Posttranscriptional regulation of de novo lipogenesis by glucose-induced O-GlcNAcylation.

O-linked ß-N-acetyl glucosamine (O-GlcNAc) is attached to proteins under glucose-replete conditions; this posttranslational modification results in molecular and physiological changes that affect cell fate. Here we show that posttranslational modification of serine/arginine-rich protein kinase 2 (SRPK2) by O-GlcNAc regulates de novo lipogenesis by regulating pre-mRNA splicing. We found that O-GlcNAc transferase O-GlcNAcylated SRPK2 at a nuclear localization signal (NLS), which triggers binding of SRPK2 to importin α. Consequently, O-GlcNAcylated SRPK2 was imported into the nucleus, where it phosphorylated serine/arginine-rich proteins and promoted splicing of lipogenic pre-mRNAs. We determined that protein nuclear import by O-GlcNAcylation-dependent binding of cargo protein to importin α might be a general mechanism in cells. This work reveals a role of O-GlcNAc in posttranscriptional regulation of de novo lipogenesis, and our findings indicate that importin α is a "reader" of an O-GlcNAcylated NLS.

BGL3 lncRNA mediates retention of the BRCA1/BARD1 complex at DNA damage sites.

Long non-coding RNAs (lncRNAs) are emerging regulators of genomic stability and human disease. However, the molecular mechanisms by which nuclear lncRNAs directly contribute to DNA damage responses remain largely unknown. Using RNA antisense purification coupled with quantitative mass spectrometry (RAP-qMS), we found that the lncRNA BGL3 binds to PARP1 and BARD1, exhibiting unexpected roles in homologous recombination. Mechanistically, BGL3 is recruited to DNA double-strand breaks (DSBs) by PARP1 at an early time point, which requires its interaction with the DNA-binding domain of PARP1. BGL3 also binds the C-terminal BRCT domain and an internal region (amino acids 127-424) of BARD1, which mediates interaction of the BRCA1/BARD1 complex with its binding partners such as HP1γ and RAD51, resulting in BRCA1/BARD1 retention at DSBs. Cells depleted for BGL3 displayed genomic instability and were sensitive to DNA-damaging reagents. Overall, our findings underscore the biochemical versatility of RNA as a mediator molecule in the DNA damage response pathway, which affects the accumulation of BRCA1/BARD1 at DSBs.

Effect of Micro-Steps on Twinning and Interfacial Segregation in Mg-Ag Alloy.

Twinning structures and their interfacial segregation play a key role in strengthening of magnesium alloys. Micro-steps are frequently existed in the incoherent twin boundaries, while the effect of them on interface and interfacial segregation is still not clear. In this work, we performed an atomic-scale microstructure analysis using high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) to explore the effect of micro-steps on twin and its interfacial segregation in Mg-Ag alloy. Diffraction pattern of the incoherent {10 1 ¯ 1} twin shows that the misorientation has a slight tilt of 5° from its theoretical angle of 125° due to the accumulated effects of the micro-steps and their misfit dislocations in twin boundaries. Most of the micro-steps in {10 1 ¯ 1} twin boundary are in the height of 2 d ( 10 1 ¯ 1 ) and 4 d ( 10 1 ¯ 1 ) , respectively, and both of them have two types according to whether there are dislocations on the micro-steps. The twin boundary is interrupted by many micro-steps, which leads to a step-line distributed interfacial segregation. Moreover, the Ag tends to segregate to dislocation cores, which results in the interruption of interfacial segregation at the micro-steps with dislocations.

The deubiquitylating enzyme USP15 regulates homologous recombination repair and cancer cell response to PARP inhibitors.

Poly-(ADP-ribose) polymerase inhibitors (PARPi) selectively kill breast and ovarian cancers with defects in homologous recombination (HR) caused by BRCA1/2 mutations. There is also clinical evidence for the utility of PARPi in breast and ovarian cancers without BRCA mutations, but the underlying mechanism is not clear. Here, we report that the deubiquitylating enzyme USP15 affects cancer cell response to PARPi by regulating HR. Mechanistically, USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1, which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BARD1-HP1γ interaction, resulting in BRCA1/BARD1 retention at DSBs. USP15 knockout mice exhibit genomic instability in vivo. Furthermore, cancer-associated USP15 mutations, with decreased USP15-BARD1 interaction, increases PARP inhibitor sensitivity in cancer cells. Thus, our results identify a novel regulator of HR, which is a potential biomarker for therapeutic treatment using PARP inhibitors in cancers.

And-1 coordinates with CtIP for efficient homologous recombination and DNA damage checkpoint maintenance.

To prevent genomic instability, cells respond to DNA lesions by blocking cell cycle progression and initiating DNA repair. Homologous recombination repair of DNA breaks requires CtIP-dependent resection of the DNA ends, which is thought to play a key role in activation of CHK1 kinase to induce the cell cycle checkpoint. But the mechanism is still not fully understood. Here, we establish that And-1, a replisome component, promotes DNA-end resection and DNA repair by homologous recombination. Mechanistically, And-1 interacts with CtIP and regulates CtIP recruitment to DNA damage sites. And-1 localizes to sites of DNA damage dependent on MDC1-RNF8 pathway, and is required for resistance to many DNA-damaging and replication stress-inducing agents. Furthermore, we show that And-1-CtIP axis is critically required for sustained ATR-CHK1 checkpoint signaling and for maintaining both the intra-S- and G2-phase checkpoints. Our findings thus identify And-1 as a novel DNA repair regulator and reveal how the replisome regulates the DNA damage induced checkpoint and genomic stability.

MiRNA-543 promotes osteosarcoma cell proliferation and glycolysis by partially suppressing PRMT9 and stabilizing HIF-1α protein.

Osteosarcoma (OS) is the most common primary bone tumor, occurring frequently in adolescents and possessing a high malignant severity. MicroRNAs play critical roles during OS development. Thus, elucidation of the involvement of specific microRNAs in the development of OS may provide novel therapeutic targets for OS treatment. Here, we showed that in the OS specimens from patients, the levels of miR-543 were significantly increased whereas the levels of PRMT9 were significantly decreased, compared to the paired normal bone tissue. Moreover, miR-543 and PRMT9 inversely correlated in the OS cell lines. Bioinformatics analyses predicted that miR-543 may target the 3'-UTR of PRMT9 mRNA to inhibit its translation, which was confirmed by luciferase-reporter assay. MiR-543 promoted OS cell proliferation in vitro and in vivo. Mechanistically, miR-543 inhibited PRMT9-enhanced cell oxidative phosphorylation, while miR-543 depletion promoted PRMT9-increased HIF-1α instability and inhibited glycolysis in OS cells. Clinically, miR-543 expression was negatively correlated with PRMT9 expression in OS tissues. Together, our data provide important evidence for glycolysis in OS development, and suggest that targeting glycolytic pathway through miR-543/PRMT9/HIF-1α axis may represent a potential therapeutic strategy to eradicate OS cells.

The Deubiquitylating Enzyme USP4 Cooperates with CtIP in DNA Double-Strand Break End Resection.

DNA end resection is a highly regulated and critical step in DNA double-stranded break (DSB) repair. In higher eukaryotes, DSB resection is initiated by the collaborative action of CtIP and the MRE11-RAD50-NBS1 (MRN) complex. Here, we find that the deubiquitylating enzyme USP4 directly participates in DSB resection and homologous recombination (HR). USP4 confers resistance to DNA damage-inducing agents. Mechanistically, USP4 interacts with CtIP and MRN via a specific, conserved region and the catalytic domain of USP4, respectively, and regulates CtIP recruitment to sites of DNA damage. We also find that USP4 autodeubiquitylation is essential for its HR functions. Collectively, our findings identify USP4 as a key regulator of DNA DSB end resection.

[The effect of microencapsulated NGF-expressing NIH-3T3 cells on bioengineered dermis function in vitro].

Nerve growth factor (NGF) can promote the regeneration of peripheral nerve as well as contraction and reepithelization of wound. We constructed a bioengineered dermis containing microencapsulated NGF-expressing NIH-3T3 cells and study the effect of the microencapsule to the bioengineered dermis and seed cells. NGF gene was transfected into NIH-3T3 cells and enclosed into alginate-poly-L-lysine-alginate (APA) microencapsulation and cultivated in vitro. Content of NGF in microencapsules culturing supernatant was measured by enzyme linked immunosorbent assay (ELISA) method. These microencapsules were co-cultured with epidermic cells and fibroblast cells. Bioengineered dermis was constructed with NGF-expressing micorencapsules as seed cells using tissue engineering method. NIH-3T3 microencapsules, empty microencapsules, normal culture media were control groups. After one week culture, the characteristics of the dermis were described by MTT test, the content of hydroxyproline (HP), HE staining and ultrastructure photograph. We found the NGF-expressing microencapsulates can secret NGF steadly after cultured 8w in vitro, promot the proliferation of epidermic cells and secret collagen of fibroblast cells. These functions can maintaine in bioengineered dermis. So NGF-expressing NIH-3T3 microencapsulates can promote the quality of bioengineered dermis.

Extraction and retrieval of potassium from water hyacinth (Eichhornia crassipes).

Studies were carried out on extraction and retrieval of potassium from water hyacinth (Eichhornia crassipes). The stem and leaf were subjected to 13 treatments. The highest rate of K removal following HCl treatment was 69.7% K. Most effective removal of suspended organic substances, Ca2+ and Mg2+ were achieved at pH approximately 13, when 88.0% of K remained in filtrate. Maximum K in precipitate following this step was achieved with tartaric acid additions at n(C4H6O6)/n(K+) of 1.72 when precipitating at 4 degrees C for 3h, which resulted in 72.3% of K removal from the solution. Over the entire process, 44.3% of K in the dried stem-leaf sample of water hyacinth was retrieved in the form of KC4H5O6. This process demonstrated the potential for use of water hyacinth as a resource of potassium to produce potassium salts and provide a valuable end use for the plant, which could be highly invasive in aquatic ecosystems.

[Influence of vacuum-assisted closure technique on expression of Bcl-2 and NGF/NGFmRNA during wound healing].

OBJECTIVE: To explore the influence of vacuum-assisted closure technique (VAC) on expression of Bcl-2 and NGF during wound healing. METHODS: Eighty Sprague-Dawley rats were randomly divided into 4 groups with 20 rats of each. Group T was the experimental group; group C1, C2 and C3 were the control groups. In group T and group C1, capsaicin was injected subcutaneously to the back of the rats to destroy the sensory nerve. VAC was employed to the wound of the rats in group T and C2 three times a day at 80 mmHg negative pressure. In all the groups, tissue samples were taken from the wound edge and granulation at 1, 3, 6, 9 and 12 days after the injury. Immunohistochemistry and in situ hybridization were used to detect the expression of Bcl-2 and NGF/NGFmRNA in the samples. RESULTS: In group C2 and C3, the expression of Bcl-2 and NGF/NGFmRNA was obvious, which increased gradually and reached the peak at the 9th day. In the process of wound healing, the expression Bcl-2 and NGF/NGFmRNA was higher in the group C2 than in group C3 (P < 0.05). The expression Bcl-2 and NGF/NGFmRNA in group T and C1 was lower than group C2 and C3 (P < 0.05). CONCLUSION: The application of the vacuum-assisted closure technique during wound healing increases the expression of the apoptosic modulation related protein Bcl-2 and affects the expression of NGF/NGFmRNA, which may promote the wound healing process.