TOPORS-mediated RAD51 SUMOylation facilitates homologous recombination repair.

Homologous recombination (HR) is critical for error-free repair of DNA double-strand breaks. Chromatin loading of RAD51, a key protein that mediates the recombination, is a crucial step in the execution of the HR repair. Here, we present evidence that SUMOylation of RAD51 is crucial for the RAD51 recruitment to chromatin and HR repair. We found that topoisomerase 1-binding arginine/serine-rich protein (TOPORS) induces the SUMOylation of RAD51 at lysine residues 57 and 70 in response to DNA damaging agents. The SUMOylation was facilitated by an ATM-induced phosphorylation of TOPORS at threonine 515 upon DNA damage. Knockdown of TOPORS or expression of SUMOylation-deficient RAD51 mutants caused reduction in supporting normal RAD51 functions during the HR repair, suggesting the physiological importance of the modification. We found that the SUMOylation-deficient RAD51 reduces the association with its crucial binding partner BRCA2, explaining its deficiency in supporting the HR repair. These findings altogether demonstrate a crucial role for TOPORS-mediated RAD51 SUMOylation in promoting HR repair and genomic maintenance.

SIAH2 regulates DNA end resection and replication fork recovery by promoting CtIP ubiquitination.

Human CtIP maintains genomic integrity primarily by promoting 5' DNA end resection, an initial step of the homologous recombination (HR). A few mechanisms have been suggested as to how CtIP recruitment to damage sites is controlled, but it is likely that we do not yet have full understanding of the process. Here, we provide evidence that CtIP recruitment and functioning are controlled by the SIAH2 E3 ubiquitin ligase. We found that SIAH2 interacts and ubiquitinates CtIP at its N-terminal lysine residues. Mutating the key CtIP lysine residues impaired CtIP recruitment to DSBs and stalled replication forks, DSB end resection, overall HR repair capacity of cells, and recovery of stalled replication forks, suggesting that the SIAH2-induced ubiquitination is important for relocating CtIP to sites of damage. Depleting SIAH2 consistently phenocopied these results. Overall, our work suggests that SIAH2 is a new regulator of CtIP and HR repair, and emphasizes that SIAH2-mediated recruitment of the CtIP is an important step for CtIP's function during HR repair.

Determining terahertz resonant peaks of biomolecules in aqueous environment.

The resonant peaks of biomolecules provide information on the molecules' physical and chemical properties. Although many biomolecules have resonant peaks in the terahertz region, it is difficult to observe their specific signals in aqueous environments. Hence, this paper proposes a method for determining these peaks. We found the specific resonant peaks of a modified nucleoside, 5-methlycytidine and modified HEK293T DNA in an aqueous solution through baseline correction. We evaluated the consistency of various fitting functions used for determining the peaks with various parameters. We separated two resonance peaks of 5-methlycytidine at 1.59 and 1.97 THz and for artificially methylated HEK293T DNA at 1.64 and 2.0 THz.

Karyopherin α-2 Mediates MDC1 Nuclear Import through a Functional Nuclear Localization Signal in the tBRCT Domain of MDC1.

Mediator of DNA damage checkpoint protein 1 (MDC1) plays a vital role in DNA damage response (DDR) by coordinating the repair of double strand breaks (DSBs). Here, we identified a novel interaction between MDC1 and karyopherin α-2 (KPNA2), a nucleocytoplasmic transport adaptor, and showed that KPNA2 is necessary for MDC1 nuclear import. Thereafter, we identified a functional nuclear localization signal (NLS) between amino acid residues 1989-1994 of the two Breast Cancer 1 (BRCA1) carboxyl-terminal (tBRCT) domain of MDC1 and demonstrated disruption of this NLS impaired interaction between MDC1 and KPNA2 and reduced nuclear localization of MDC1. In KPNA2-depleted cells, the recruitment of MDC1, along with the downstream signaling p roteins Ring Finger Protein 8 (RNF8), 53BP1-binding protein 1 (53BP1), BRCA1, and Ring Finger Protein 168 (RNF168), to DNA damage sites was abolished. Additionally, KPNA2-depleted cells had a decreased rate of homologous recombination (HR) repair. Our data suggest that KPNA2-mediated MDC1 nuclear import is important for DDR signaling and DSB repair.

Examining the Relationship between Individualism and Pro-Environmental Behavior: The Moderating Role of Social Cohesion.

Combining Hofstede's cultural dimensions, value-belief-norm theory, and social exchange theory, this study explores the impact of individualism and social cohesion on pro-environmental behavior (PEB) as well as the moderating role of social cohesion in the individualism-PEB link in the context of Korean society. Using the 2021 Korean General Social Survey and multiple linear regression analyses, we found that individualism is negatively related to PEB, whereas social cohesion is positively related to PEB. Further analysis showed that social cohesion attenuates the negative relationship between individualism and PEB. Our findings suggest that although individuals with high levels of individualism are less likely to perform PEB than those with a high level of collectivism, social cohesion is a valuable community resource that encourages them to engage in eco-friendly activities even when they seek to achieve person-oriented goals and pursue their own interests. The implications and contributions of these findings regarding environmental psychology are discussed.

CtIP Regulates Mitotic Spindle Assembly by Modulating the TPX2-Aurora A Signaling Axis.

CtBP-interacting protein (CtIP) plays a critical role in controlling the homologous recombination-mediated DNA double-stranded break (DSB) repair pathway through DNA end resection, and recent studies suggest that it also plays a role in mitosis. However, the mechanism by which CtIP contributes to mitosis regulation remains elusive. Here, we show that depletion of CtIP leads to a delay in anaphase progression resulting in misaligned chromosomes, an aberrant number of centrosomes, and defects in chromosome segregation. Additionally, we demonstrate that CtIP binds and colocalizes with Targeting protein for Xklp2 (TPX2) during mitosis to regulate the recruitment of TPX2 to the spindle poles. Furthermore, depletion of CtIP resulted in both a lower concentration of Aurora A, its downstream target, and very low microtubule intensity at the spindle poles, suggesting an important role for the CtIP-TPX2-Auroa A complex in microtubule dynamics at the centrosomal spindles. Our findings reveal a novel function of CtIP in regulating spindle dynamics through interactions with TPX2 and indicate that CtIP is involved in the proper execution of the mitotic program, where deregulation may lead to chromosomal instability.

The novel role of mast cells in the microenvironment of acute myocardial infarction.

Mast cells are multifunctional cells containing various mediators, such as cytokines, tryptase, and histamine, and they have been identified in infarct myocardium. Here, we elucidated the roles of mast cells in a myocardial infarction (MI) rat model. We studied the physiological and functional roles of mast cell granules (MCGs), isolated from rat peritoneal fluid, on endothelial cells, neonatal cardiomyocytes, and infarct heart (1-hour occlusion of left coronary artery followed by reperfusion). The number of mast cells had two peak time points of appearance in the infarct region at 1day and 21days after MI induction in rats (p<0.05 in each compared with sham-operated heart). Simultaneous injection of an optimal dose of MCGs modulated the microenvironment and resulted in the increased infiltration of macrophages and decreased apoptosis of cardiomyocytes without change in the mast cell number in infarct myocardium. Moreover, MCG injection attenuated the progression of MI through angiogenesis and preserved left ventricular function after MI. MCG-treated cardiomyocytes were more resistant to hypoxic injury through phosphorylation of Akt, and MCG-treated endothelial cells showed enhanced migration and tube formation. We have shown that MCGs have novel cardioprotective roles in MI via the prolonged survival of cardiomyocytes and the induction of angiogenesis.

Transformation of terahertz vibrational modes of cytosine under hydration.

Cytosine and cytosine monohydrate are representative biomolecules for investigating the effect of hydrogen bonds in deoxyribonucleic acid. To better understand intermolecular interactions, such as hydrogen bonds, between nucleobases it is necessary to identify the low-frequency vibrational modes associated with intermolecular interactions and crystalline structures. In this study, we investigated the characteristic low-frequency vibrational modes of cytosine and cytosine monohydrate using terahertz time-domain spectroscopy (THz-TDS). The crystal geometry was obtained by the powder X-ray diffraction technique. The optimized atomic positions and the normal modes in the terahertz region were calculated using density functional theory (DFT), which agreed well with the experimental results. We found that overall terahertz absorption peaks of cytosine and cytosine monohydrate consist of collective vibrations mixed with intermolecular and intramolecular vibrations in mode character analysis, and that the most intense peaks of both samples involve remarkable intermolecular translational vibration. These results indicate that THz-TDS combined with DFT calculations including mode character analysis can be an effective method for understanding how water molecules contribute to the characteristics of the low-frequency vibrational modes by intermolecular vibrations with hydrogen bonding in biological and biomedical applications.

Protective Mechanisms of Avocado Oil Extract Against Ototoxicity.

Despite the excellent antimicrobial activity of aminoglycoside antibiotics, permanent inner ear damage associated with the use of these drugs has resulted in the need to develop strategies to address the ototoxic risk given their widespread use. In a previous study, we showed that avocado oil protects ear hair cells from damage caused by neomycin. However, the detailed mechanism by which this protection occurs is still unclear. Here, we investigated the auditory cell-protective mechanism of enhanced functional avocado oil extract (DKB122). RNA sequencing followed by pathway analysis revealed that DKB122 has the potential to enhance the expression of detoxification and antioxidant genes associated with glutathione metabolism (Hmox4, Gsta4, Mgst1, and Abcc3) in HEI-OC1 cells. Additionally, DKB122 effectively decreased ROS levels, resulting in the inhibition of apoptosis in HEI-OC1 cells. The expression of the inflammatory genes that encode chemokines and interleukins was also downregulated by DKB122 treatment. Consistent with these results, DKB122 significantly inhibited p65 nuclear migration induced by TNF-α or LPS in HEI-OC1 cells and THP-1 cells and the expression of inflammatory chemokine and interleukin genes induced by TNF-α was significantly reduced. Moreover, DKB122 treatment increased LC3-II and decreased p62 in HEI-OC1 cells, suggesting that DKB122 increases autophagic flux. These results suggest that DKB122 has otoprotective effects attributable to its antioxidant activity, induction of antioxidant gene expression, anti-inflammatory activity, and autophagy activation.

Photoactivation of N-retinylidene-N-retinylethanolamine compromises autophagy in retinal pigmented epithelial cells.

As a part of the aging process, N-retinylidene-N-retinylethanolamine (A2E) accumulates in the retina to activate autophagy in retinal pigmented epithelial cells. However, the effect of A2E photoactivation on autophagy, which is more clinically relevant, still remains unclear. Here, we investigated the effect of blue light (BL)-activated A2E on autophagy in human retinal pigmented epithelial cells, ARPE-19. A significant increase in LC3-II protein was observed when BL was irradiated on ARPE-19 cells containing A2E. The mammalian target of rapamycin (mTOR) pathway was examined to verify whether autophagy was activated, but no change in AKT, mTOR, and 4EBP phosphorylation was observed. Transcription factor EB (TFEB) target gene expression, which is another pathway involved in autophagy, was also not altered by A2E and BL. However, intracellular p62 protein levels were significantly increased, which represented the inhibition of autophagic flux. To investigate the mechanism of the suppressed autophagic flux, the lysosomal state was observed. After BL irradiation, lysosomal damage was induced in A2E-treated ARPE-19 cells, and this phenomenon was prevented by treatment with the antioxidant, N-acetylcysteine. Our results suggest that A2E photoactivation compromises autophagy in ARPE-19 cells and that reactive oxygen species (ROS) play an important role in this process.

Avocado Oil Extract Modulates Auditory Hair Cell Function through the Regulation of Amino Acid Biosynthesis Genes.

Sensorineural hearing loss (SNHL) is one of the most common causes of disability, affecting over 466 million people worldwide. However, prevention or therapy of SNHL has not been widely studied. Avocado oil has shown many health benefits but it has not yet been studied in regards to SNHL. Therefore, we aimed to investigate the efficacy of avocado oil on SNHL in vitro and in vivo and elucidate its mode of action. For the present study, we used enhanced functional avocado oil extract (DKB122). DKB122 led to recovery of otic hair cells in zebrafish after neomycin-induced otic cell damage. Also, DKB122 improved auditory sensory transmission function in a mouse model of noise induced-hearing loss and protected sensory hair cells in the cochlea. In addition, RNA sequencing was performed to elucidate the mechanism involved. KEGG pathway enrichment analysis of differentially expressed genes showed that DKB122 protected House Ear Institute-Organ of Corti 1 (HEI-OC1) cells against neomycin-related alterations in gene expression due to oxidative stress, cytokine production and protein synthesis.

Author Correction: ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability.

This Article contains errors in Fig. 3, Fig. 4 and Fig. 7, for which we apologize. In Fig. 3, panel 'b', the 0.5 hour time point after Ku55933 treatment images were inadvertently replaced with duplicates of the 3 hour time point after Ku55933 treatment images in Fig. 3b. Additionally, in panel 'b', the 0.5 hour time point after Nu7026 treatment images were inadvertently replaced with duplicates of the 180 min time point after siMDC1 treatment images in Fig. 3d. In Fig. 4, panel 'g', RNF168 foci in U2OS cell images were inadvertently replaced with duplicates of RNF168 foci in HeLa cell images in Fig. 4f. In Fig. 7, panel 'b', the DAPI images 0.5 hours after IR under siID3 treatment were inadvertently replaced with DAPI images of a different field of view from the same experiment. Additionally, in panel 'i', the shID3 mock-treated GFP-ID3 cells image was inadvertently replace with duplications of the shID3 mock-treated GFP-ID3 cells image in Fig. 7g.

ID3 regulates the MDC1-mediated DNA damage response in order to maintain genome stability.

MDC1 plays a critical role in the DNA damage response (DDR) by interacting directly with several factors including γ-H2AX. However, the mechanism by which MDC1 is recruited to damaged sites remains elusive. Here, we show that MDC1 interacts with a helix-loop-helix (HLH)-containing protein called inhibitor of DNA-binding 3 (ID3). In response to double-strand breaks (DSBs) in the genome, ATM phosphorylates ID3 at serine 65 within the HLH motif, and this modification allows a direct interaction with MDC1. Moreover, depletion of ID3 results in impaired formation of ionizing radiation (IR)-induced MDC1 foci, suppression of γ-H2AX-bound MDC1, impaired DSB repair, cellular hypersensitivity to IR, and genomic instability. Disruption of the MDC1-ID3 interaction prevents accumulation of MDC1 at sites of DSBs and suppresses DSB repair. Thus, our study uncovers an ID3-dependent mechanism of recruitment of MDC1 to DNA damage sites and suggests that the ID3-MDC1 interaction is crucial for DDR.MDC1 is a key component of the DNA damage response and interacts with several factors such as γ-H2AX. Here the authors show that MDC1 interacts with ID3, facilitating MDC1 recruitment to sites of damage and repair of breaks.

MicroRNA-22 Suppresses DNA Repair and Promotes Genomic Instability through Targeting of MDC1.

MDC1 is critical component of the DNA damage response (DDR) machinery and orchestrates the ensuring assembly of the DDR protein at the DNA damage sites, and therefore loss of MDC1 results in genomic instability and tumorigenicity. However, the molecular mechanisms controlling MDC1 expression are currently unknown. Here, we show that miR-22 inhibits MDC1 translation via direct binding to its 3' untranslated region, leading to impaired DNA damage repair and genomic instability. We demonstrated that activated Akt1 and senescence hinder DDR function of MDC1 by upregulating endogenous miR-22. After overexpression of constitutively active Akt1, homologous recombination was inhibited by miR-22-mediated MDC1 repression. In addition, during replicative senescence and stress-induced premature senescence, MDC1 was downregulated by upregulating miR-22 and thereby accumulating DNA damage. Our results demonstrate a central role of miR-22 in the physiologic regulation of MDC1-dependent DDR and suggest a molecular mechanism for how aberrant Akt1 activation and senescence lead to increased genomic instability, fostering an environment that promotes tumorigenesis.

Origin of restenosis after drug-eluting stent implantation in hyperglycemia is inflammatory cells and thrombus.

AIMS: The cellular and molecular mechanisms and safety after drug-eluting stent (DES) implantation in diabetic patients are still poorly understood; therefore, in this study, we evaluated the pathologic responses of the sirolimus-eluting stent (SES) or paclitaxel-eluting stent (PES) in a type I diabetes mellitus (DM) rat model. METHODS: The type I DM rat model was manipulated by intra-peritoneal streptozotocin injection. Two weeks later, DES was implanted in the aorta of rats with hyperglycemia or not as a control. Four weeks after DES implantation, the stented aorta was isolated and histomorphometric analysis was performed. RESULTS: On histomorphometric analysis, increased thrombus, inflammatory cell infiltration, and neointimal hyperplasia (NIH) without change of the smooth muscle cell number after DES implantation were observed in DM rats compared with non-DM (NDM) rats. Furthermore, delayed coverage of mature endothelial cells defined as a von Willebrand factor expression and increased immature endothelial cells as a c-kit expression after DES implantation were observed in DM rats compared with NDM rats. Increased fibrin deposition and decreased hyaluronic acid accumulation at NIH after DES implantation were also observed in DM rats compared with NDM rats. CONCLUSIONS: In conclusion, the main mechanism of restenosis after DES implantation under hyperglycemic conditions was initial thrombus with changes of the extracellular matrix rather than SMC proliferation. These results provided a therapeutic clue for the selection of DES and application of combination therapy using anti-thrombotic and anti-inflammatory drugs in diabetic patients.

Enhanced angiogenesis mediated by vascular endothelial growth factor plasmid-loaded thermo-responsive amphiphilic polymer in a rat myocardial infarction model.

Thermo-responsive hydrogel-mediated gene transfer may be preferred for the muscle, because the release of DNA into the surrounding tissue can be controlled by the 3-dimensional structure of the hydrogel. Such a system for the controlled release of a therapeutic gene may extend the duration of gene expression. Here, a thermo-responsive, biodegradable polymeric hydrogel was synthesized for local gene transfer in the heart. Initially, the luciferase gene was delivered into mouse heart. The intensity of gene expression assessed by optical imaging was closely correlated with the expressed protein concentration measured by luciferase assay in homogenized heart. Polymeric hydrogel-based gene transfer enhanced gene expression up to 4 fold, compared with naked plasmid, and displayed 2 bi-modal expression profiles with peaks at 2 days and around 25 days after local injection. Histological analyses showed that gene expression was initially highest in the myocardium, whereas lower and longer expression was seen mainly in fibrotic or inflammatory cells that infiltrated the injury site during injection. Next, a rat myocardial infarction model was made for 1 week, and human vascular endothelial growth factor (hVEGF) plasmid was injected into the infarct area with an amphiphilic thermo-responsive polymer. Enhanced and sustained hVEGF expression in the infarct region mediated by amphiphilic thermo-responsive polymer increased capillary density and larger vessel formation, thus enabling effective angiogenesis.