Synthesis and evaluation of pentacyclic triterpenoids conjugates as novel HBV entry inhibitors targeting NTCP receptor.

Chronic liver diseases caused by hepatitis B virus (HBV) are the accepted main cause leading to liver cirrhosis, hepatic fibrosis, and hepatic carcinoma. Sodium taurocholate cotransporting polypeptide (NTCP), a specific membrane receptor of hepatocytes for triggering HBV infection, is a promising target against HBV entry. In this study, pentacyclic triterpenoids (PTs) including glycyrrhetinic acid (GA), oleanolic acid (OA), ursolic acid (UA) and betulinic acid (BA) were modified via molecular hybridization with podophyllotoxin respectively, and resulted in thirty-two novel conjugates. The anti-HBV activities of conjugates were evaluated in HepG2.2.15 cells. The results showed that 66% of the conjugates exhibited lower toxicity to the host cells and had significant inhibitory effects on the two HBV antigens, especially HBsAg. Notably, the compounds BA-PPT1, BA-PPT3, BA-PPT4, and UA-PPT3 not only inhibited the secretion of HBsAg but also suppressed HBV DNA replication. A significant difference in the binding of active conjugates to NTCP compared to the HBV PreS1 antigen was observed by SPR assays. The mechanism of action was found to be the competitive binding of these compounds to the NTCP 157-165 epitopes, blocking HBV entry into host cells. Molecular docking results indicated that BA-PPT3 interacted with the amino acid residues of the target protein mainly through π-cation, hydrogen bond and hydrophobic interaction, suggesting its potential as a promising HBV entry inhibitor targeting the NTCP receptor.

DcGST1, encoding a glutathione S-transferase activated by DcMYB7, is the main contributor to anthocyanin pigmentation in purple carrot.

The color of purple carrot taproots mainly depends on the anthocyanins sequestered in the vacuoles. Glutathione S-transferases (GSTs) are key enzymes involved in anthocyanin transport. However, the precise mechanism of anthocyanin transport from the cytosolic surface of the endoplasmic reticulum (ER) to the vacuoles in carrots remains unclear. In this study, we conducted a comprehensive analysis of the carrot genome, leading to the identification of a total of 41 DcGST genes. Among these, DcGST1 emerged as a prominent candidate, displaying a strong positive correlation with anthocyanin pigmentation in carrot taproots. It was highly expressed in the purple taproot tissues of purple carrot cultivars, while it was virtually inactive in the non-purple taproot tissues of purple and non-purple carrot cultivars. DcGST1, a homolog of Arabidopsis thaliana TRANSPARENT TESTA 19 (TT19), belongs to the GSTF clade and plays a crucial role in anthocyanin transport. Using the CRISPR/Cas9 system, we successfully knocked out DcGST1 in the solid purple carrot cultivar 'Deep Purple' ('DPP'), resulting in carrots with orange taproots. Additionally, DcMYB7, an anthocyanin activator, binds to the DcGST1 promoter, activating its expression. Compared with the expression DcMYB7 alone, co-expression of DcGST1 and DcMYB7 significantly increased anthocyanin accumulation in carrot calli. However, overexpression of DcGST1 in the two purple carrot cultivars did not change the anthocyanin accumulation pattern or significantly increase the anthocyanin content. These findings improve our understanding of anthocyanin transport mechanisms in plants, providing a molecular foundation for improving and enhancing carrot germplasm.

Mutation of the gene encoding the PHD-type transcription factor SAB23 confers submergence tolerance in rice.

Submergence is a major constraint on rice production in South and Southeast Asia. In this study, we determined that a gene of the Sub1A-binding protein family, SAB23, encodes a plant homeodomain (PHD)-type transcription factor that has a novel function of negatively regulating submergence tolerance in rice. The T-DNA insertion mutant sab23 displayed reduced plant height, delayed seed maturation, and lower percentage seed set. Importantly, this mutant also exhibited enhanced submergence tolerance. In addition, CRISPR/Cas9 knock out of SAB23 resulted in a significant reduction in the content of the gibberellin GA4 and a dramatic increase in the content of GA1 in the plants. SAB23 binds to the promoter of CYTOCHROME P450 714B2 (CYP714B2), which encodes a GA13-oxidase that catalyses the conversion of GA53 to GA19. Disruption of SAB23 function led to increased CYP714B2 transcription, and overexpression of CYP714B2 produced phenotypes similar to those of the SAB23-knockout plants. Taken together, our results reveal that SAB23 negatively regulates rice submergence tolerance by modulating CYP714B2 expression, which has significant potential for use in future breeding.

Highly Biocompatible Polyester-Based Piezoelectric Elastomer with Antitumor and Antibacterial Activity for Ultrasound-Enhanced Piezoelectric Therapy.

Currently, the use of piezoelectric materials to provide sustainable and noninvasive bioelectric stimulation to eradicate tumor cells and accelerate wound healing has raised wide attention. The development of a multifunctional piezoelectric elastomer with the ability to perform in situ tumor therapy as well as wound repair is of paramount importance. However, current piezoelectric materials have a large elastic modulus and limited stretchability, making it difficult to match with the dynamic curvature changes of the wound. Therefore, by copolymerizing lactic acid, butanediol, sebacic acid, and itaconic acid to develop a piezoelectric elastomer (PLBSIE), we construct a new ultrasound-activated PLBSIE-based tumor/wound unified therapeutic platform. Excitedly, it showed outstanding piezoelectric performance and high stretchability, and the separated carrier could react with water to generate highly cytotoxic reactive oxygen species (ROS), contributing to effectively killing tumor cells and eliminating bacteria through piezoelectric therapy. In addition, ultrasound-triggered piezoelectric effects could promote the migration and differentiation of wound-healing-related cells, thus accelerating wound healing. Herein, such a piezoelectric elastomer exerted a critical role in postoperative tumor-induced wound therapy and healing with the merits of possessing multifunctional abilities. Taken together, the developed ultrasound-activated PLBSIE will offer a comprehensive treatment for postoperative osteosarcoma therapy.

Generating colorful carrot germplasm through metabolic engineering of betalains pigments.

Betalains are tyrosine-derived plant pigments exclusively found in the Caryophyllales order and some higher fungi and generally classified into two groups: red-violet betacyanins and yellow-orange betaxanthins. Betalains attract great scientific and economic interest because of their relatively simple biosynthesis pathway, attractive colors and health-promoting properties. Co-expressing two core genes BvCYP76AD1 and BvDODA1 with or without a glycosyltransferase gene MjcDOPA5GT allowed the engineering of carrot (an important taproot vegetable) to produce a palette of unique colors. The highest total betalains content, 943.2 µg·g-1 DW, was obtained in carrot taproot transformed with p35S:RUBY which produces all of the necessary enzymes for betalains synthesis. Root-specific production of betalains slightly relieved tyrosine consumption revealing the possible bottleneck in betalains production. Furthermore, a unique volcano-like phenotype in carrot taproot cross-section was created by vascular cambium-specific production of betalains. The betalains-fortified carrot in this study is thus anticipated to be used as functional vegetable and colorful carrot germplasm in breeding to promote health.

A MYB activator, DcMYB11c, regulates carrot anthocyanins accumulation in petiole but not taproot.

The first domesticated carrots were thought to be purple carrots rich in anthocyanins. The anthocyanins biosynthesis in solid purple carrot taproot was regulated by DcMYB7 within P3 region containing a gene cluster of six DcMYBs. Here, we described a MYB gene within the same region, DcMYB11c, which was highly expressed in the purple pigmented petioles. Overexpression of DcMYB11c in 'Kurodagosun' (KRDG , orange taproot carrot with green petioles) and 'Qitouhuang' (QTHG , yellow taproot carrot with green petioles) resulted in deep purple phenotype in the whole carrot plants indicating anthocyanins accumulation. Knockout of DcMYB11c in 'Deep Purple' (DPPP , purple taproot carrot with purple petioles) through CRISPR/Cas9-based genome editing resulted in pale purple phenotype due to the dramatic decrease of anthocyanins content. DcMYB11c could induce the expression of DcbHLH3 and anthocyanins biosynthesis genes to jointly promote anthocyanins biosynthesis. Yeast one-hybrid assay (Y1H) and dual-luciferase reporter assay (LUC) revealed that DcMYB11c bound to the promoters of DcUCGXT1 and DcSAT1 and directly activated the expression of DcUCGXT1 and DcSAT1 responsible for anthocyanins glycosylation and acylation, respectively. Three transposons were present in the carrot cultivars with purple petioles but not in the carrot cultivars with green petioles. We revealed the core factor, DcMYB11c, involved in anthocyanins pigmentation in carrot purple petioles. This study provides new insights into precise regulation mechanism underlying anthocyanins biosynthesis in carrot. The orchestrated regulation mechanism in carrot might be conserved across the plant kingdom and useful for other researchers working on anthocyanins accumulation in different tissues.

Differential hydroxylation efficiency of the two non-heme carotene hydroxylases: DcBCH1, rather than DcBCH2, plays a major role in carrot taproot.

Carotene hydroxylase plays an important role in catalyzing the hydroxylation of carotene to xanthopylls, including two types: non-heme carotene hydroxylase (BCH type) and heme-containing cytochrome P450 hydroxylase (P450 type). Two BCH-encoding genes were annotated in the carrot genome. However, the role of BCHs and whether there are functional interactions between the duplicated BCHs in carrot remains unclear. In this study, two BCH encoding genes, DcBCH1 and DcBCH2, were cloned from carrot. The relative expression level of DcBCH1 was much higher than that of DcBCH2 in carrot taproots with different carotene accumulation levels. Overexpression of DcBCH1 in 'KRD' (high carotene accumulated) carrot changed the taproot color from orange to yellow, accompanied by substantial reductions in α-carotene and ß-carotene. There was no obvious change in taproot color between transgenic 'KRD' carrot overexpressing DcBCH2 and control carrot. Simultaneously, the content of α-carotene in the taproot of DcBCH2-overexpressing carrot decreased, but the content of ß-carotene did not change significantly in comparison with control carrot. Using the CRISPR/Cas9 system to knock out DcBCH1 in 'KRD' carrot lightened the taproot color from orange to pink-orange; the content of α-carotene in the taproot increased slightly, while the ß-carotene content was still significantly decreased, compared with control carrot. In DcBCH1-knockout carrot, the transcript level of DcBCH2 was significantly increased. These results indicated that in carrot taproot, DcBCH1 played the main function of BCH enzyme, which could hydroxylate α-carotene and ß-carotene; DcBCH1 and DcBCH2 had functional redundancy, and these two DcBCHs could partially compensate for each other.

Genome-Wide Identification and Evolution Analysis of R2R3-MYB Gene Family Reveals S6 Subfamily R2R3-MYB Transcription Factors Involved in Anthocyanin Biosynthesis in Carrot.

The taproot of purple carrot accumulated rich anthocyanin, but non-purple carrot did not. MYB transcription factors (TFs) condition anthocyanin biosynthesis in many plants. Currently, genome-wide identification and evolution analysis of R2R3-MYB gene family and their roles involved in conditioning anthocyanin biosynthesis in carrot is still limited. In this study, a total of 146 carrot R2R3-MYB TFs were identified based on the carrot transcriptome and genome database and were classified into 19 subfamilies on the basis of R2R3-MYB domain. These R2R3-MYB genes were unevenly distributed among nine chromosomes, and Ka/Ks analysis suggested that they evolved under a purified selection. The anthocyanin-related S6 subfamily, which contains 7 MYB TFs, was isolated from R2R3-MYB TFs. The anthocyanin content of rhizodermis, cortex, and secondary phloem in 'Black nebula' cultivar reached the highest among the 3 solid purple carrot cultivars at 110 days after sowing, which was approximately 4.20- and 3.72-fold higher than that in the 'Deep purple' and 'Ziwei' cultivars, respectively. The expression level of 7 MYB genes in purple carrot was higher than that in non-purple carrot. Among them, DcMYB113 (DCAR_008994) was specifically expressed in rhizodermis, cortex, and secondary phloem tissues of 'Purple haze' cultivar, with the highest expression level of 10,223.77 compared with the control 'DPP' cultivar at 70 days after sowing. DcMYB7 (DCAR_010745) was detected in purple root tissue of 'DPP' cultivar and its expression level in rhizodermis, cortex, and secondary phloem was 3.23-fold higher than that of secondary xylem at 110 days after sowing. Our results should be useful for determining the precise role of S6 subfamily R2R3-MYB TFs participating in anthocyanin biosynthesis in carrot.

Long Noncoding RNA LINC00909 Induces Epithelial-Mesenchymal Transition and Contributes to Osteosarcoma Tumorigenesis and Metastasis.

Background: Osteosarcoma (OS) is a malignant tumor that is highly metastatic with a high mortality rate. Although mounting evidence suggests that LINC00909 is strongly associated with the malignant progression of various tumors, the exact role of LINC00909 in OS remains unknown. Therefore, the current study was designed to investigate the relationship between LINC00909 and the malignant progression of OS. Methods: LINC00909 expression was measured in OS cell lines and clinical specimens using RT-qPCR assays. The effects of LINC00909 on OS proliferation, invasion, and migration were calculated both in vitro and in vivo. Apart from that, bioinformatics analyses, FISH, RIP, and luciferase reporter assays were carried out to investigate the downstream target of LINC00909. Rescue experiments were also conducted to investigate the potential mechanisms of action of competitive endogenous RNAs (ceRNAs). Results: In this study, we found that LINC00909 was highly expressed in OS cell lines and clinical specimens. In vivo and in vitro experiments demonstrated that LINC00909 induces epithelial-to-mesenchymal transition (EMT) and contributes to OS tumorigenesis and metastasis. FISH, RIP, and luciferase assays indicated that miR-875-5p is a direct target of LINC00909. Moreover, HOXD9 was validated as the downstream target of miR-875-5p in a luciferase reporter assay and western blotting experiments. Rescue experiments revealed that HOXD9 reversed the effect of LV-sh-LINC00909 on OS cells by positively regulating the PI3K/AKT/mTOR signaling pathway. Conclusion: Collectively, LINC00909 induces EMT and contributes to OS tumorigenesis and metastasis through the PI3K/AKT/mTOR pathway by binding to miR-875-5p to upregulate HOXD9 expression. Targeting the LINC00909/miR-875-5p/HOXD9 axis may have potential in treating OS.

Presence of ginsenoside re during in vitro maturation protects porcine oocytes from heat stress.

Heat stress (HS) affects the development of porcine gametes and embryos negatively, induces the decrease of reproductive ability significantly, threatens global pig production. Ginsenoside Re (GRe), is a main bioactive component of ginseng, shows very specific anti-apoptotic, antioxidant and anti-inflammatory activities. To investigate the alleviating effect of GRe on the in vitro maturation of porcine oocyte under the HS, the polar body extrusion rate, intracellular levels of reactive oxygen species (ROS) and glutathione (GSH), ATP content, mitochondrial membrane potential (MMP) were assessed. For the current study, porcine cumulus-oocyte complexes (COCs) randomly divided into four groups: the control, GRe, HS and HS + GRe group. The results showed that HS inhibited the cumulus cell expansion and polar body extrusion rate, the levels of GSH and MMP, the ATP content, the gene expression of Nrf2 of porcine oocytes and the parthenogenetic activation (PA) embryo development competence, but GRe treatment could partly neutralize these adverse effects. Furthermore, HS increased the ROS formation and percentage of apoptosis, the gene expression of HSP90, CASP3 and CytoC of porcine oocytes, but GRe could weaken the effect on Cyto C and BAX expression induced by HS. Taken together, these results showed that the presence of GRe during in vitro maturation protects porcine oocytes from HS. These findings lay a foundation for GRe may be used as a potential protective drug to protect porcine oocytes against HS damage.

Extracellular vesicles derived from human umbilical cord mesenchymal stem cells alleviate osteoarthritis of the knee in mice model by interacting with METTL3 to reduce m6A of NLRP3 in macrophage.

BACKGROUND: Osteoarthritis (OA) is a prevalent degenerative joint disease that not only significantly impairs the quality of life of middle-aged and elderly individuals but also imposes a significant financial burden on patients and society. Due to their significant biological properties, extracellular vesicles (EVs) have steadily received great attention in OA treatment. This study aimed to investigate the influence of EVs on chondrocyte proliferation, migration, and apoptosis and their protective efficacy against OA in mice. METHODS: The protective impact of EVs derived from human umbilical cord mesenchymal stem cells (hucMSCs-EVs) on OA in mice was investigated by establishing a mouse OA model by surgically destabilizing the medial meniscus (DMM). Human chondrocytes were isolated from the cartilage of patients undergoing total knee arthroplasty (TKA) and cultured with THP-1 cells to mimic the in vivo inflammatory environment. Levels of inflammatory factors were then determined in different groups, and the impacts of EVs on chondrocyte proliferation, migration, apoptosis, and cartilage extracellular matrix (ECM) metabolism were explored. N6-methyladenosine (m6A) level of mRNA and methyltransferase-like 3 (METTL3) protein expression in the cells was also measured in addition to microRNA analysis to elucidate the molecular mechanism of exosomal therapy. RESULTS: The results indicated that hucMSCs-EVs slowed OA progression, decreased osteophyte production, increased COL2A1 and Aggrecan expression, and inhibited ADAMTS5 and MMP13 overexpression in the knee joint of mice via decreasing pro-inflammatory factor secretion. The in vitro cell line analysis revealed that EVs enhanced chondrocyte proliferation and migration while inhibiting apoptosis. METTL3 is responsible for these protective effects. Further investigations revealed that EVs decreased the m6A level of NLRP3 mRNA following miR-1208 targeted binding to METTL3, resulting in decreased inflammatory factor release and preventing OA progression. CONCLUSION: This study concluded that hucMSCs-EVs inhibited the secretion of pro-inflammatory factors and the degradation of cartilage ECM after lowering the m6A level of NLRP3 mRNA with miR-1208 targeting combined with METTL3, thereby alleviating OA progression in mice and providing a novel therapy for clinical OA treatment.

AgGMP encoding GDP-D-mannose pyrophosphorylase from celery enhanced the accumulation of ascorbic acid and resistance to drought stress in Arabidopsis.

Ascorbic acid (AsA) is an important nutrient in celery, the conversion of D-mannose-1-P to GDP-D-mannose catalyzed by GDP-D-mannose pyrophosphorylase (GMPase) represents the first committed step in the biosynthesis of AsA. To clarify the function of the AgGMP gene of celery, the AgGMP gene was cloned from celery cv. 'Jinnan Shiqin' . It contains an open reading frame (ORF) with the length of 1,086 bp, encoding 361 amino acids. AgGMP protein was highly conserved among different plant species. Phylogenetic analysis demonstrated that the GMP proteins from celery and carrot belonged to the same branch. AgGMP protein was mainly composed of three α-helixes and certain random coils. No signal peptide was found in the AgGMP protein. The subcellular localization indicated that the AgGMP protein was located in the cytoplasm. The relative expression levels of AgGMP in 'Jinnan Shiqin' were significantly up-regulated at 2 h and 4 h under drought stress treatments. AsA contents in transgenic Arabidopsis lines hosting AgGMP gene were higher than that in wild type plants, and the root lengths were also longer in the MS medium containing 300 mM mannitol. The present study provides useful evidence for the functional involvement of AgGMP in regulating AsA accumulation and response to drought stress in celery.

Ruxolitinib attenuates secondary injury after traumatic spinal cord injury.

Excessive inflammation post-traumatic spinal cord injury (SCI) induces microglial activation, which leads to prolonged neurological dysfunction. However, the mechanism underlying microglial activation-induced neuroinflammation remains poorly understood. Ruxolitinib (RUX), a selective inhibitor of JAK1/2, was recently reported to inhibit inflammatory storms caused by SARS-CoV-2 in the lung. However, its role in disrupting inflammation post-SCI has not been confirmed. In this study, microglia were treated with RUX for 24 hours and then activated with interferon-γ for 6 hours. The results showed that interferon-γ-induced phosphorylation of JAK and STAT in microglia was inhibited, and the mRNA expression levels of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-1ß, interleukin-6, and cell proliferation marker Ki67 were reduced. In further in vivo experiments, a mouse model of spinal cord injury was treated intragastrically with RUX for 3 successive days, and the findings suggest that RUX can inhibit microglial proliferation by inhibiting the interferon-γ/JAK/STAT pathway. Moreover, microglia treated with RUX centripetally migrated toward injured foci, remaining limited and compacted within the glial scar, which resulted in axon preservation and less demyelination. Moreover, the protein expression levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 were reduced. The neuromotor function of SCI mice also recovered. These findings suggest that RUX can inhibit neuroinflammation through inhibiting the interferon-γ/JAK/STAT pathway, thereby reducing secondary injury after SCI and producing neuroprotective effects.

Exosomes derived from hypoxia preconditioned mesenchymal stem cells laden in a silk hydrogel promote cartilage regeneration via the miR-205-5p/PTEN/AKT pathway.

Tissue engineering has promising prospects for cartilage regeneration. However, there remains an urgent need to harvest high quality seed cells. Bone marrow mesenchymal cells (BMSCs), and in particular their exosomes, might promote the function of articular chondrocytes (ACs) via paracrine mechanisms. Furthermore, preconditioned BMSCs could provide an enhanced therapeutic effect. BMSCs naturally exist in a relatively hypoxic environment (1%-5% O2); however, they are usually cultured under higher oxygen concentrations (21% O2). Herein, we hypothesized that hypoxia preconditioned exosomes (H-Exos) could improve the quality of ACs and be more conducive to cartilage repair. In our study, we compared the effects of exosomes derived from BMSCs preconditioned with hypoxia and normoxia (N-Exos) on ACs, demonstrating that H-Exos significantly promoted the proliferation, migration, anabolism and anti-inflammation effects of ACs. Furthermore, we confirmed that hypoxia preconditioning upregulated the expression of miR-205-5p in H-Exos, suggesting that ACs were promoted via the miR-205-5p/PTEN/AKT pathway. Finally, an injectable silk fibroin (SF) hydrogel containing ACs and H-Exos (SF/ACs/H-Exos) was utilized to repair cartilage defects and effectively promote cartilage regeneration in vivo. The application of SF/ACs/H-Exos hydrogel in cartilage regeneration therefore has promising prospects. STATEMENT OF SIGNIFICANCE: Cartilage tissue engineering (CTE) has presented a promising prospect. However, the quality of seed cells is an important factor affecting the repair efficiency. Our study demonstrates for the first time that the exosomes derived from hypoxia preconditioned BMSCs (H-Exos) effectively promote the proliferation, migration and anabolism of chondrocytes and inhibit inflammation through miR-205-5p/PTEN/AKT pathway. Furthermore, we fabricated an injectable silk fibrion (SF) hydrogel to preserve and sustained release H-Exos. A complex composed of SF hydrogel, H-Exos and chondrocytes can effectively promote the regeneration of cartilage defects. Therefore, this study demonstrates that hypoxia pretreatment could optimize the therapeutic effects of BMSCs-derived exosomes, and the combination of exosomes and SF hydrogel could be a promising therapeutic method for cartilage regeneration.

AgMYB12, a novel R2R3-MYB transcription factor, regulates apigenin biosynthesis by interacting with the AgFNS gene in celery.

KEY MESSAGE: Overexpression of AgMYB12 in celery improved the accumulation of apigenin by interacting with the AgFNS gene. Celery is a common vegetable, and its essential characteristic is medicine food homology. A natural flavonoid and a major pharmacological component in celery, apigenin plays an important role in human health. In this study, we isolated a novel R2R3-MYB transcription factor that regulates apigenin accumulation from the celery cultivar 'Jinnan Shiqin' through yeast one-hybrid screening and designated it as AgMYB12. The AgMYB12 protein was located in the nucleus. It showed transcriptional activation activity and bound specifically to the promoter of AgFNS, a gene involved in apigenin biosynthesis. Phylogenetic tree analysis demonstrated that AgMYB12 belongs to the flavonoid branch. It contains two flavonoid-related motifs, SG7 and SG7-2, and shared a highly conserved R2R3 domain with flavonoid-related MYBs. The homologous overexpression of AgMYB12 induced the up-regulation of AgFNS gene expression and accumulation of apigenin and luteolin in celery. Additionally, the expression levels of apigenin biosynthesis-related genes, including AgPAL, AgCHI, AgCHS, Ag4CL, and AgC4H, increased in transgenic celery plants. These results indicated that AgMYB12 acted as a positive regulator of apigenin biosynthesis and activated the expression of AgFNS gene. The current study provides new information about the regulation mechanism of apigenin metabolism in celery and offers a strategy for cultivating the plants with high apigenin content.

1,25-Dihydroxyvitamin D Inhibits Osteoarthritis by Modulating Interaction Between Vitamin D Receptor and NLRP3 in Macrophages.

BACKGROUND: Osteoarthritis (OA) is the most prevalent chronic joint disease globally. Loss of extracellular matrix (ECM) by chondrocytes is a classic feature of OA. Inflammatory cytokines, such as interleukin-1ß (IL-1ß) and interleukin-18 (IL-18), secreted mainly by macrophages, promote expression of matrix degrading proteins and further aggravate progression of OA. 1,25-dihydroxyvitamin D (1,25VD) modulates inflammation thus exerting protective effects on cartilage tissue. However, the underlying mechanisms of 1,25VD activity have not been fully elucidated. METHODS: The destabilization of the medial meniscus (DMM)-induced mice model of OA was established to investigate the protective effects of 1,25VD by micro-CT and Safranin-O and Fast Green staining. And the co-culture system between THP-1 cells and primary chondrocytes was constructed to explore the effects of vitamin D receptor (VDR) and 1,25VD on chondrogenic proliferation, apoptosis, and migration. The immunofluorescence staining and Western blot analysis were used to detect the expressions of ECM proteins and matrix degradation-associated proteases. Enzyme-linked immunosorbent assay (ELISA) was used to examine the expression levels of inflammatory cytokines. RESULTS: The findings of the study showed that 1,25VD prevented cartilage degeneration and osteophyte formation by inhibiting secretion of inflammatory cytokines in OA mice model. These protective effects were exerted through the vitamin D receptor (VDR). Further studies showed that 1,25VD increased ubiquitination level of NLRP3 by binding to VDR, resulting in decrease in IL-1ß and IL-18 secretion. These findings indicate that 1,25VD binds to VDR thus preventing chondrogenic ECM degradation by modulating macrophage NLRP3 activation and secretion of inflammatory cytokines, thus alleviating OA progression. CONCLUSION: Here, our study suggests that 1,25VD, targeting to VDR, prevents chondrogenic ECM degradation through regulating macrophage NLRP3 activation and inflammatory cytokines secretion, thereby alleviating OA. These findings provide information on a novel molecular mechanism for application of 1,25VD as OA therapy.

Integrative genome, transcriptome, microRNA, and degradome analysis of water dropwort (Oenanthe javanica) in response to water stress.

Water dropwort (Liyang Baiqin, Oenanthe javanica (BI.) DC.) is an aquatic perennial plant from the Apiaceae family with abundant protein, dietary fiber, vitamins, and minerals. It usually grows in wet soils and can even grow in water. Here, whole-genome sequencing of O. javanica via HiSeq 2000 sequencing technology was reported for the first time. The genome size was 1.28 Gb, including 42,270 genes, of which 93.92% could be functionally annotated. An online database of the whole-genome sequences of water dropwort, Water dropwortDB, was established to share the results and facilitate further research on O. javanica (database homepage: http://apiaceae.njau.edu.cn/waterdropwortdb ). Water dropwortDB offers whole-genome and transcriptome sequences and a Basic Local Alignment Search Tool. Comparative analysis with other species showed that the evolutionary relationship between O. javanica and Daucus carota was the closest. Twenty-five gene families of O. javanica were found to be expanded, and some genetic factors (such as genes and miRNAs) related to phenotypic and anatomic differentiation in O. javanica under different water conditions were further investigated. Two miRNA and target gene pairs (miR408 and Oja15472, miR171 and Oja47040) were remarkably regulated by water stress. The obtained reference genome of O. javanica provides important information for future work, thus making in-depth genetic breeding and gene editing possible. The present study also provides a foundation for the understanding of the O. javanica response to water stress, including morphological, anatomical, and genetic differentiation.

AgZDS, a gene encoding ζ-carotene desaturase, increases lutein and ß-carotene contents in transgenic Arabidopsis and celery.

ζ-Carotene desaturase (ZDS) is one of the key enzymes regulating carotenoids biosynthesis and accumulation. Celery transgenic efficiency is low and it is difficult to obtain transgenic plants. The study on ZDS was limited in celery. Here, the AgZDS gene was cloned from celery and overexpressed in Arabidopsis thaliana and celery to verify its function. The AgZDS has typical characteristic of ZDS protein and is highly conserved in higher plants. Phylogenetic analysis showed that AgZDS has the closest evolutionary relationship with ZDSs from Solanum lycopersicum, Capsicum annuum and Tagetes erecta. Overexpression of AgZDS gene in A. thaliana and celery resulted in increased accumulations of lutein and ß-carotene and up-regulated the expression levels of the genes involved in carotenoids biosynthesis. The contents of lutein and ß-carotene in two lines, AtL1 and AgL5, were the highest in transgenic A. thaliana and celery, respectively. The relative expression levels of 5 genes (AtPDS, AtZISO, AtZEP, AtNCED3, and AtCCD4) were up-regulated compared to the wild type plants. The relative expression levels of most genes in carotenoids biosynthesis pathway, such as AgPDS, AgCRTISO1, and AgZISO, were up-regulated in transgenic celery plants. The antioxidant capacity of A. thaliana and photosynthetic capacity of celery were also enhanced. This research is the first report on the function of structure gene related to carotenoid biosynthesis in transgenic celery plants. The findings in this study demonstrated the roles of AgZDS in regulating carotenoids metabolism of celery, which laid a potential foundation for quality improvement of celery.

Circular RNA-Related CeRNA Network and Prognostic Signature for Patients with Osteosarcoma.

INTRODUCTION: Osteosarcoma (OSA) is characterized by its relatively high morbidity in children and adolescents. Patients usually have advanced disease at the time of diagnosis, resulting in poor outcomes. This study focused on building a circular RNA-based ceRNA network to develop a reliable model for OSA risk prediction. METHODS: We used the Gene Expression Omnibus (GEO) datasets to explore the expression patterns of circRNA, miRNA, and mRNA in OSA. The prognostic value of circRNA host genes was assessed with data from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database using Kaplan-Meier survival analysis. We established a circRNA-related ceRNA network and annotated its biological functions. Next, we developed a prognostic risk signature based on mRNAs extracted from the ceRNA network. We also developed a prognostic model and constructed a nomogram to enhance the prediction of OSA prognosis. RESULTS: We identified 166 DEcircRNAs, 233 DEmiRNAs, and 1317 DEmRNAs and used them to create a circRNA-related ceRNA network. We then established a prognostic risk model consisting of four genes (MLLT11, TNFRSF11B, SLC7A7, and PARVA). Moreover, we found that inhibition of MLLT11 and SLC7A7 blocked OSA cell proliferation and migration in in vitro experiments. CONCLUSION: Our study identifies crucial prognostic genes and provides a circRNA-related ceRNA network for OSA, which will contribute to the elucidation of the molecular mechanisms underlying the oncogenesis and development of OSA.