Emotional Distress of the COVID-19 Cluster Infection on Health Care Workers Working at a National Hospital in Korea.

BACKGROUND: Frontline healthcare workers responding to coronavirus disease 2019 (COVID-19) inevitably face tremendous psychological burden. Thus, the present study aimed to identify the psychological impact and the factors contributing to the likely increase in emotional distress of healthcare workers. METHODS: The participants include a total of 99 healthcare workers at Bugok National Hospital. Psychometric scales were used to assess emotional distress (12-item General Health Questionnaire; GHQ-12), depression symptoms (Patient Health Questionnaire-9; PHQ-9), and post-traumatic stress disorder-related symptoms (Impact of Events Scale-Revised; IES-R). A supplementary questionnaire was administered to investigate the experience of healthcare workers exposed to COVID-19-infected patients. Based on the results of GHQ-12 survey, participants were categorized into two groups: distress and non-distress. All the assessed scores were compared between the two groups. A logistic regression model was constructed to identify factors associated with emotional distress. RESULTS: Emotional distress was reported by 45.3% (n = 45) of all participants. The emotionally distressed group was more likely to be female, manage close contacts, have higher scores on PHQ-9 and IES-R, feel increased professional risk, and report that proper infection control training was not provided. Female gender, managing close contacts, higher scores on PHQ-9, and a feeling that proper infection control training was not provided were associated with emotional distress in logistic regression. CONCLUSION: Frontline healthcare workers face tremendous psychological burden during the COVID-19 pandemic. Therefore, appropriate psychological interventions should be provided to the HCWs engaged in the management of COVID-19-infected patients.

[The Effects of an Acceptance-Commitment Therapy Based Stress Management Program on Hospitalization Stress, Self-Efficacy and Psychological Well-Being of Inpatients with Schizophrenia].

PURPOSE: The purpose of this study was to construct an acceptance-commitment therapy (ACT)-based stress management program for inpatients with schizophrenia and to examine its effects on hospitalization stress, self-efficacy, and psychological well-being. METHODS: A non-equivalent control group pretest-posttest design was used. Participants were 44 inpatients with a diagnosis of schizophrenia. The experimental group (n=22) received the ACT-based stress management program twice a week for a total of four weeks. The control group (n=22) received the usual care from their primary health care providers. The study was carried out from August 7 to September 1, 2017, and data were analyzed using IBM SPSS/WIN 22.0 with a Chi-square test, Fisher's exact test, and an independent t-test. RESULTS: The experimental group showed a significant decrease in hospitalization stress (t=5.09, p<.001) and an increase in self-efficacy (t=2.44, p=.019). However, there was no significant difference in psychological well-being between the two groups (t=0.13, p=.894). CONCLUSION: The results of this study suggest that the ACT-based stress management program can be used as an effective mental health nursing intervention for hospitalization stress and self-efficacy for inpatients with schizophrenia.

Perturbation of NCOA6 leads to dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a progressive heart disease characterized by left ventricular dilation and contractile dysfunction. Although many candidate genes have been identified with mouse models, few of them have been shown to be associated with DCM in humans. Germline depletion of Ncoa6, a nuclear hormone receptor coactivator, leads to embryonic lethality and heart defects. However, it is unclear whether Ncoa6 mutations cause heart diseases in adults. Here, we report that two independent mouse models of NCOA6 dysfunction develop severe DCM with impaired mitochondrial function and reduced activity of peroxisome proliferator-activated receptor δ (PPARδ), an NCOA6 target critical for normal heart function. Sequencing of NCOA6-coding regions revealed three independent nonsynonymous mutations present in 5 of 50 (10%) patients with idiopathic DCM (iDCM). These data suggest that malfunction of NCOA6 can cause DCM in humans.

Monitoring of non-point source pollutants load from a mixed forest land use.

The aim of this study was to determine the unit load of NPS (non-point source) pollutants including organic variables such as BOD (biochemical oxygen demand), COD (chemical oxygen demand) and DOC (dissolved organic carbon), nitrogen and phosphorus constituents, and suspended solids (SS) and their event mean concentration (EMC) of runoff flows from a water-shed of mixed forest land use by intensive field experiments. Field monitoring for continuous measurements of rainfall, flow, and water quality was conducted over 12 storm events during 2008-2009 using automated and manual sampling methods. The EMCs of individual runoff event were estimated for each water quality constituent based on the flow rate and concentration data of runoff discharge. The average EMCs of BOD, COD, DOC, SS, TN (total nitrogen), NH4(+)-N, NO3(-)-N, TP (total phosphorus), PO4(3-)-P from the mixed forest land were 1.794, 3.498, 1.462, 10.269, 0.862, 0.044, 0.634, 0.034, and 0.005 mg/L, respectively. The annual unit loads of BOD, COD, DOC, SS, TN, NH4(+)-N, NO3(-)-N, TP and PO4(3-)-P were estimated as 66.9, 133.2, 55.5, 429.8, 36.5, 1.6, 26.9, 1.3 and 0.1 kg/(ha x yr), respectively. In addition, affecting parameters on the EMCs were investigated by statistic analysis of the field data. As a result, significant correlations with precipitation, rainfall intensity, and total runoff flows were found in most constituents.

RXR heterodimerization allosterically activates LXR binding to the second NR box of activating signal co-integrator-2.

ASC-2 (activating signal co-integrator-2) is a transcriptional co-activator that mediates the transactivation of NRs (nuclear receptors) via direct interactions with these receptors. ASC-2 contains two separate NR-interaction domains harbouring a core signature motif, LXXLL (where X is any amino acid), named the NR box. Although the first NR box (NR box-1) of ASC-2 interacts with many different NRs, the second NR box (NR box-2) specifically interacts with only LXR (liver X receptor), whose transactivation in vivo requires heterodimerization with RXR (retinoid X receptor). Interestingly, RXR has been shown to enhance the LXR transactivation, even in the absence of LXR ligand via a unique mechanism of allosteric regulation. In the present study we demonstrate that LXR binding to an ASC-2 fragment containing NR box-2 (Co4aN) is enhanced by RXR and even further by liganded RXR. We also identified specific residues in Co4aN involved in its interaction with LXR that were also required for the ASC-2-mediated transactivation of LXR in mammalian cells. Using these mutants, we found that the Co4aN-LXR interaction surface is not altered by the presence of RXR and RXR ligand and that the Ser(1490) residue is the critical determinant for the LXR-specific interaction of Co4aN. Notably the NR box-2, but not the NR box-1, is essential for ASC-2-mediated transactivation of LXR in vivo and for the interaction between LXR-RXR and ASC-2 in vitro. These results indicate that RXR does not interact directly with NR box-1 of ASC-2, but functions as an allosteric activator of LXR binding to NR box-2 of ASC-2.

One- plus two-hybrid system, a novel yeast genetic selection for specific missense mutations disrupting protein/protein interactions.

To facilitate analysis of protein/protein interaction interfaces, we devised a novel yeast genetic screening method, named the "one- plus two-hybrid system," for the efficient selection of missense mutations that specifically disrupt known protein/protein interactions. This system modifies the standard yeast two-hybrid system to allow the operation of dual reporter systems within the same cell. The one-hybrid system is first used to select the intact interacting partner (prey), resulting in the positive selection of informative missense mutants from a large library of randomly generated mutant alleles. Then in a second screening step, interaction-defective prey mutants for a given protein are selected using the two-hybrid reporter system among the isolated missense mutants. We used this method to characterize the interactions between unliganded nuclear receptors (NRs) and the conserved motif within the bipartite NR interaction domains (IDs) of the NR corepressor (N-CoR) and identified the specific residues of N-CoR-IDs required either generally for optimal NR binding or to interact with a particular NR. This efficient and rapid method should allow us to quickly analyze a large number of interaction interfaces.

Regulation of MLL1 H3K4 methyltransferase activity by its core components.

Histone H3 Lys4 (H3K4) methylation is a prevalent mark associated with transcription activation. A common feature of several H3K4 methyltransferase complexes is the presence of three structural components (RbBP5, Ash2L and WDR5) and a catalytic subunit containing a SET domain. Here we report the first biochemical reconstitution of a functional four-component mixed-lineage leukemia protein-1 (MLL1) core complex. This reconstitution, combined with in vivo assays, allows direct analysis of the contribution of each component to MLL1 enzymatic activity and their roles in transcriptional regulation. Moreover, taking clues from a crystal structure analysis, we demonstrate that WDR5 mediates interactions of the MLL1 catalytic unit both with the common structural platform and with the histone substrate. Mechanistic insights gained from this study can be generalized to the whole family of SET1-like histone methyltransferases in mammals.

Regulation of insulin secretion and beta-cell mass by activating signal cointegrator 2.

Activating signal cointegrator 2 (ASC-2) is a transcriptional coactivator of many nuclear receptors (NRs) and other transcription factors and contains two NR-interacting LXXLL motifs (NR boxes). In the pancreas, ASC-2 is expressed only in the endocrine cells of the islets of Langerhans, but not in the exocrine cells. Thus, we examined the potential role of ASC-2 in insulin secretion from pancreatic beta-cells. Overexpressed ASC-2 increased glucose-elicited insulin secretion, whereas insulin secretion was decreased in islets from ASC-2+/- mice. DN1 and DN2 are two dominant-negative fragments of ASC-2 that contain NR boxes 1 and 2, respectively, and block the interactions of cognate NRs with the endogenous ASC-2. Primary rat islets ectopically expressing DN1 or DN2 exhibited decreased insulin secretion. Furthermore, relative to the wild type, ASC-2+/- mice showed reduced islet mass and number, which correlated with increased apoptosis and decreased proliferation of ASC-2+/- islets. These results suggest that ASC-2 regulates insulin secretion and beta-cell survival and that the regulatory role of ASC-2 in insulin secretion appears to involve, at least in part, its interaction with NRs via its two NR boxes.

Characterization of activating signal cointegrator-2 as a novel transcriptional coactivator of the xenobiotic nuclear receptor constitutive androstane receptor.

Activating signal cointegrator-2 (ASC-2) is a recently isolated transcriptional coactivator protein for a variety of different transcription factors, including many members of the nuclear receptor superfamily. In this report, we demonstrate that ASC-2 also serves as a coactivator of the xenobiotic nuclear receptor constitutive androstane receptor (CAR). First, transcriptional activation by CAR was enhanced by cotransfected ASC-2 in CV-1 and HeLa cells. In contrast, CAR transactivation was significantly impaired in HepG2 cells stably expressing specific small interfering RNA directed against ASC-2. Consistent with these results, chromatin immunoprecipitation experiments revealed that ASC-2 is recruited to the known CAR target genes in a ligand-dependent manner. Secondly, CAR specifically interacted with the first LXXLL motif of ASC-2, and these interactions were stimulated by CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene and repressed by CAR inverse agonist androstanol, suggesting that this motif may mediate the interactions of ASC-2 and CAR in vivo. In support of this idea, DN1, a fragment of ASC-2 encompassing the first LXXLL motif, suppressed CAR transactivation, and coexpressed ASC-2 but not other LXXLL-type coactivators such as thyroid hormone receptor-associated protein 220 reversed this repression. Finally, CAR was recently found to play a pivotal role in effecting the severe acetaminophen-induced liver damage. Interestingly, transgenic mice expressing DN1 were resistant to the acetaminophen-induced hepatotoxicity and expression of a series of the known CAR target genes was specifically repressed in these transgenic mice. Taken together, these results strongly suggest that ASC-2 is a bona fide coactivator of the xenobiotic nuclear receptor CAR and mediate the specific xenobiotic response by CAR in vivo.

Mammalian two-hybrid assay for detecting protein-protein interactions in vivo.

Mammalian two-hybrid assay is a convenient, powerful tool to investigate protein-protein interactions in vivo. In particular, this method has a major advantage over the better known yeast version in that one can study interactions between mammalian proteins that may not fold correctly in yeast or that require post-translational modification or external stimulation that are not present in yeast.

Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes.

The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones.

Interactions between activating signal cointegrator-2 and the tumor suppressor retinoblastoma in androgen receptor transactivation.

Activating signal cointegrator-2 (ASC-2), a cancer-amplified transcription coactivator of nuclear receptors and numerous other transcription factors, was previously shown to contain two LXXLL motifs, each of which interacts with a distinct set of nuclear receptors. In this work, we showed that ASC-2 has an indirect, separate binding site for androgen receptor (AR). Interestingly, this region overlapped with the direct interaction interfaces with the tumor suppressor retinoblastoma (Rb). Although ASC-2 alone stimulated AR transactivation in cotransfections of HeLa cells, ectopic expression of Rb effected ASC-2 to act as a transcription coactivator of AR in Rb-null Saos2 cells. These results, along with the previous report in which AR was shown to directly interact with Rb (Yeh, S., Miyamoto, H., Nishimura, K., Kang, H., Ludlow, J., Hsiao, P., Wang, C., Su, C., and Chang C. (1998) Biochem. Biophys. Res. Commun. 248, 361-367), suggest that the AR-ASC-2 interactions in vivo may involve Rb. Thus, ASC-2 appears to contain at least three distinct nuclear receptor interaction domains.

Interaction and functional cooperation of the cancer-amplified transcriptional coactivator activating signal cointegrator-2 and E2F-1 in cell proliferation.

Activating signal cointegrator-2 (ASC-2), a novel coactivator, is amplified in several cancer cells and known to interact with mitogenic transcription factors, including serum response factor, activating protein-1, and nuclear factor-kappaB, suggesting the physiological role of ASC-2 in the promotion of cell proliferation. Here, we show that the expression pattern of ASC-2 was correlated with that of E2F-1 for protein increases at G(1) and S phase. Furthermore, cells stably overexpressing ASC-2 had an increased cell proliferation profile. These results prompted us to examine the functional interaction of ASC-2 and E2F-1. Biochemical evidence of protein interaction indicated that the transactivation domain of E2F-1 interacted with the COOH-terminal region of ASC-2. The importance of the E2F-1-ASC-2 interaction was supported by the demonstration that the coexpression of ASC-2 and E2F-1 synergistically transactivated E2F-1-driven gene transcription and the acetylation of E2F-1 protein was necessary for ASC-2-mediated transcriptional coactivation. Interestingly, overexpression of ASC-2 increased the endogenous protein level of E2F-1 in cells, resulting from the prolonged protein stability of E2F-1. Taken together, these results suggest that the cancer-amplified transcriptional coactivator ASC-2 may promote cell proliferation through enhancement of E2F-1-dependent transactivation of the expression of genes associated with cell cycle progression that may be available to favor tumor growth in vivo.

Expression of MIS in the testis is downregulated by tumor necrosis factor alpha through the negative regulation of SF-1 transactivation by NF-kappa B.

The expression of Mullerian inhibiting substance (MIS), a key molecule in sex differentiation and reproduction, is tightly regulated. It has been suggested that meiotic germ cells repress MIS expression in testicular Sertoli cells, although the substance responsible for this cell-cell communication remains unknown. Here, we present the cytokine tumor necrosis factor alpha (TNF-alpha) as a strong candidate for such a substance and its downstream molecular events. TNF-alpha inhibited MIS expression in testis organ cultures, and TNF-alpha(-/-) testes showed high and prolonged MIS expression. Furthermore, in transient-transfection assays TNF-alpha suppressed the MIS promoter that was activated by steroidogenic factor 1 (SF-1), one of the major transcription factors that regulate MIS expression. The modulation of SF-1 transactivation by TNF-alpha is through the activation of NF-kappa B, which subsequently interacts with SF-1 and represses its transactivation. The physical association of NF-kappa B with SF-1 was shown by yeast two-hybrid protein interaction, glutathione S-transferase pull-down, and coimmunoprecipitation (ChIP) analyses. ChIP assays also revealed that endogenous NF-kappa B, as well as SF-1, is recruited to the MIS promoter upon TNF-alpha signaling. SF-1-bound NF-kappa B subsequently recruits histone deacetylases to inhibit the SF-1-activated gene expression. These results may identify, for the first time, the responsible substance and its action mechanism underlying the repression of MIS expression by meiotic germ cells in the testis.

Activating signal cointegrator 2 required for liver lipid metabolism mediated by liver X receptors in mice.

Activating signal cointegrator 2 (ASC-2), a cancer-amplified transcriptional coactivator of nuclear receptors and many other transcription factors, contains two LXXLL-type nuclear receptor interaction domains. Interestingly, the second LXXLL motif is highly specific to the liver X receptors (LXRs). In cotransfection, DN2, an ASC-2 fragment encompassing this motif, exerts a potent dominant-negative effect on transactivation by LXRs, which is rescued by ectopic coexpression of the full-length ASC-2 but not by other LXXLL-type coactivators, such as SRC-1 and TRAP220. In contrast, DN2/m, in which the LXXLL motif is mutated to LXXAA to abolish the interactions with LXRs, is without any effect. Accordingly, expression of DN2, but not DN2/m, in transgenic mice results in phenotypes that are highly homologous to those previously observed with LXRalpha(-/-) mice, including a rapid accumulation of large amounts of cholesterol and down-regulation of the known lipid-metabolizing target genes of LXRalpha in the liver upon being fed a high-cholesterol diet. These results identify ASC-2 as a physiologically important transcriptional coactivator of LXRs and demonstrate its pivotal role in the liver lipid metabolism.

Dynamic inhibition of nuclear receptor activation by corepressor binding.

Nuclear receptors adopt dramatically different conformations in the presence or absence of ligand, and such liganded (holo) and unliganded (apo) receptors are specifically recognized by transcriptional coactivators and corepressors, respectively. These two states likely exist in dynamic equilibrium, contrary to the conventional model of static off and on conformations. First, corepressor SMRT [for silencing mediator of thyroid hormone receptor (TR) and retinoic acid receptor (RAR)] inhibits the interaction of coactivator steroid receptor coactivator-1 with liganded TR/RAR. Second, SMRT enables receptors to adopt apo-form even in the presence of ligand, as demonstrated with limited proteolyses and decreased binding of radiolabeled retinoid to RAR. Finally, chromatin immunoprecipitation results indicate that SMRT and steroid receptor coactivator-1 dynamically compete for receptor bindings in vivo in the presence of ligand. These results suggest that corepressor binding can drive receptors to adopt the apo-state, even in the presence of ligand, and inhibit activated liganded (holo) nuclear receptors in vivo.

Activating signal cointegrator 2 belongs to a novel steady-state complex that contains a subset of trithorax group proteins.

Many transcription coactivators interact with nuclear receptors in a ligand- and C-terminal transactivation function (AF2)-dependent manner. These include activating signal cointegrator 2 (ASC-2), a recently isolated transcriptional coactivator molecule, which is amplified in human cancers and stimulates transactivation by nuclear receptors and numerous other transcription factors. In this report, we show that ASC-2 belongs to a steady-state complex of approximately 2 MDa (ASC-2 complex [ASCOM]) in HeLa nuclei. ASCOM contains retinoblastoma-binding protein RBQ-3, alpha/beta-tubulins, and trithorax group proteins ALR-1, ALR-2, HALR, and ASH2. In particular, ALR-1/2 and HALR contain a highly conserved 130- to 140-amino-acid motif termed the SET domain, which was recently implicated in histone H3 lysine-specific methylation activities. Indeed, recombinant ALR-1, HALR, and immunopurified ASCOM exhibit very weak but specific H3-lysine 4 methylation activities in vitro, and transactivation by retinoic acid receptor appears to involve ligand-dependent recruitment of ASCOM and subsequent transient H3-lysine 4 methylation of the promoter region in vivo. Thus, ASCOM may represent a distinct coactivator complex of nuclear receptors. Further characterization of ASCOM will lead to a better understanding of how nuclear receptors and other transcription factors mediate transcriptional activation.

Multiple developmental defects derived from impaired recruitment of ASC-2 to nuclear receptors in mice: implication for posterior lenticonus with cataract.

ASC-2, a recently isolated transcriptional coactivator molecule, stimulates transactivation by multiple transcription factors, including nuclear receptors. We generated a potent dominant negative fragment of ASC-2, encompassing the N-terminal LXXLL motif that binds a broad range of nuclear receptors. This fragment, termed DN1, specifically inhibited endogenous ASC-2 from binding these receptors in vivo, whereas DN1/m, in which the LXXLL motif was mutated to LXXAA to abolish the receptor interactions, was inert. Interestingly, DN1 transgenic mice but not DN1/m transgenic mice exhibited severe microphthalmia and posterior lenticonus with cataract as well as a variety of pathophysiological phenotypes in many other organs. Our results provide a novel insight into the molecular and histopathological mechanism of posterior lenticonus with cataract and attest to the importance of ASC-2 as a pivotal transcriptional coactivator of nuclear receptors in vivo.

Activating signal cointegrator 1 is highly expressed in murine testicular Leydig cells and enhances the ligand-dependent transactivation of androgen receptor.

Activating signal cointegrator 1 (ASC-1) has been recently reported as a coactivator of some nuclear receptors. In the present study, we have analyzed the expression of ASC-1 in the mouse testis and investigated its capacity to modulate the transcriptional activity of androgen receptor (AR). We found that although ASC-1 mRNA was ubiquitously expressed at a low level in mouse tissues, a couple of testis-specific mRNAs were expressed in the adult testis. Cloning of one testis-specific variant revealed that the ubiquitous and testis-specific transcripts of ASC-1 share at least the same open reading frame. The expression of the testis-specific ASC-1 mRNAs was developmentally regulated, and the onset of their expression coincided with the initiation of spermatogenesis. In situ hybridization of mouse testis with ASC-1 antisense probe demonstrated predominant expression of ASC-1 in the interstitial Leydig cells that express AR. Moreover, yeast two-hybrid tests and glutathione S-transferase pull-down assays revealed that ASC-1 associates directly with AR and that the hinge domain of AR and a putative zinc-finger motif of ASC-1 are major determinants for their interaction. Transient transfection assays performed by expressing ASC-1 in combination with AR and an androgen-responsive reporter gene showed that ASC-1 moderately alters the induction of the reporter gene. Taken together, these results suggest that ASC-1 may function as an AR coregulator and have a role in testicular functions.

Defining requirements for heterodimerization between the retinoid X receptor and the orphan nuclear receptor Nurr1.

Nurr1, an orphan nuclear receptor mainly expressed in the central nervous system, is essential for the development of the midbrain dopaminergic neurons. Nurr1 binds DNA as a monomer and exhibits constitutive transcriptional activity. Nurr1 can also regulate transcription as a heterodimer with the retinoid X receptor (RXR) and activate transcription in response to RXR ligands. However, the specific physiological roles of Nurr1 monomers and RXR-Nurr1 heterodimers remain to be elucidated. The aim of this study was to define structural requirements for RXR-Nurr1 heterodimerization. Several amino acid substitutions were introduced in both Nurr1 and RXR in the I-box, a region previously shown to be important for nuclear receptor dimerization. Single amino acid substitutions introduced in either Nurr1 or RXR abolished heterodimerization. Importantly, heterodimerization-deficient Nurr1 mutants exhibited normal activities as monomers. Thus, by introducing specific amino acid substitutions in Nurr1, monomeric and heterodimeric properties of Nurr1 can be distinguished. Interestingly, substitutions in the RXR I-box differentially affected heterodimerization with Nurr1, retinoic acid receptor, thyroid hormone receptor, and constitutive androstane receptor demonstrating that the dimerization interfaces in these different heterodimers are functionally unique. Furthermore, heterodimerization between RXR and Nurr1 had a profound influence on the constitutive activity of Nurr1, which was diminished as a result of RXR interaction. In conclusion, our data show unique structural and functional properties of RXR-Nurr1 heterodimers and also demonstrate that specific mutations in Nurr1 can abolish heterodimerization without affecting other essential functions.