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Abstract Background Ewing sarcoma (ES) is an aggressive childhood bone and soft tissue cancer. KIAA1429 is one type of N6-methyladenosine (m6A) writer that plays a tumor-progressive role in various cancers, but the role of KIAA1429 in ES remains to be elucidated. The aim of the study was to investigate the role of KIAA1429 in ES. Methods We performed a multi-omic screen including CRISPR-Cas9 functional genomic and transcriptomic approaches, and identified that KIAA1429 played a significant role in ES progression. Gene knockdown, quantitative real-time PCR (Q-RT-PCR), immunoblotting, CellTiter-Glo assays, clonogenic assays, a subcutaneous xenograft model and immunohistochemistry were used to assess the functional role of KIAA1429 in ES. We mainly conducted RNA sequencing (RNA-seq) in ES cells to analyze the downstream regulatory mechanism of KIAA1429. An integrative analysis of chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq indicated the upstream regulatory mechanism of KIAA1429. Results In vitro and in vivo CRISPR-Cas9 knockout screening identified KIAA1429 as an ES-dependent gene. Genetic suppression of KIAA1429 inhibited ES cell proliferation and tumorigenicity both in vitro and in vivo. Further studies revealed that KIAA1429 promotes ES tumorigenesis by regulating the ribosome-associated cell cycle and cancer-related inflammation. Interestingly, we found that STAT3 was a target of KIAA1429 and that a STAT3 inhibitor reduced KIAA1429 transcript levels, indicating positive feedback between KIAA1429 and STAT3. Finally, we found that NKX2-2 bound to the KIAA1429 promoter and transactivated KIAA1429. Conclusion Our study systematically analyzed ES-dependent epigenetic/transcriptional regulatory genes and identified KIAA1429 as a biomarker of tumor progression in ES, providing a potential therapeutic target for treating ES.

Arabinogalactan-proteins (AGPs) are cell wall glycoproteins that make up a relatively small component of the extracellular matrix of plants yet have significant influence on wall mechanics and signalling. Present in walls of algae, bryophytes and angiosperms, AGPs have a wide range of functional roles, from signalling, cell expansion and division, embryogenesis, responses to abiotic and biotic stress, plant growth and development. AGPs interact with and influence wall matrix components and plasma membrane proteins to regulate developmental pathways and growth responses, yet the exact mechanisms remain elusive. Comprising a large gene family that is highly diverse, from minimally to highly glycosylated members, varying in their glycan heterogeneity, can be plasma membrane bound or secreted into the extracellular matrix, have members that are highly tissue specific to those with constitutive expression; all these factors have made it extremely challenging to categorise AGPs many qualities and roles. Here we attempt to define some key features of AGPs and their biological functions.

IntroductionFasciclin-like arabinogalactan-proteins (FLAs) are a family of multi-domain glycoproteins present at the cell surface and walls of plants. Arabidopsis thaliana FLA12 and homologs in cotton, Populus, and flax have been shown to play important functions regulating secondary cell wall (SCW) development. FLA12 has been shown to have distinct roles from the closely related FLA11 that also functions during SCW development. The promoter and domain features of FLA12 that regulate functional specificity have not been well characterized.MethodsIn this study, promoter swap experiments of FLA11 and FLA12 were investigated. Mutation of proposed functional regions within FLA12 were used to investigate the role of post-translational modifications on sub-cellular location and trafficking. Domain swap experiments between FLA11 and FLA12 were performed to identify regions of functional specificity.ResultsPromote swap experiments showed that FLA12 is differentially expressed in both stem and rosette leaves compared to FLA11. Post-translational modifications, in particular addition of the glycosylphosphatidylinositol-anchor (GPI-anchor), were shown to be important for FLA12 location at the plasma membrane (PM)/cell wall interface. Domain swap experiments between FLA11 and FLA12 showed that the C-terminal arabinogalactan (AG) glycan motif acts as a key regulatory region differentiating FLA12 functions from FLA11.DiscussionUnderstanding of FLA12 promoter and functional domains has provided new insights into the regulation of SCW development and functional specificity of FLAs for plant growth and development.

Abstract Background Neuroblastoma (NB) is the most common extracranial solid tumor occurring during childhood and high-risk NB patients have a poor prognosis. The amplified MYCN gene serves as an important determinant of a high risk of NB. Methods We performed an integrative screen using public NB tissue and cell line data, and identified that SMAD9 played an important role in high-risk NB. An investigation of the super-enhancers database (SEdb) and chromatin immunoprecipitation sequencing (ChIP-seq) dataset along with biological experiments of incorporating gene knockdown and CRISPR interference (CRISPRi) were performed to identify upstream regulatory mechanism of SMAD9. Gene knockdown and rescue, quantitative real-time PCR (Q-RT-PCR), cell titer Glo assays, colony formation assays, a subcutaneous xenograft model and immunohistochemistry were used to determine the functional role of SMAD9 in NB. An integrative analysis of ChIP-seq data with the validation of CRISPRi and dual-luciferase reporter assays and RNA sequencing (RNA-seq) data with Q-RT-PCR validation was conducted to analyze the downstream regulatory mechanism of SMAD9. Results High expression of SMAD9 was specifically induced by the transcription factors including MYCN, PHOX2B, GATA3 and HAND2 at the enhancer region. Genetic suppression of SMAD9 inhibited MYCN-amplified NB cell proliferation and tumorigenicity both in vitro and in vivo. Further studies revealed that SMAD9 bound to the MYCN promoter and transcriptionally regulate MYCN expression, with MYCN reciprocally binding to the SMAD9 enhancer and transactivating SMAD9, thus forming a positive feedback loop along with the MYCN-associated cancer cell cycle. Conclusion This study delineates that SMAD9 forms a positive transcriptional feedback loop with MYCN and represents a unique tumor-dependency for MYCN-amplified neuroblastoma.

The cell wall is one of the defining features of plants, controlling cell shape, regulating growth dynamics and hydraulic conductivity, as well as mediating plants interactions with both the external and internal environments. Here we report that a putative mechanosensitive Cys-protease DEFECTIVE KERNEL1 (DEK1) influences the mechanical properties of primary cell walls and regulation of cellulose synthesis. Our results indicate that DEK1 is an important regulator of cellulose synthesis in epidermal tissue of Arabidopsis thaliana cotyledons during early post-embryonic development. DEK1 is involved in regulation of cellulose synthase complexes (CSCs) by modifying their biosynthetic properties, possibly through interactions with various cellulose synthase regulatory proteins. Mechanical properties of the primary cell wall are altered in DEK1 modulated lines with DEK1 affecting both cell wall stiffness and the thickness of the cellulose microfibril bundles in epidermal cell walls of cotyledons.

BackgroundBiliary atresia (BA) is a childhood liver disease characterized by fibrous obstruction and obstruction of the extrahepatic biliary system and is one of the most common and serious biliary disorders in infants. Significant inflammation and fibrosis of the liver and biliary tract are the most prominent features, regardless of the initial damage to the BA. Abnormalities in innate or adaptive immunity have been found in human patients and mouse models of BA. We previously reported that children with BA had abnormal lipid metabolism, including free serum carnitine.ObjectiveTo study gene and protein expression levels of the hepatic peroxisome proliferator-activated receptor-α (PPARα) signaling pathway and farnesoid X receptor (FXR) in BA and BA fibrosis, and assess their clinical values.MethodsLow expression of PPARα and NR1H4 (FXR) in BA were validated in the Gene Expression Omnibus database. Functional differences were determined by gene set enrichment analysis based on of PPARα and NR1H4 expression. BA patients from GSE46960 were divided into two clusters by using consensus clustering according to PPARα, NR1H4, and SMAD3 expression levels, and immunoinfiltration analysis was performed. Finally, 58 cases treated in our hospital were used for experimental verification. (IHC: 10 Biliary atresia, 10 choledochal cysts; PCR: 10 Biliary atresia, 14 choledochal cysts; WB: 10 Biliary atresia, 4 choledochal cysts).ResultsBioinformatics analysis showed that the expression of PPARα, CYP7A1 and NR1H4 (FXR) in the biliary atresia group was significantly lower than in the control group. More BA-specific pathways, including TGFβ signaling pathway, P53 signaling pathway, PI3K-AKT-mTOR signaling pathway, etc., are enriched in BA patients with low PPARα and NR1H4 expression. In addition, low NR1H4 expression is abundant in inflammatory responses, IL6/STAT3 signaling pathways, early estrogen responses, IL2 STAT5 signaling pathways, and TGFβ signaling pathways. The TGFβ signaling pathway was significant in both groups. According to the expression of PPARα, NR1H4 and SMAD3, a key node in TGFβ pathway, BA patients were divided into two clusters using consensus clustering. In cluster 2, SMAD3 expression was high, and PPARα and NR1H4 expression were low. In contrast to cluster 1, immune cell infiltration was higher in cluster 2, which was confirmed by immunohistochemistry. The mRNA and protein levels of PPARα and NR1H4 in BA patients were lower than in the control group by immunohistochemistry, Western blot analysis and real-time PCR.ConclusionsThe downregulation of PPARα and NR1H4 (FXR) signaling pathway may be closely related to biliary atresia.

Lipid remodeling of Glycosylphosphatidylinositol (GPI) anchors is required for their maturation and may influence the localization and function of GPI-anchored proteins (GPI-APs). Maturation of GPI-anchors is well characterized in animals and fungi but very little is known about this process in plants. In yeast, the GPI-lipid remodeling occurs entirely at the ER and is initiated by the remodeling enzyme Bst1p (Post-Glycosylphosphatidylinositol Attachment to Proteins inositol deacylase 1 -PGAP1- in mammals and Arabidopsis). Next, the remodeling enzyme Per1p (Post-Glycosylphosphatidylinositol Attachment to Proteins phospholipase 3 -PGAP3- in mammals) removes a short, unsaturated fatty acid of phosphatidylinositol (PI) that is replaced with a very long-chain saturated fatty acid or ceramide to complete lipid remodeling. In mammals, lipid remodeling starts at the ER and is completed at the Golgi apparatus. Studies of the Arabidopsis PGAP1 gene showed that the lipid remodeling of the GPI anchor is critical for the final localization of GPI-APs. Here we characterized loss-of-function mutants of Arabidopsis Per1/PGAP3 like genes (AtPGAP3A and AtPGAP3B). Our results suggest that PGAP3A function is required for the efficient transport of GPI-anchored proteins from the ER to the plasma membrane/cell wall. In addition, loss of function of PGAP3A increases susceptibility to salt and osmotic stresses that may be due to the altered localization of GPI-APs in this mutant. Furthermore, PGAP3B complements a yeast strain lacking PER1 gene suggesting that PGAP3B and Per1p are functional orthologs. Finally, subcellular localization studies suggest that PGAP3A and PGAP3B cycle between the ER and the Golgi apparatus.

Wall-associated kinases/kinase-likes (WAKs/WAKLs) are plant cell surface sensors. A variety of studies have revealed the important functions of WAKs/WAKLs in regulating cell expansion and defense in cells with primary cell walls. Less is known about their roles during the development of the secondary cell walls (SCWs) that are present in xylem vessel (XV) and interfascicular fiber (IF) cells. In this study, we used RNA-seq data to screen Arabidopsis thaliana WAKs/WAKLs members that may be involved in SCW development and identified WAKL8 as a candidate. We obtained T-DNA insertion mutants wakl8-1 (inserted at the promoter region) and wakl8-2 (inserted at the first exon) and compared the phenotypes to wild-type (WT) plants. Decreased WAKL8 transcript levels in stems were found in the wakl8-2 mutant plants, and the phenotypes observed included reduced stem length and thinner walls in XV and IFs compared with those in the WT plants. Cell wall analysis showed no significant changes in the crystalline cellulose or lignin content in mutant stems compared with those in the WT. We found that WAKL8 had alternative spliced versions predicted to have only extracellular regions, which may interfere with the function of the full-length version of WAKL8. Our results suggest WAKL8 can regulate SCW thickening in Arabidopsis stems.

The predominant Fascilin 1 (FAS1)-containing proteins in plants belong to the Fasciclin-Like Arabinogalactan-protein (FLA) family of extracellular glycoproteins. In addition to FAS1 domains, these multi-domain FLA proteins contain glycomotif regions predicted to direct addition of large arabinogalactan (AG) glycans and many contain signal sequences for addition of a glycosylphosphatidylinositol (GPI)-anchor to tether them to the plasma membrane. FLAs are proposed to play both structural and signaling functions by forming a range of interactions in the plant extracellular matrix, similar to FAS1-containing proteins in animals. FLA group B members contain two FAS1 domains and are not predicted to be GPI-anchored. None of the group B members have been functionally characterized or their sub-cellular location resolved, limiting understanding of their function. We investigated the group B FLA16 in Arabidopsis that is predominantly expressed in inflorescence tissues. FLA16 is the most highly expressed FLA in the stem after Group A members FLA11 and FLA12 that are stem specific. A FLA16-YFP fusion protein driven by the endogenous putative FLA16 promoter in wild type background showed expression in cells with secondary cell walls, and FLA16 displayed characteristics of cell wall glycoproteins with moderate glycosylation. Investigation of a fla16 mutant showed loss of FLA16 leads to reduced stem length and altered biomechanical properties, likely as a result of reduced levels of cellulose. Immuno-labeling indicated support for FLA16 location to the plasma-membrane and (apoplastic) cell wall of interfascicular stem fiber cells. Together these results indicate FLA16, a two-FAS1 domain FLAs, plays a role in plant secondary cell wall synthesis and function.

Abstract Hepatocellular carcinoma (HCC) is one of the most malignant tumors in the world, and patients with HCC face a poor prognosis. The conventional therapeutic strategies for HCC have undergone a challenge-riddled evolution owing to side effects and unsatisfactory efficacy. Here, aiming to provide a new method of HCC elimination, we formulated a novel multifunctional nanocapsule (PFP@PLGA/Cu12Sb4S13, PPCu) with applications in contrast-enhanced ultrasound imaging (CEUS) and photothermal therapy (PTT). These PPCu were successfully constructed with an average diameter of 346 nm (polydispersity index, PDI = 0.276). The reinforced contrast ratio of these PPCu was determined by CEUS, revealing their promising applications in image-guided monitoring of HCC treatment. Furthermore, the excellent photoabsorption and biocompatibility indicated by organ H&E staining indicated that PPCu meet quality expectations for use as photothermal transduction agent (PTA). PPCu treatment at 50 °C and higher temperatures efficiently repressed the proliferation, induced the apoptosis and decreased the motility of HCC cells. These effects might have been results of RAS/MAPK/MT-CO1 signaling pathway inhibition. In summary, PPCu were constructed to integrate CEUS and PTT successfully into therapy, which can lead to HCC elimination through RAS/MAPK/MT-CO1 signaling pathway repression.

Background: Oligogalacturonic acids (OGs) are oligomers of alpha-1,4-linked galacturonosyl residues that are released from cell walls by the hydrolysis of polygalacturonic acids upon fruit ripening and under abiotic/biotic stress. OGs may induce ethylene production and fruit ripening, however, the mechanism(s) behind these processes is unknown. Results: Tomato cultivar 'Ailsa Craig' (AC) and mutant Neverripe, ripening inhibitor, non-ripening, and colorless non-ripening fruits were treated with OGs at different stages. Only AC fruits at mature green stage 1 showed an advanced ripening phenomenon, although transient ethylene production was detected in all of the tomato fruits. Ethylene synthesis genes LeACS2 and LeACO1 were rapidly up-regulated, and the phosphorylated LeACS2 protein was detected after OGs treatment. Protein kinase/phosphatase inhibitors significantly affected the ripening process induced by the OGs. As a potential receptor of OGs, LeWAKL2 was also up-regulated in their presence. Conclusions: We demonstrated that OGs promoted tomato fruit ripening by inducing ethylene synthesis through the regulation of LeACS2 at transcriptional and post-translational levels. [ABSTRACT FROM AUTHOR]