Crucial roles of the BRCA1-BARD1 E3 ubiquitin ligase activity in homology-directed DNA repair.

The tumor-suppressor breast cancer 1 (BRCA1) in complex with BRCA1-associated really interesting new gene (RING) domain 1 (BARD1) is a RING-type ubiquitin E3 ligase that modifies nucleosomal histone and other substrates. The importance of BRCA1-BARD1 E3 activity in tumor suppression remains highly controversial, mainly stemming from studying mutant ligase-deficient BRCA1-BARD1 species that we show here still retain significant ligase activity. Using full-length BRCA1-BARD1, we establish robust BRCA1-BARD1-mediated ubiquitylation with specificity, uncover multiple modes of activity modulation, and construct a truly ligase-null variant and a variant specifically impaired in targeting nucleosomal histones. Cells expressing either of these BRCA1-BARD1 separation-of-function alleles are hypersensitive to DNA-damaging agents. Furthermore, we demonstrate that BRCA1-BARD1 ligase is not only required for DNA resection during homology-directed repair (HDR) but also contributes to later stages for HDR completion. Altogether, our findings reveal crucial, previously unrecognized roles of BRCA1-BARD1 ligase activity in genome repair via HDR, settle prior controversies regarding BRCA1-BARD1 ligase functions, and catalyze new efforts to uncover substrates related to tumor suppression.

The chromatin remodeler RSF1 coordinates epigenetic marks for transcriptional repression and DSB repair.

DNA lesions impact on local transcription and the damage-induced transcriptional repression facilitates efficient DNA repair. However, how chromatin dynamics cooperates with these two events remained largely unknown. We here show that histone H2A acetylation at K118 is enriched in transcriptionally active regions. Under DNA damage, the RSF1 chromatin remodeling factor recruits HDAC1 to DSB sites. The RSF1-HDAC1 complex induces the deacetylation of H2A(X)-K118 and its deacetylation is indispensable for the ubiquitination of histone H2A at K119. Accordingly, the acetylation mimetic H2A-K118Q suppressed the H2A-K119ub level, perturbing the transcriptional repression at DNA lesions. Intriguingly, deacetylation of H2AX at K118 also licenses the propagation of γH2AX and recruitment of MDC1. Consequently, the H2AX-K118Q limits DNA repair. Together, the RSF1-HDAC1 complex controls the traffic of the DNA damage response and transcription simultaneously in transcriptionally active chromatins. The interplay between chromatin remodelers and histone modifiers highlights the importance of chromatin versatility in the maintenance of genome integrity.

USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing.

Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.

De novo phosphorylation of H2AX by WSTF regulates transcription-coupled homologous recombination repair.

Histone H2AX undergoes a phosphorylation switch from pTyr142 (H2AX-pY142) to pSer139 (γH2AX) in the DNA damage response (DDR); however, the functional role of H2AX-pY142 remains elusive. Here, we report a new layer of regulation involving transcription-coupled H2AX-pY142 in the DDR. We found that constitutive H2AX-pY142 generated by Williams-Beuren syndrome transcription factor (WSTF) interacts with RNA polymerase II (RNAPII) and is associated with RNAPII-mediated active transcription in proliferating cells. Also, removal of pre-existing H2AX-pY142 by ATM-dependent EYA1/3 phosphatases disrupts this association and requires for transcriptional silencing at transcribed active damage sites. The following recovery of H2AX-pY142 via translocation of WSTF to DNA lesions facilitates transcription-coupled homologous recombination (TC-HR) in the G1 phase, whereby RAD51 loading, but not RPA32, utilizes RNAPII-dependent active RNA transcripts as donor templates. We propose that the WSTF-H2AX-RNAPII axis regulates transcription and TC-HR repair to maintain genome integrity.

Improved Multilayered (Bi,Sc)O3-(Pb,Ti)O3 Piezoelectric Energy Harvesters Based on Impedance Matching Technique.

As a piezoelectric material, (Bi,Sc)O3-(Pb,Ti)O3 ceramics have been tested and analyzed for sensors and energy harvester applications owing to their relatively high Curie temperature and high piezoelectric coefficient. In this work, we prepared optimized (Bi,Sc)O3-(Pb,Ti)O3 piezoelectric materials through the conventional ceramic process. To increase the output energy, a multilayered structure was proposed and designed, and to obtain the maximum output energy, impedance matching techniques were considered and tested. By varying and measuring the energy harvesting system, we confirmed that the output energies were optimized by varying the load resistance. As the load resistance increased, the output voltage became saturated. Then, we calculated the optimized output power using the electric energy formula. Consequently, we identified the highest output energy of 5.93 µW/cm2 at 3 MΩ for the quadruple-layer harvester and load resistor using the impedance matching system. We characterized and improved the electrical properties of the piezoelectric energy harvesters by introducing impedance matching and performing the modeling of the energy harvesting component. Modeling was conducted for the piezoelectric generator component by introducing the mechanical force dependent voltage sources and load resistors and piezoelectric capacitor connected in parallel. Moreover, the generated output voltages were simulated by introducing an impedance matching technique. This work is designed to explain the modeling of piezoelectric energy harvesters. In this model, the relationship between applied mechanical force and output energy was discussed by employing experimental results and simulation.

Ring finger protein 126 (RNF126) suppresses ionizing radiation-induced p53-binding protein 1 (53BP1) focus formation.

Cells have evolved sophisticated mechanisms to maintain genomic integrity in response to DNA damage. Ionizing radiation (IR)-induced DNA damage results in the formation of IR-induced foci (iRIF) in the nucleus. The iRIF formation is part of the DNA damage response (DDR), which is an essential signaling cascade that must be strictly regulated because either the loss of or an augmented DDR leads to loss of genome integrity. Accordingly, negative regulation of the DDR is as critical as its activation. In this study, we have identified ring finger protein 126 (RNF126) as a negative regulator of the DDR from a screen of iRIF containing 53BP1. RNF126 overexpression abolishes not only the formation of 53BP1 iRIF but also of RNF168, FK2, RAP80, and BRCA1. However, the iRIF formation of γH2AX, MDC1, and RNF8 is maintained, indicating that RNF126 acts between RNF8 and RNF168 during the DDR. In addition, RNF126 overexpression consistently results in the loss of RNF168-mediated H2A monoubiquitination at lysine 13/15 and inhibition of the non-homologous end joining capability. Taken together, our findings reveal that RNF126 is a novel factor involved in the negative regulation of DDR, which is important for sustaining genomic integrity.

Energy Harvesting Through Wasted Thermal Energy by Light Grid Sources.

In this study, emitted light energy and the recycling of thermal energy from the arrays of a light emitting diode system were investigated. A light grid system is composed of the array of high power LED chips, thermoelement and heat sink. High power LED source has an advantage of high luminous efficiency, which combined with wasted thermal energy. Thermal energy loss can be regarded wasted energy. However, this wasting thermal energy can be effectively converted to the electrical energy from thermoelement and heat sink of a light grid system. By introducing the light grid system, the optical energy and thermal energy can be more effectively managed. In particular, we have intensively studied energy conversion efficiency of light grid system and energy harvesting characteristic through thermal energy.

Relaxation-Related Piezoelectric and Dielectric Behavior of Bi(Mg,Ti)O3⁻PbTiO3 Ceramic.

Piezoelectric and dielectric materials have attracted much attention for their functional device applications. Despite its excellent piezoelectric properties, the content of lead in piezoelectric materials should be restricted to prevent future environmental problems. Therefore, reduced lead content in piezoelectric materials with similar piezoelectric properties is favorable. In our research, piezoelectric materials with decreased lead content will be studied and discussed. Even though the lead content is decreased in Bi(Mg0.5Ti0.5)O3-PbTiO3 ceramics, they show piezoelectric properties similar to that of lead zirconate titanate (PZT)-based materials. We believe this high piezoelectric behavior is related to the relaxation behavior of Bi(Mg0.5Ti0.5)O3-PbTiO3 (BMT-PT) ceramics. In this study, 0.62Bi(Mg0.5Ti0.5)O3-0.38PbTiO3 ceramics were prepared by the conventional sintering process. These piezoelectric ceramics were sintered at varying temperatures of 975-1100 °C. Crystallinity and structural properties were analyzed and discussed. X-ray diffraction pattern analysis demonstrated that the optimal sintering temperature was around 1075 °C. A very high Curie temperature of 447 °C was recorded for 0.62BMT-0.38PT ceramics sintered at 1075 °C. For the first time, we found that the origin of the high Curie temperature, d33, and the dielectric constant is the relaxation behavior of different dipoles in 0.62BMT-0.38PT ceramics.

Pyroelectric and Piezoelectric Properties of (1-x)Na0.5K0.5NbO3-xBiScO3 Lead-Free Ceramics.

In this research, the pyroelectric and piezoelectric properties of (1 - x)Na0.5K0.5NbO3-xBiScO3 ceramics were investigated and analyzed. (Na, K)NbO3 based (1 - x)Na0.5K0.5NbO3-xBiScO3 ceramics were prepared by a conventional mixed oxide method. As the substituent, BiScO3 material enhanced the sintering mechanism of NKN ceramic, which improved the density, pyroelectric and piezoelectric properties, without any structural distortion. In this study, the structural dependent improved piezoelectric properties of (1 - x)Na0.5K0.5NbO3-xBiScO3 ceramics were investigated with various sintering temperatures. Also, the pyroelectric properties of (1 - x)Na0.5K0.5NbO3-xBiScO3 ceramics were observed up to 200 °C for the devices applications. The crystalline structures of the (1-x)Na0.5K0.5NbO3-x BiScO3 ceramics were measured by X-ray diffraction (XRD). The microstructure was examined by field emission scanning electron microscopy (FE-SEM). In addition, piezo-electric charge coefficient d33 and pyroelectric coefficient will be discussed.

Angle Dependent 3-D Directional Patterning of Flexible Films by Infrared Laser.

Laser direct patterning (LDP) technology has attracted great attention due to its process simplification and environmental friendliness. It is replacing the existing photo-lithography technology. Infrared (IR) laser equipment also has advantages such as low cost and long duration, although it is difficult to implement fine line width compared to ultraviolet (UV) or excimer laser. Therefore, it is very important to realize 3-D patterning system based on cheap infrared laser system. In this paper, 3-D infrared laser direct patterning system was designed by introducing an etching process on Ag paste/ITO/PET flexible film. Such 3-D patterned Ag paste/ITO/PET flexible films showed feasibility and effectiveness of 3-D laser directional patterning technologies. Etch ratio of the fabricated Ag paste/ITO/PET flexible film after 3-D infrared laser direct patterning was then systematically investigated.

Recycled Thermal Energy from High Power Light Emitting Diode Light Source.

In this research, the recycled electrical energy from wasted thermal energy in high power Light Emitting Diode (LED) system will be investigated. The luminous efficiency of lights has been improved in recent years by employing the high power LED system, therefore energy efficiency was improved compared with that of typical lighting sources. To increase energy efficiency of high power LED system further, wasted thermal energy should be re-considered. Therefore, wasted thermal energy was collected and re-used them as electrical energy. The increased electrical efficiency of high power LED devices was accomplished by considering the recycled heat energy, which is wasted thermal energy from the LED. In this work, increased electrical efficiency will be considered and investigated by employing the high power LED system, which has high thermal loss during the operating time. For this research, well designed thermoelement with heat radiation system was employed to enhance the collecting thermal energy from the LED system, and then convert it as recycled electrical energy.

Plasma micoRNA-122 as a predictive marker for treatment response following transarterial chemoembolization in patients with hepatocellular carcinoma.

BACKGROUND AND AIM: Circulating microRNA (miR)-122 has recently been investigated as a potential biomarker of various hepatic diseases, such as chronic hepatitis and hepatocellular carcinoma (HCC). We investigated the association between plasma miR-122 levels and the treatment outcomes following transarterial chemoembolization (TACE) in HCC patients. METHODS: We included 177 HCC patients treated with TACE in the study; TACE refractoriness and liver transplantation-free survival were evaluated during follow up. Pretreatment plasma miR-122 levels were assessed using quantitative real-time polymerase chain reaction. Relative quantification of miR-122 expression (fold change) was determined using the 2(-ΔΔCt) method. MiR-16 was used as an internal control for the normalization of miRNA data. RESULTS: During the mean follow up of 22.4 (range, 1-79) months, 112 (69.5%) patients exhibited TACE refractoriness. Multivariate analyses showed that tumor number (hazard ratio [HR], 2.51; 95% confidence interval [CI], 1.43-4.41; P = 0.001) and tumor size (HR, 2.65; 95% CI, 1.62-4.32; P = 0.000) can independently predict overall TACE refractoriness. High miR-122 expression (> 100) was associated with early TACE refractoriness (within 1 year; HR, 2.77; 95% CI, 1.12-6.86; P = 0.028), together with tumor number (HR, 22.73; 95% CI, 2.74-188.66; P = 0.004) and tumor size (HR, 4.90; 95% CI, 1.99-12.06; P = 0.001). Univariate analyses showed that high miR-122 expression tends to be associated with poor liver transplantation-free survival (HR, 1.42; 95% CI, 0.95-2.11; P = 0.085). However, it was statistically insignificant in multivariate analysis. CONCLUSION: High expression levels of plasma miR-122 are associated with early TACE refractoriness in HCC patients treated with TACE.

Hepatitis B virus X protein activates the ATM-Chk2 pathway and delays cell cycle progression.

Genetic instability is intimately associated with tumour development. In particular, liver cancers associated with hepatitis B virus (HBV) exhibit high genetic instability; however, our understanding of the underlying molecular mechanisms remains limited. In this study, we found that γ-H2AX, a marker of DNA double-strand breaks (DSBs), and the levels of phospho-Chk2 (p-Chk2, the activated form) were significantly elevated in HBV-associated hepatocellular carcinomas and neighbouring regenerating nodules. Likewise, introduction of the pHBV or pMyc-HBx plasmids into cells induced accumulation of γ-H2AX foci and increased the p-Chk2 level. In these cells, inhibitory phosphorylation of Cdc25C phosphatase (Ser(216)) and CDK1 (Tyr(15)) was elevated; consequently, cell-cycle progression was delayed at G2/M phase, suggesting that activation of the ATM-Chk2 pathway by the HBV X protein (HBx) induces cell-cycle delay. Accordingly, inhibition of ataxia telangiectasia mutated (ATM) by caffeine or siRNA abolished the increase in the p-Chk2 level and restored the delayed CDK1 kinase activity in ChangX cells. We also found that cytoplasmic HBx, but not nuclear HBx, induced reactive oxygen species (ROS) production and led to the accumulation of γ-H2AX foci and the increased p-Chk2 level. Together, these data indicate that HBx-induced ROS accumulation induces DNA damage that activates the ATM-Chk2 pathway. Our findings provide insight into the mechanisms of HBV pathogenesis.

Autophagy inhibition enhances silibinin-induced apoptosis by regulating reactive oxygen species production in human prostate cancer PC-3 cells.

Silibinin is a major bioactive component of silymarin and has anticancer effects on cancer cell line and has been used as a supportive therapy for chronic inflammatory liver condition. These anticancer effects of silibinin have been demonstrated both in vitro and in vivo cancer models. Although various evidences showed apoptosis signaling pathways by silibinin, there is no report to address the clearly mechanism of silibinin-induced autophagy in prostate cancer PC-3 cells. Our study showed that silibinin triggered autophagy through up-regulation of microtubule-associated protein 1 light chain 3 (LC3)-II, formation of acidic vesicular organelles (AVO) and punctuate of GFP-LC3, which was inhibited by 3-methyladenine (3-MA), an inhibitor of specific autophagy. In addition, silibinin induced autophagy through production of reactive oxygen species (ROS). Inhibition of ROS with diphenyleneiodonium (DPI), a ROS inhibitor, attenuated silibinin-triggered autophagy. Inhibition of autophagy with 3-MA enhanced the silibinin-induced apoptosis through the regulation of caspase-3 and PARP. These results suggested that silibinin induced autophagy by regulating ROS and its mechanism played a protective role against apoptosis in PC-3 cells.

Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis.

Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic cell death mechanism characterized by endoplasmic reticulum and mitochondria dilation. Intriguingly, oncogenic HRas sensitizes non-transformed cells to VCP inhibition-mediated paraptosis. The susceptibility of cancer cells to VCP inhibition is attributed to the non-attenuation and recovery of protein synthesis under proteotoxic stress. Mechanistically, mTORC2/Akt activation and eIF3d-dependent translation contribute to translational rebound and amplification of proteotoxic stress. Furthermore, the ATF4/DDIT4 axis augments VCP inhibition-mediated paraptosis by activating Akt. Given that hyperactive Akt counteracts chemotherapeutic-induced apoptosis, VCP inhibition presents a promising therapeutic avenue to exploit Akt-associated vulnerabilities in cancer cells by triggering paraptosis while safeguarding normal cells.

HBxAPα/Rsf-1-mediated HBx-hBubR1 interactions regulate the mitotic spindle checkpoint and chromosome instability.

Hepatitis B virus (HBV) X protein (HBx), encoded by the HBV genome, is involved in the development of HBV-mediated liver cancer, whose frequency is highly correlated with chromosomal instability (CIN). We reported previously that HBx induces mitotic checkpoint dysfunction by targeting the human serine/threonine kinase BubR1 (hBubR1). However, the underlying mechanism remained unresolved. Here, we show that HBx protein-associated protein α (HBxAPα)/Rsf-1 associates with hBubR1 and HBx in the chromatin fraction during mitosis. Depletion of HBxAPα/Rsf-1 abolished the interaction between HBx and hBubR1, indicating that HBxAPα/Rsf-1 mediates these interactions. Knockdown of HBxAPα/Rsf-1 with small interfering RNA did not affect the recruitment of hBubR1 to kinetochores; however, it did significantly impair HBx targeting to kinetochores. A deletion mutant analysis revealed that two Kunitz domains of HBx, the Cdc20-binding domain of hBubR1 and full-length of HBxAPα/Rsf-1 were essential for these interactions. Thus, binding of HBx to hBubR1, stabilized by HBxAPα/Rsf-1, significantly attenuated hBubR1 binding to Cdc20 and consequently increased the rate of mitotic aberrations. Collectively, our data show that the HBx impairs hBubR1 function and induces CIN through HBxAPα/Rsf-1, providing a novel mechanism for induction of genomic instability by a viral pathogen in hepatocarcinogenesis.

Pipernonaline from Piper longum Linn. induces ROS-mediated apoptosis in human prostate cancer PC-3 cells.

The antiproliferation effects of pipernonaline, a piperine derivative, were investigated on human prostate cancer PC-3 cells. It inhibited growth of androgen independent PC-3 and androgen dependent LNCaP prostate cells in a dose-dependent (30-90 µM) and time-dependent (24-48 h) manner. The growth inhibition of PC-3 cells was associated with sub-G(1) and G(0)/G(1) accumulation, confirmed by the down-regulation of CDK2, CDK4, cyclin D1 and cyclin E, which are correlated with G(1) phase of cell cycle. Pipernonaline up-regulated cleavage of procaspase-3/PARP, but did not change expression of proapoptotic bax and antiapoptotic bcl-2 proteins. Its caspase-3 activation was confirmed by the caspase-3 assay kit. In addition, pipernonaline caused the production of reactive oxygen species (ROS), increase of intracellular Ca(2+), and mitochondrial membrane depolarization, which these phenomena were reversed by N-acetylcysteine, a ROS scavenger. The results suggest that pipernonaline exhibits apoptotic properties through ROS production, which causes disruption of mitochondrial function and Ca(2+) homeostasis and leads to its downstream events including activation of caspase-3 and cleavage of PARP in PC-3 cells. This is the first report of pipernonaline toward the anticancer activity of prostate cancer cells, which provides a role for candidate agent as well as the molecular basis for human prostate cancer.

Phosphorylation of Ran-binding protein-1 by Polo-like kinase-1 is required for interaction with Ran and early mitotic progression.

Polo-like kinase-1 (Plk1) is essential for progression of mitosis and localizes to centrosomes, central spindles, midbody, and kinetochore. Ran, a small GTPase of the Ras superfamily, plays a role in microtubule dynamics and chromosome segregation during mitosis. Although Ran-binding protein-1 (RanBP1) has been reported as a regulator of RanGTPase for its mitotic functions, the action mechanism between Ran and RanBP1 during mitosis is still unknown. Here, we demonstrated in vitro and in vivo phosphorylation of RanBP1 by Plk1 as well as the importance of phosphorylation of RanBP1 in the interaction between Plk1 and Ran during early mitosis. Both phosphorylation-defective and N-terminal deletion mutant constructs of RanBP1 disrupted the interaction with Ran, and depletion of Plk1 also disrupted the formation of a complex between Ran and RanBP1. In addition, the results from both ectopic expression of phosphorylation-defective mutant construct and a functional complementation on RanBP1 deficiency with this mutant indicated that phosphorylation of RanBP1 by Plk1 might be crucial to microtubule nucleation and spindle assembly during mitosis.

Transcriptional regulation and chromatin dynamics at DNA double-strand breaks.

In eukaryotic cells, DNA damage can occur at any time and at any chromatin locus, including loci at which active transcription is taking place. DNA double-strand breaks affect chromatin integrity and elicit a DNA damage response to facilitate repair of the DNA lesion. Actively transcribed genes near DNA lesions are transiently suppressed by crosstalk between DNA damage response factors and polycomb repressive complexes. Epigenetic modulation of the chromatin environment also contributes to efficient DNA damage response signaling and transcriptional repression. On the other hand, RNA transcripts produced in the G1 phase, as well as the active chromatin context of the lesion, appear to drive homologous recombination repair. Here, we discuss how the ISWI family of chromatin remodeling factors coordinates the DNA damage response and transcriptional repression, especially in transcriptionally active regions, highlighting the direct modulation of the epigenetic environment.

Synthesis of a nitrogen doped reduced graphene oxide based ceramic polymer composite nanofiber film for wearable device applications.

In this study, piezoelectric composite nanofiber films were fabricated by introducing nitrogen-doped-reduced-graphene-oxide as a conductive material to a P(VDF-TrFE) polymer and a BiScO3-PbTiO3 ceramic composite employing an electrospinning process. Nitrogen was doped/substituted into rGO to remove or compensate defects formed during the reduction process. Electro-spinning process was employed to extract piezoelectric composite nanofiber films under self-poling condition. Interdigital electrodes was employed to make planner type energy harvesters to collect electro-mechanical energy applied to the flexible energy harvester. From the piezoelectric composite with interdigital electrode, the effective dielectric permittivity extracted from the conformal mapping method. By introducing BS-PT ceramics and N-rGO conductors to the P(VDF-TrFE) piezoelectric composite nanofiber films, the effective dielectric permittivity was improved from 8.2 to 15.5. This improved effective dielectric constant probably come from the increased electric flux density due to the increased conductivity. Fabricated interdigital electrode using this thin composite nanofiber film was designed and tested for wearable device applications. An external mechanical force of 350 N was applied to the composite nanofiber-based energy harvester with interdigital electrodes at a rate of 0.6 Hz, the peak voltage and current were 13 V and 1.25 µA, respectively. By optimizing the device fabrication, the open-circuit voltage, stored voltage, and generated output power obtained were 12.4 V, 3.78 V, and 6.3 µW, respectively.