Boosting genome editing in plants with single transcript unit surrogate reporter systems.

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of editing events. In this study, we introduce multiple single transcript unit surrogate reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems use the same single guide RNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are used to restore functional reporter genes after genome editing, including efficient single-strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-base editor systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are shown to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.

Iron Oxide Nanoparticles Engineered Macrophage-Derived Exosomes for Targeted Pathological Angiogenesis Therapy.

Engineering exosomes with nanomaterials usually leads to the damage of exosomal membrane and bioactive molecules. Here, pathological angiogenesis targeting exosomes with magnetic imaging, ferroptosis inducing, and immunotherapeutic properties is fabricated using a simple coincubation method with macrophages being the bioreactor. Extremely small iron oxide nanoparticle (ESIONPs) incorporated exosomes (ESIONPs@EXO) are acquired by sorting the secreted exosomes from M1-polarized macrophages induced by ESIONPs. ESIONPs@EXO suppress pathological angiogenesis in vitro and in vivo without toxicity. Furthermore, ESIONPs@EXO target pathological angiogenesis and exhibit an excellent T1-weighted contrast property for magnetic resonance imaging. Mechanistically, ESIONPs@EXO induce ferroptosis and exhibit immunotherapeutic ability toward pathological angiogenesis. These findings demonstrate that a pure biological method engineered ESIONPs@EXO using macrophages shows potential for targeted pathological angiogenesis therapy.

Copper oxide nanoparticles suppress retinal angiogenesis via inducing endothelial cell cuproptosis.

Background: Copper oxide nanoparticles (CuO NPs) exhibit antitumor activity; however, their potential as an antiangiogenesis agent is unknown. Materials & methods: The antiangiogenesis properties of CuO NPs were evaluated in vitro and in vivo and the underlying mechanism was examined using RNA sequencing and metabolomic analyses. Results: CuO NPs inhibited endothelial cell function in vitro. They also mitigated retinal vasculature development and alleviated pathological retinal angiogenesis in vivo. RNA sequencing and metabolomic analyses revealed that CuO NPs disrupt the tricarboxylic acid cycle and induce cuproptosis, which was further supported by evaluating cuproptosis-related metabolites and proteins. Conclusion: CuO NPs may be an effective antiangiogenic agent for the treatment of retinal angiogenesis.

Introducing gradient Er ions and oxygen defects into SrCoO3 for regulating structural, electrical and magnetic transport properties.

The SrCoO3-δ system has broad application potential due to its diverse crystal structures, oxidation stoichiometric ratio, and significant electrical and magnetic properties. However, it faces the challenges of a complex crystal structure and oxygen defect control in this material system. Herein, we introduce oxygen defects into SrCoO3-δvia Er doping to regulate the structural, electrical and magnetic transport properties. Sr1-xErxCoO3-δ (x = 0-0.25) undergoes an evolution of structure and oxygen content (measured using the iodometric method) from hexagonal SrCoO2.626 (H + Co3O4) to cubic perovskite Sr0.9Er0.1CoO2.689 (CP) and finally to ordered tetragonal Sr0.8Er0.2CoO2.635 (OT). Among the three phases, Sr0.9Er0.1CoO2.689 (CP) exhibits the lowest resistivity, only 4.06 mΩ cm at room temperature, which is attributed to its high three-dimensional symmetry, overlap of O 2p and Co 3d orbitals at high oxygen ion concentration. Further introduction of Er ions and oxygen defects promotes the transformation from low spin Co4+ (LS, t52ge0g, S = 1/2) to high spin Co3+ (HS, t42ge2g, S = 2), and from the CoO6 octahedron (low magnetic moment transformation) to the CoO4.25 tetrahedron (high magnetic moment). The oxygen-deficient CoO4.25 layer appears, which can enhance the ordering of A sites and oxygen vacancies, and the CP phase transforms into room-temperature ferromagnetic Sr0.8Er0.2CoO2.635 (OT, TC∼330 K). Er ions provide unpaired electrons in the 2f orbital, which results in a strong magnetization of Sr0.8Er0.2CoO2.635 (OT, 4.66 µB/Co) at low temperatures.

Aurora kinase B disruption suppresses pathological retinal angiogenesis by affecting cell cycle progression.

PURPOSE: The detrimental effects of pathological angiogenesis on the visual function are indisputable. Within a prominent role in chromosome segregation and tumor progression, aurora kinase B (AURKB) assumes a prominent role. However, its role in pathological retinal angiogenesis remains unclear. This study explores this latent mechanism. METHODS: To inhibit AURKB expression, we designed specific small interfering RNAs targeting AURKB and transfected them into vascular endothelial cells. Barasertib was selected as the AURKB inhibitor. The anti-angiogenic effects of both AURKB siRNA and barasertib were assessed in vitro by cell proliferation, transwell migration, and tube formation. To evaluate the angiogentic effects of AURKB in vivo, neonatal mice were exposed to 75% oxygen followed by normoxic repositioning to establish an oxygen-induced retinopathy (OIR) model. Subsequently, phosphate-buffered saline and barasertib were administered into OIR mice via intravitreal injection. The effects of AURKB on cell cycle proteins were determined by western blot analysis. RESULTS: We found that AURKB was overexpressed during pathological angiogenesis. AURKB siRNA and barasertib significantly inhibited endothelial cell proliferation, migration, and tube formation in vitro. Furthermore, AURKB inhibition attenuated retinal angiogenesis in the OIR model. A possible mechanism is the disruption of cell cycle by AURKB inhibition. CONCLUSION: In conclusion, AURKB significantly influenced pathological retinal angiogenesis, thereby presenting a promising therapeutic target in ocular neovascular diseases.

Thermal and electrical transport properties of two-dimensional Dirac graphenylene: a first-principles study.

The development of high performance two-dimensional thermoelectric (TE) materials is crucial for enhancing the conversion of waste heat into electricity and for achieving the transition to new energy. In recent years, two-dimensional Dirac materials with high carrier mobility and non-trivial topological properties have been expected to extend the application of carbon-based materials in the TE field. However, research on the TE properties of two-dimensional Dirac materials is still scarce, and the relevant physical mechanisms that affect the TE figure of merit of the materials are still unclear. Therefore, we carefully selected a typical and experimentally synthesized Dirac structure, graphenylene, and systematically studied its thermal transport and electrical transport properties using density functional theory (DFT) and Boltzmann transport theory. The results show that the ZT value of graphenylene exhibits an extremely significant anisotropy. There is a significant discrepancy in the figure of merit (ZT) values of n-type and p-type systems at the optimum doping concentration, i.e., the ZT value of the n-type system (0.49) is one order of magnitude greater than that of the p-type system (0.06). Graphenylene exhibits excellent electronic performance due to its unique electronic band structure and has an extremely high conductivity (for the n-type system, electrical conductivity at room temperature is 109 S m-1). Interestingly, graphenylene has an unusually higher ZT at low temperature (0.5 at 300 K) than at high temperature (0.3 at 800 K) for n-type doping along the x-axis, contrary to the conventional view that higher ZT values exist in the high temperature range. This work provides a deep insight into the TE properties of two-dimensional Dirac carbon materials and offers new perspectives for enhancing the TE performance and application of carbon-based nanomaterials.

Endothelial Cell-Derived Lactate Triggers Bone Mesenchymal Stem Cell Histone Lactylation to Attenuate Osteoporosis.

Blood vessels play a role in osteogenesis and osteoporosis; however, the role of vascular metabolism in these processes remains unclear. The present study finds that ovariectomized mice exhibit reduced blood vessel density in the bone and reduced expression of the endothelial glycolytic regulator pyruvate kinase M2 (PKM2). Endothelial cell (EC)-specific deletion of Pkm2 impairs osteogenesis and worsens osteoporosis in mice. This is attributed to the impaired ability of bone mesenchymal stem cells (BMSCs) to differentiate into osteoblasts. Mechanistically, EC-specific deletion of Pkm2 reduces serum lactate levels secreted by ECs, which affect histone lactylation in BMSCs. Using joint CUT&Tag and RNA sequencing analyses, collagen type I alpha 2 chain (COL1A2), cartilage oligomeric matrix protein (COMP), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and transcription factor 7 like 2 (TCF7L2) as osteogenic genes regulated by histone H3K18la lactylation are identified. PKM2 overexpression in ECs, lactate addition, and exercise restore the phenotype of endothelial PKM2-deficient mice. Furthermore, serum metabolomics indicate that patients with osteoporosis have relatively low lactate levels. Additionally, histone lactylation and related osteogenic genes of BMSCs are downregulated in patients with osteoporosis. In conclusion, glycolysis in ECs fuels BMSC differentiation into osteoblasts through histone lactylation, and exercise partially ameliorates osteoporosis by increasing serum lactate levels.

Multiple-phase evolution and electrical transport of Sr4-xYxCo4O12-δ (x = 0-1.0): an ordered phase transition process.

The transition metal oxide (TMO) SrCoO3-δ family with rich structural diversity has been widely studied in the phase transition and energy application fields. We report the multiple-phase structure evolution, phase transitions during sintering, and electrical transport of A-site doped Sr4-xYxCo4O12-δ (x = 0-1.0) ceramics. Sr6Co5O15 (x = 0) adopts a hexagonal structure (H), Sr4-xYxCo4O12-δ (x = 0.2-0.4) ceramics adopts a cubic perovskite (CP) structure, and Sr4-xYxCo4O10.5+δ' (x = 0.8-1.0) ceramics adopts an ordered-tetragonal (OT) structure; moreover, their phase transitions during the sintering processing of samples are systematically investigated. Combining the thermal analysis and X-ray diffraction results, the exothermic peak and weight gain of Sr3YCo4O10.5 (x = 1.0, T) at 1042 °C are considered to correspond to an ordered phase transition (T → OT) occurring. Finally, a systematic phase schema of the Sr4-xYxCo4O12-δ (x = 0-1.0) state dependence on the Y content and sintering temperature is obtained. The high-energy Y-O bond stabilizes the high-temperature CP structure (x = 0.2-0.4) and induces a structural evolution from the CP to OT structure (x = 0.8-1.0). In addition, all Sr4-xYxCo4O12-δ (x = 0-1.0) ceramics show semiconductive electrical transport behavior. Sr6Co5O15 (H) with a one-dimensional chain structure has the highest resistivity, while Sr3.8Y0.2Co4O12-δ (CP) with a three-dimensional corner-sharing structure exhibits the lowest resistivity, and Sr4-xYxCo4O12-δ (x = 0.2-1.0) ceramics show an increasing tendency in resistivity due to the hole carrier Co4+ converting to Co3+. We studied multiple-phase evolution and ordered phase transition in Sr4-xYxCo4O12-δ (x = 0-1.0) ceramics through Y-O bonding.

Exosomes incorporated with black phosphorus quantum dots attenuate retinal angiogenesis via disrupting glucose metabolism.

Black phosphorus quantum dots (BPQDs) have shown potential in tumor therapy, however, their anti-angiogenic functions have not been studied. Although BPQDs are easily degraded to non-toxic phosphrous, the reported toxicity, poor stability, and non-selectivity largely limit their further application in medicine. In this study, a vascular targeting, biocompatible, and cell metabolism-disrupting nanoplatform is engineered by incorporating BPQDs into exosomes modified with the Arg-Gly-Asp (RGD) peptide (BPQDs@RGD-EXO nanospheres, BREs). BREs inhibit endothelial cells (ECs) proliferation, migration, tube formation, and sprouting in vitro. The anti-angiogenic role of BREs in vivo is evaluated using mouse retinal vascular development model and oxygen-induced retinopathy model. Combined RNA-seq and metabolomic analysis reveal that BREs disrupt glucose metabolism, which is further confirmed by evaluating metabolites, ATP production and the c-MYC/Hexokinase 2 pathway. These BREs are promising anti-angiogenic platforms for the treatment of pathological retinal angiogenesis with minimal side effects.

Role of glucose metabolism in ocular angiogenesis (Review).

Glucose metabolism, the major source of energy, plays a crucial role in physiological cell function and the maintenance of homeostasis. Glucose acts as the predominant source of metabolic fuel in the generation of ATP and is involved in biosynthesis and epigenetics. Thus, glucose metabolism maintains a key role in cell function, homeostasis, energy generation, biosynthesis and epigenetics. An increasing number of studies have revealed that glucose metabolism is intricately involved in angiogenesis, with the disruption of angiogenesis contributing to several vascular diseases. Ocular vascular diseases are common ophthalmological disorders, and the prevalence of these disorders is increasing annually. Ocular vascular diseases largely occur from abnormal congenital development or acquired disturbances to the vasculature. Thus, identifying the process of occurrence and development of physiological and pathological angiogenesis is of utmost importance, and this involves understanding the inseparable role of intercellular communications between vascular cells. Although vascular endothelial growth factor (VEGF) is a well­recognized therapeutic target for the management of ocular vascular diseases, VEGF­based therapy fails to achieve the desired therapeutic effects in several cases, partly due to drug resistance and non­compliance. In the present review, current knowledge on the processes and roles of glucose metabolism in governing both physiological and pathological ocular angiogenesis are summarized, highlighting vascular glucose metabolism as a promising strategy for maintaining or restoring the physiological functions of the vasculature, thus potentially ameliorating ocular vascular diseases.

A 215-bp indel at intron I of BoFLC2 affects flowering time in Brassica oleracea var. capitata during vernalization.

KEY MESSAGE: In response to cold, a 215-bp deletion at intron I of BoFLC2 slows its silencing activity by feedback to the core genes of the PHD-PRC2 complex, resulting in late flowering in cabbage. Cabbage is a plant-vernalization-responsive flowering type. In response to cold, BoFLC2 is an important transcription factor, which allows cabbage plants to remain in the vegetative phase. However, there have been few reports on the detailed and functional effects of genetic variation in BoFLC2 on flowering time in cabbage. Herein, BoFLC2E and BoFLC2L, cloned from extremely early and extremely late flowering cabbages, respectively, exhibited a 215-bp indel at intron I, three non-synonymous SNPs and a 3-bp indel at exon II. BoFLC2L was found to be related to late flowering, as verified in 40 extremely early/late flowering accessions, a diverse set of cabbage inbred lines and two F2 generations by using indel-FLC2 marker. Among the genetic variation of BoFLC2, the 215-bp deletion at intron I was the main reason for the delayed flowering time, as verified in the transgenic progenies of seed-vernalization-responsive Arabidopsis thaliana (Col) and rapid cycler B. oleracea (TO1000, boflc2). This is the first report to show that the intron I indel of BoFLC2 affects the flowering time of cabbage. Although the intron I 215-bp indel between BoFLC2E and BoFLC2L did not cause alternative splicing, it slowed BoFLC2L silencing during vernalization and feedback to the core genes of the PHD-PRC2 complex, resulting in their lower transcription levels. Our study not only provides an effective molecular marker-assisted selective strategy for identifying bolting-resistant resources and breeding improved varieties in cabbage, but also provides an entry point for exploring the mechanisms of flowering time in plant-vernalization-responsive plants.

Graphene quantum dots rescue angiogenic retinopathy via blocking STAT3/Periostin/ERK signaling.

BACKGROUND: Pathological retinal angiogenesis resulting from a variety of ocular diseases including oxygen induced retinopathy, diabetic retinopathy and ocular vein occlusion, is one of the major reasons for vision loss, yet the therapeutic option is limited. Multiple nanoparticles have been reported to alleviate angiogenic retinopathy. However, the adverse effect cannot be ignored due to the relatively large scale. Graphene quantum dots (GQDs) have shown potential in drug delivery and have been proved biocompatible. In this study, Graphene quantum dots are extensively investigated for their application in angiogenic retinopathy therapy. RESULTS: We showed that GQDs were biocompatible nanomaterials in vitro and in vivo. The nanoparticles have a dose-dependent inhibitory effect on proliferation, migration, tube formation and sprouting of human umbilical vein endothelial cells (HUVECs). Further data show that GQDs could inhibit pathological retinal neovascularization in an oxygen-induced retinopathy (OIR) model. The data of RNA sequencing suggested that periostin is involved in this process. GQDs inhibit the expression of periostin via STAT3, and further regulated cell cycle-related protein levels through ERK pathway. The signaling pathway was conformed in vivo using OIR mouse model. CONCLUSIONS: The present study indicated that GQDs could be a biocompatible anti-angiogenic nanomedicine in the treatment of pathological retinal neovascularization via disrupting periostin/ERK pathway and subsequent cell cycle.

Identification of Candidate Genes for Clubroot-Resistance in Brassica oleracea Using Quantitative Trait Loci-Sequencing.

Clubroot caused by Plasmodiophora brassicae is a devastating disease of cabbage (Brassica oleracea). To identify quantitative trait loci (QTLs) for clubroot resistance (CR) in B. oleracea, genomic resequencing was carried out in two sets of extreme pools, group I and group II, which were constructed separately from 110 and 74 F2 cloned lines derived from the cross between clubroot-resistant (R) cabbage "GZ87" (against race 4) and susceptible (S) cabbage "263." Based on the QTL-sequencing (QTL-Seq) analysis of group I and group II, three QTLs (i.e., qCRc7-2, qCRc7-3, and qCRc7-4) were determined on the C07 chromosome. RNA-Seq and qRT-PCR were conducted in the extreme pools of group II before and after inoculation, and two potential candidate genes (i.e., Bol037115 and Bol042270), which exhibiting upregulation after inoculation in the R pool but downregulation in the S pool, were identified from the three QTLs on C07. A functional marker "SWU-OA" was developed from qCRc7-4 on C07, exhibiting ∼95% accuracy in identifying CR in 56 F2 lines. Our study will provide valuable information on resistance genes against P. brassicae and may accelerate the breeding process of B. oleracea with CR.

Comparative analysis of the clinical outcomes between wavefront-guided and conventional femtosecond LASIK in myopia and myopia astigmatism.

AIM: To compare the clinical outcomes of wavefront guided femtosecond LASIK (WFG LASIK) and conventional femtosecond LASIK (NWFG LASIK) in eyes with myopia and myopia astigmatism. METHODS: This was a retrospective, nonrandomized, comparative investigation enrolling 236 eyes of 122 patients (18-50y) with low & moderate and high myopia. The WFG group including 97 eyes (50 patients) undergone WFG LASIK and the NWFG group including 139 eyes (72 patients) undergone conventional LASIK. Mean efficacy index, high order aberrations (HOAs), pupil size and the quality of visual questionnaire were evaluated 6mo postoperatively. RESULTS: There is no difference between WFG group (-0.054±0.049 in logMAR) and NWFG group (-0.040±0.056) in uncorrected distance visual acuity (UDVA) postoperatively. The myopia astigmatism is higher in WFG group than that in NWFG group (P<0.05). However, the mean efficacy index (MEI) in the WFG group (1.09±0.106) is better than that in the NWFG group (1.036±0.124; P<0.001). Increased HOAs were observed in NWFG group (0.30±0.196) than that in WFG group (0.146±0.188; P<0.001). The pupil size is larger in WFG group (5.15±0.76 mm) than that in NWFG group (4.32±0.52 mm). The patients are satisfied with the clinical surgery, yet WFG group showed better visual quality using the questionnaire survey. Meanwhile, high myopia would result in worse MEI, HOAs and visual quality than low & moderate myopia. CONCLUSION: WFG and NWFG FS-LASIK are both effective and safe procedures to correct low & moderate and high myopia, but WFG FS-LASIK gives a better postoperative MEI, aberrometric control and predictable outcome. Meanwhile, WFG FS-LASIK is better than NWFG FS-LASIK in correction of myopia astigmatism. Low & moderate myopia allow better clinical outcomes than high myopia using any surgical method.

Metabolic flexibility maintains proliferation and migration of FGFR signaling-deficient lymphatic endothelial cells.

Metabolic flexibility is the capacity of cells to alter fuel metabolism in response to changes in metabolic demand or nutrient availability. It is critical for maintaining cellular bioenergetics and is involved in the pathogenesis of cardiovascular disease and metabolic disorders. However, the regulation and function of metabolic flexibility in lymphatic endothelial cells (LECs) remain unclear. We have previously shown that glycolysis is the predominant metabolic pathway to generate ATP in LECs and that fibroblast growth factor receptor (FGFR) signaling controls lymphatic vessel formation by promoting glycolysis. Here, we found that chemical inhibition of FGFR activity or knockdown of FGFR1 induces substantial upregulation of fatty acid ß-oxidation (FAO) while reducing glycolysis and cellular ATP generation in LECs. Interestingly, such compensatory elevation was not observed in glucose oxidation and glutamine oxidation. Mechanistic studies show that FGFR blockade promotes the expression of carnitine palmitoyltransferase 1A (CPT1A), a rate-limiting enzyme of FAO; this is achieved by dampened extracellular signal-regulated protein kinase activation, which in turn upregulates the expression of the peroxisome proliferator-activated receptor alpha. Metabolic analysis further demonstrates that CPT1A depletion decreases total cellular ATP levels in FGFR1-deficient rather than wildtype LECs. This result suggests that FAO, which makes a negligible contribution to cellular energy under normal conditions, can partially compensate for energy deficiency caused by FGFR inhibition. Consequently, CPT1A silencing potentiates the effect of FGFR1 knockdown on impeding LEC proliferation and migration. Collectively, our study identified a key role of metabolic flexibility in modulating the effect of FGFR signaling on LEC growth.

Development of Ogura CMS restorers in Brassica oleracea subspecies via direct RfoB gene transformation.

KEY MESSAGE: The Ogura CMS RfoB restorer developing via RfoB gene transformation was utilized to produce specific morphological Ogura CMS restorers and clubroot resistance lines in Brassica oleracea subspecies. Brassica oleracea vegetables including cabbage, cauliflower, kohlrabi, Brussels sprouts and Chinese kale are morphologically very different despite being members of the same species. The Ogura cytoplasmic male sterility (CMS) system is the most stable strategy for the hybrid breeding of these species. However, this limits the utilization of some excellent genes due to the lack of fertile restorer genes in the system. Herein, to efficaciously use Ogura CMS, the Ogura CMS RfoB restorer was produced by transforming the modified RfoB restorer gene into the Ogura CMS line 'CMS2016' of B. oleracea var. capitata. This gene was shown to recover fertility of natural Ogura CMS lines in B. oleracea subspecies and create transient Ogura CMS RfoB restorers such as the clubroot resistance Ogura CMS RfoB restorer. Interestingly, clubroot resistant individuals without transgenic elements were screened in the progenies of hybrids between B. oleracea inbred lines and the clubroot resistance Ogura CMS RfoB restorer. In addition, 18 different morphological Ogura CMS restorers were developed to specifically recover fertile of Ogura CMS cultivars in B. oleracea subspecies.

l-tetrahydropalmatine suppresses osteoclastogenesis in vivo and in vitro via blocking RANK-TRAF6 interactions and inhibiting NF-κB and MAPK pathways.

Bone homeostasis is delicately orchestrated by osteoblasts and osteoclasts. Various pathological bone loss situations result from the overactivated osteoclastogenesis. Receptor activator of nuclear factor κB ligand (RANKL)-activated NF-κB and MAPK pathways is vital for osteoclastogenesis. Here, we for the first time explored the effects of l-tetrahydropalmatine (l-THP), an active alkaloid derived from corydalis, on the formation and function of osteoclasts in vitro and in vivo. In RAW264.7 cells and bone marrow monocytes cells (BMMCs), l-THP inhibited osteoclastic differentiation at the early stage, down-regulated transcription level of osteoclastogenesis-related genes and impaired osteoclasts functions. Mechanically, Western blot showed that l-THP inhibited the phosphorylation of P50, P65, IκB, ERK, JNK and P38, and the electrophoretic mobility shift assay (EMSA) revealed that DNA binding activity of NF-κB was suppressed, ultimately inhibiting the expression of nuclear factor of activated T cells (NFATc1). Besides, Co-immunoprecipitation indicated that l-THP blocked the interactions of RANK and TNF receptor associated factor 6 (TRAF6) at an upstream site. In vivo, l-THP significantly inhibited ovariectomy-induced bone loss and osteoclastogenesis in mice. Collectively, our study demonstrated that l-THP suppressed osteoclastogenesis by blocking RANK-TRAF6 interactions and inhibiting NF-κB and MAPK pathways. l-THP is a promising agent for treating osteoclastogenesis-related diseases such as post-menopausal osteoporosis.

Cuprous oxide nanoparticles reduces hypertrophic scarring by inducing fibroblast apoptosis.

BACKGROUND: Less apoptosis and excessive growth of fibroblasts contribute to the progression of hypertrophic scar formation. Cuprous oxide nanoparticles (CONPs) could have not only inhibited tumor by inducing apoptosis and inhibiting proliferation of tumor cells, but also promoted wound healing. The objective of this study was to further explore the therapeutic effects of CONPs on hypertrophic scar formation in vivo and in vitro. METHODS: In vivo, a rabbit ear scar model was established on New Zealand albino rabbits. Six full-thickness and circular wounds (10 mm diameter) were made to each ear. Following complete re-epithelization observed on postoperative day 14, an intralesional injection of CONPs or 5% glucose solution was conducted to the wounds. The photo and ultrasonography of each wound were taken every week and scars were harvested on day 35 for further histomorphometric analysis. In vitro, the role of CONPs in human hypertrophic scar fibroblasts (HSFs) apoptosis and proliferation were evaluated by Tunnel assay, Annexin V/PI staining, cell cycle analysis, and EdU proliferation assay. The endocytosis of CONPs by fibroblasts were detected through transmission electron microscopy (TEM) and the mitochondrial membrane potential and ROS production were also detected. RESULTS: In vivo, intralesional injections of CONPs could significantly improve the scar appearance and collagen arrangement, and decreased scar elevation index (SEI). In vitro, CONPs could prominently inhibit proliferation and induce apoptosis in HSFs in a concentration-dependent manner. In addition, CONPs could be endocytosed into mitochondria，damage the mitochondrial membrane potential and increase ROS production. CONCLUSION: CONPs possessed the therapeutic potential in the treatment of hypertrophic scar by inhibiting HSFs proliferation and inducing HSFs apoptosis.

Reversal of Osteoporotic Activity by Endothelial Cell-Secreted Bone Targeting and Biocompatible Exosomes.

Exosomes, also known as extracellular vesicles, are naturally occurring, biocompatible, and bioacive nanoparticles ranging from 40 to 150 nm in diameter. Bone-secreted exosomes play important roles in bone homeostasis, the interruption of which can lead to diseases such as osteoporosis, rheumatoid arthritis, and osteopetrosis. Though the relationship between vascular and bone homeostasis has been recognized recently, the role of vascular endothelial cell (EC)-secreted exosomes (EC-Exos) in bone homeostasis is not well understood. Herein, we found that EC-Exos show more efficient bone targeting than osteoblast-derived exosomes or bone marrow mesenchymal stem cell-derived exosomes. We also found that EC-Exos can be internalized by bone marrow-derived macrophages (BMMs) to alter their morphology. EC-Exos can inhibit osteoclast activity in vitro and inhibit osteoporosis in an ovariectomized mouse model. Sequencing of exosome miRNA revealed that miR-155 was highly expressed in EC-Exos-treated BMMs. The miR-155 level in EC-Exos was much higher than that in BMMs and ECs, indicating that miR-155 was endogenous cargo of EC-derived vesicles. Blockage of BMMs miR-155 levels reversed the suppression by EC-Exos of osteoclast induction, confirming that exosomal miR-155 may have therapeutic potential against osteoporosis. Taken together, our findings suggest that EC-Exos may be utilized as a bone targeting and nontoxic nanomedicine for the treatment of bone resorption disorders.

CRISPR/Cas9-mediated multiple gene editing in Brassica oleracea var. capitata using the endogenous tRNA-processing system.

Cabbage (Brassica oleracea var. capitata) is a biennial plant with strong self-incompatibility and an obligate requirement for prolonged vernalization by exposure to low temperatures to induce flowering. These characteristics significantly increase the difficulty of exploiting novel germplasm induced by physical or chemical mutagens. In this study, we report a CRISPR/Cas9 gene-editing system based on endogenous tRNA processing to induce high efficiency and inheritable mutagenesis in cabbage. Using the phytoene desaturase gene BoPDS, the S-receptor kinase gene BoSRK, and the male-sterility-associated gene BoMS1 as the target genes, multisite and multiple gene mutations were achieved using a construct with tandemly arrayed tRNA-sgRNA architecture to express multiple sgRNAs. The BoSRK3 gene mutation suppressed self-incompatibility completely, converting the self-incompatible line into a self-compatible line. In addition, the BoMS1 gene mutation produced a completely male-sterile mutant, which was highly cross compatible with its nonmutant isoline at the flowering stage as a result of a simultaneous BoSRK3 gene mutation, enabling the economic propagation of the male-sterile line through bee-mediated cross-pollination. Interestingly, higher site mutation efficiency was detected when a guide sequence was inserted into a location in the tandemly arrayed tRNA-sgRNA architecture that was distal from the upstream Pol III promoter. In addition, mutation sites were also detected in the paralogous genes of the BoPDS and BoSRK genes that had fully consistent sequences or base mismatches but beyond the "seed" region in the spacer sequence compared with the target sgRNAs. Collectively, our results demonstrate that the CRISPR/Cas9 system, coupled with an endogenous tRNA-processing system, is an efficient tool to improve cabbage traits.