts OVX-Induced osteoporosis in mice.

Bone and mineral metabolism homeostasis accounts for the maintenance of normal skeletal remodeling. However, with aging and changes in hormone levels, over-activated osteoclasts disrupt homeostasis, induce osteoporosis, and even cause osteoporotic fractures, leading to an enormous economic burden. Despite the rapid development of pharmacological therapy for osteoporosis, safer and more effective treatments remain to be explored. Here, we demonstrate that Mulberroside A (Mul-A), a natural component extracted from mulberry bark and branches, effectively suppresses osteoclastogenesis in vitro and counteracts bone loss caused by ovariectomy (OVX). The mechanism underlying this effect involves the repression of autophagic flux during osteoclastogenesis by Mul-A, which can be attributed to the restrained expression of microphthalmia-related transcription factor (Mitf) and its nuclear translocation. Importantly, Mitf overexpression partially reverses the inhibitory effects of Mul-A on autophagy and osteoclastogenesis. Moreover, applying two autophagy agonizts, rapamycin and Torin 1, attenuates the osteoclastogenic regulatory role of Mul-A. Collectively, our study demonstrates that Mul-A damages osteoclast differentiation and ameliorates osteoporosis caused by estrogen deficiency by modulation of Mitf-associated autophagy, indicating its therapeutic potential against osteoporosis.

[Arthroscopic assistance of latissimus dorsi tendon transposition for the treatment of unrepairable rotator cuff tear].

OBJECTIVE: To explore clinical effect of arthroscopy-assisted rotator cuff tendon transfer in treating irreparable rotator cuff tears (IRCT). METHODS: From May 2015 to May 2018, 23 patients with unrepairable rotator cuff tears were treated with arthroscopy-assisted rotator cuff tendon transfer, and 21 patients were followed up finally, including 8 males and 13 females, aged from 48 to 82 years old with an average of(64.3±9.1) years old;the courses of disease ranged from 6 to 36 months with an average of (14.0±6.4) months. American Rotator and Elbow Surgeons Score(ASES) and Constant-Murley score were used to evaluate clinical efficacy before surgery and at the latest follow-up. RESULTS: All 21 patients were followed up for 36 to 54 months with an average of (39.4±4.4) months. Axillary incision of 1 patient was redness, swelling and exudation after surgery, which healed after 3 weeks of dressing change, and exudate culture was negative. At the latest follow-up, MRI showed partial tearing of the metastatic tendon in 2 patients, but pain and movement of the affected shoulder were still better than before surgery. ASES increased from preoperative (41.0±9.6) scores to the latest follow-up (75.6±14.0) scores, and had statistical difference (t=10.50, P<0.01). Constant-Murley score increased from (49.8±7.1) scores before operation to (67.5±11.6) scores at the latest follow-up (t=11.27, P<0.01). CONCLUSION: Arthroscopic assisted latissimus dorsalis tendon transposition restores physiological and anatomical structure of glenohumeral joint by reconstructing balance of horizontal and vertical couples of shoulder joint, thus achieving the stability of the shoulder joint, relieving shoulder pain and improving shoulder joint function.

Metabolite asymmetric dimethylarginine (ADMA) functions as a destabilization enhancer of SOX9 mediated by DDAH1 in osteoarthritis.

Osteoarthritis (OA) is a degenerative disease with a series of metabolic changes accompanied by many altered enzymes. Here, we report that the down-regulated dimethylarginine dimethylaminohydrolase-1 (DDAH1) is accompanied by increased asymmetric dimethylarginine (ADMA) in degenerated chondrocytes and in OA samples. Global or chondrocyte-conditional knockout of ADMA hydrolase DDAH1 accelerated OA development in mice. ADMA induces the degeneration and senescence of chondrocytes and reduces the extracellular matrix deposition, thereby accelerating OA progression. ADMA simultaneously binds to SOX9 and its deubiquitinating enzyme USP7, blocking the deubiquitination effects of USP7 on SOX9 and therefore leads to SOX9 degradation. The ADMA level in synovial fluids of patients with OA is increased and has predictive value for OA diagnosis with good sensitivity and specificity. Therefore, activating DDAH1 to reduce ADMA level might be a potential therapeutic strategy for OA treatment.

USP1 inhibition suppresses the progression of osteosarcoma via destabilizing TAZ.

Mutations and altered expression of deubiquitinating enzymes (DUBs) profoundly influence tumor progression. Ubiquitin-specific protease 1 (USP1) is a well-characterized human DUB reportedly overexpressed in and associated with maintaining the mesenchymal stem cell status of osteosarcoma (OS); however, the potential mechanisms of USP1 in OS remain poorly understood. In this study, we identified that USP1 directly interacts with Transcriptional Co-Activator With PDZ-Binding Motif (TAZ) in OS cell lines, and with mechanistic analysis indicating that the anti-OS effects of USP1 inhibition could be partially attributed to TAZ instability, with its reduced nuclear accumulation responsible for a subsequent decrease in the expression of downstream genes associated with the Hippo signaling pathway. Moreover, pharmacological inhibition USP1 by ML323 presented the similar effects on Hippo signaling pathway and suppressed OS growth and metastasis both in vitro and in vivo. Taken together, our results revealed a novel molecular mechanism underlying the function of USP1 in OS and a potential role of ML323 as a therapeutic strategy for the clinical treatment of OS.

DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling.

Osteoporosis-related fractures, such as femoral neck and vertebral fractures, are common in aged people, resulting in increased disability rate and health-care costs. Thus, it is of great importance to clarify the mechanism of osteoclast-related osteoporosis and find effective ways to avoid its complication. In this study, gene expression profile analysis and real-time polymerase chain reaction revealed that DUSP6 expression was suppressed in human and mice osteoporosis cases. In vitro experiments confirmed that DUSP6 overexpression prevented osteoclastogenesis, whereas inhibition of DUSP6 by small interference RNA or with a chemical inhibitor, (E/Z)-BCI, had the opposite effect. (E/Z)-BCl significantly accelerated the bone loss process in vivo by enhancing osteoclastogenesis. Bioinformatics analyses and in vitro experiments indicated that miR-181a was an upstream regulator of DUSP6. Moreover, miR-181a positively induced the differentiation and negatively regulated the apoptosis of osteoclasts via DUSP6. Furthermore, downstream signals by ERK2 and SMAD2 were also found to be involved in this process. Evaluation of ERK2-deficiency bone marrow-derived macrophages confirmed the role of ERK2 signaling in the DUSP6-mediated osteoclastogenesis. Additionally, immunoprecipitation assays confirmed that DUSP6 directly modified the phosphorylation status of SMAD2 and the subsequent nuclear transportation of NFATC1 to regulate osteoclast differentiation. Altogether, this study demonstrated for the first time the role of miRNA-181a/DUSP6 in the progression of osteoporosis via the ERK2 and SMAD2 signaling pathway. Hence, DUSP6 may represent a novel target for the treatment of osteoclast-related diseases in the future.

Senescence-Specific Expression of RAmy1A Accelerates Non-structural Carbohydrate Remobilization and Grain Filling in Rice (Oryza sativa L.).

Remobilization of pre-anthesis NSCs (non-structural carbohydrates) is significant for effective grain filling in rice (Oryza sativa L.). However, abundant starch particles as an important component of NSCs are still present in the leaf sheath and stem at the late stage of grain filling. There are no studies on how bioengineering techniques can be used to improve the efficiency of NSC remobilization. In this study, RAmy1A was expressed under the senescence-specific promoter of SAG12, which was designed to degrade starch in the leaf sheath and stem during grain filling. RAmy1A mRNA successfully accumulated in the leaf, stem, and sheath of transgenic plants after anthesis. At the same time, the starch and total soluble sugar content in the leaf, stem, and leaf sheath were obviously decreased during the grain-filling period. The photosynthetic rate of transgenic lines was higher than that of the wild types by an average of 4.0 and 9.9%, at 5 and 10 days after flowering, respectively. In addition, the grain-filling rate of transgenic lines was faster than that of the wild types by an average of 26.09%. These results indicate an enhanced transport efficiency of NSCs from source tissues in transgenic rice. Transgenic rice also displayed accelerated leaf senescence, which was hypothesized to contribute to decreased grain weight.

Oxidative Stress-Induced Hypermethylation of KLF5 Promoter Mediated by DNMT3B Impairs Osteogenesis by Diminishing the Interaction with ß-Catenin.

Aims: Emerging evidence suggests that the pathogenesis of osteoporosis, characterized by impaired osteogenesis, is shifting from estrogen centric to oxidative stress. Our previous studies have shown that the zinc-finger transcription factor krüppel-like factor 5 (KLF5) plays a key role in the degeneration of nucleus pulposus and cartilage. However, its role in osteoporosis remains unknown. We aimed to investigate the effect and mechanism of KLF5 on osteogenesis under oxidative stress. Results: First, KLF5 was required for osteogenesis and stimulated osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). KLF5 was hypermethylated and downregulated in ovariectomy-induced osteoporosis mice and in BMSCs treated with H2O2. Interestingly, DNA methyltransferases 3B (DNMT3B) upregulation mediated the hypermethylation of KLF5 induced by oxidative stress, thereby impairing osteogenic differentiation. The inhibition of KLF5 hypermethylation using DNMT3B siRNA or 5-AZA-2-deoxycytidine (5-AZA) protected osteogenic differentiation of BMSCs from oxidative stress. Regarding the downstream mechanism, KLF5 induced ß-catenin expression. More importantly, KLF5 promoted the nuclear translocation of ß-catenin, which was mediated by the armadillo repeat region of ß-catenin. Consistently, oxidative stress-induced KLF5 hypermethylation inhibited osteogenic differentiation by reducing the expression and nuclear translocation of ß-catenin. Innovation: We describe the novel effect and mechanism of KLF5 on osteogenesis under oxidative stress, which is linked to osteoporosis for the first time. Conclusion: Our results suggested that oxidative stress-induced hypermethylation of KLF5 mediated by DNMT3B impairs osteogenesis by diminishing the interaction with ß-catenin, which is likely to contribute to osteoporosis. Targeting the hypermethylation of KLF5 might be a new strategy for the treatment of osteoporosis. Antioxid. Redox Signal. 35, 1-20.

Cyclic Digestion and Ligation-Mediated PCR Used for Flanking Sequence Walking.

Ligation-mediated PCR (LM-PCR) is a classical method for isolating flanking sequences; however, it has a common limitation of reduced success rate owing to the circularization or multimerization of target restriction fragments including the known sequence. To address this limitation, we developed a novel LM-PCR method, termed Cyclic Digestion and Ligation-Mediated PCR (CDL-PCR). The novelty of this approach involves the design of new adapters that cannot be digested after being ligated with the restriction fragment, and cyclic digestion and ligation may be manipulated to block the circularization or multimerization of the target restriction fragments. Moreover, to improve the generality and flexibility of CDL-PCR, an adapter precursor sequence was designed, which could be digested to prepare 12 different adapters at low cost. Using this method, the flanking sequences of T-DNA insertions were obtained from transgenic rice and Arabidopsis thaliana. The experimental results demonstrated that CDL-PCR is an efficient and flexible method for identifying the flanking sequences in transgenic rice and Arabidopsis thaliana.