The therapeutic effects of vitamin D3 administration on the embryo implantation.

Various adjuvants have been tested clinically for patients with problems with embryo implantation during in vitro fertilization (IVF)-embryo transfer (ET). Vitamin D3, an essential modulator of various physiological processes, has received attention as an important adjuvant for successful pregnancy, as many studies have shown a strong association between vitamin D deficiency and implantation failure and fetal growth restriction. However, vitamin D has been widely utilized in different protocols, resulting in non-reproducible and debatable outcomes. In the present study, we demonstrated that cyclic intrauterine administration of vitamin D3 increased endometrial receptivity and angiogenesis, which could be attributed to increased recruitment of uterus-resident natural killer cells. In particular, cyclic treatment of vitamin D3 promoted stable attachment of the embryo onto endometrial cells in vitro, suggesting its merit during the early stage of embryo implantation to support the initial maternal-fetal interactions. Our findings suggest that women with repeated implantation failure may benefit from the use of vitamin D3 as a risk-free adjuvant prior to IVF-ET procedures to improve the uterine environment, and make it favorable for embryo implantation.

RAD51 separation of function mutation disables replication fork maintenance but preserves DSB repair.

Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.

Endometrial organoids: a reservoir of functional mitochondria for uterine repair.

Background: Asherman's syndrome (AS) is a dreadful gynecological disorder of the uterus characterized by intrauterine adhesion with severe fibrotic lesions, resulting in a damaged basalis layer with infertility. Despite extensive research on overcoming AS, evidence-based effective and reproducible treatments to improve the structural and functional morphology of the AS endometrium have not been established. Methods: Endometrial organoids generated from human or mouse endometrial tissues were transplanted into the uterine cavity of a murine model of AS to evaluate their transplantable feasibility to improve the AS uterine environment. The successful engraftment of organoid was confirmed by detection of human mitochondria and cytosol (for human endometrial organoid) or enhanced green fluorescent protein signals (for mouse endometrial organoid) in the recipient endometrium. The therapeutic effects mediated by organoid transplantation were examined by the measurements of fibrotic lesions, endometrial receptivity and angiogenesis, and fertility assessment by recording the number of implantation sites and weighing the fetuses and placenta. To explore the cellular and molecular mechanisms underlying the recovery of AS endometrium, we evaluated the status of mitochondrial movement and biogenetics in organoid transplanted endometrium. Results: Successfully engrafted endometrial organoids with similar morphological and molecular features to the parental tissues dramatically repaired the AS-induced damaged endometrium, significantly reducing fibrotic lesions and increasing fertility outcomes in mice. Moreover, dysfunctional mitochondria in damaged tissues, which we propose might be a key cellular feature of the AS endometrium, was fully recovered by functional mitochondria transferred from engrafted endometrial organoids. Endometrial organoid-originating mitochondria restored excessive collagen accumulation in fibrotic lesions and shifted uterine metabolic environment to levels observed in the normal endometrium. Conclusions: Our findings suggest that endometrial organoid-originating mitochondria might be key players to mediate uterine repair resulting in fertility enhancement by recovering abrogated metabolic circumstance of the endometrium with AS. Further studies addressing the clinical applicability of endometrial organoids may aid in identifying new therapeutic strategies for infertility in patients with AS.

Licochalcone D Inhibits Skin Epidermal Cells Transformation through the Regulation of AKT Signaling Pathways.

Cell transformation induced by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is a critical event in cancer initiation and progression, and understanding the underlying mechanisms is essential for the development of new therapeutic strategies. Licorice extract contains various bioactive compounds, which have been reported to have anticancer and anti-inflammatory effects. This study investigated the cancer preventive efficacy of licochalcone D (LicoD), a chalcone derivative in licorice extract, in EGF and TPA-induced transformed skin keratinocyte cells. LicoD effectively suppressed EGF-induced cell proliferation and anchorage-independent colony growth. EGF and TPA promoted the S phase of cell cycle, while LicoD treatment caused G1 phase arrest and down-regulated cyclin D1 and up-regulated p21 expression associated with the G1 phase. LicoD also induced apoptosis and increased apoptosis-related proteins such as cleaved-caspase-3, cleaved-caspase-7, and Bax (Bcl-2-associated X protein). We further investigated the effect of LicoD on the AKT signaling pathway involved in various cellular processes and found decreased p-AKT, p-GSK3ß, and p-NFκB expression. Treatment with MK-2206, an AKT pharmacological inhibitor, suppressed EGF-induced cell proliferation and transformed colony growth. In conclusion, this study demonstrated the potential of LicoD as a preventive agent for skin carcinogenesis.

New Sesquiterpene Glycosides from the Flowers of Aster koraiensis and Their Inhibition Activities on EGF- and TPA-Induced Cell Transformation.

In total, four new eudesmane-type sesquiterpene glycosides, askoseosides A-D (1-4), and 18 known compounds (5-22) were isolated from the flowers of Aster koraiensis via chromatographic techniques. Chemical structures of the isolated compounds were identified by spectroscopic/spectrometric methods, including NMR and HRESIMS, and the absolute configuration of the new compounds (1 and 2) was performed by electronic circular dichroism (ECD) studies. Further, the anticancer activities of the isolated compounds (1-22) were evaluated using the epidermal growth factor (EGF)-induced as well as the 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced cell transformation assay. Among the 22 compounds, compounds 4, 9, 11, 13-15, 17, 18, and 22 significantly inhibited both EGF- and TPA-induced colony growth. In particular, askoseoside D (4, EGF: 57.8%; TPA: 67.1%), apigenin (9, EGF: 88.6%; TPA: 80.2%), apigenin-7-O-ß-d-glucuronopyranoside (14, EGF: 79.2%; TPA: 70.7%), and 1-(3',4'-dihydroxycinnamoyl) cyclopentane-2,3-diol (22, EGF: 60.0%; TPA: 72.1%) showed higher potent activities.

ZNF212 promotes genomic integrity through direct interaction with TRAIP.

TRAIP is a key factor involved in the DNA damage response (DDR), homologous recombination (HR) and DNA interstrand crosslink (ICL) repair. However, the exact functions of TRAIP in these processes in mammalian cells are not fully understood. Here we identify the zinc finger protein 212, ZNF212, as a novel binding partner for TRAIP and find that ZNF212 colocalizes with sites of DNA damage. The recruitment of TRAIP or ZNF212 to sites of DNA damage is mutually interdependent. We show that depletion of ZNF212 causes defects in the DDR and HR-mediated repair in a manner epistatic to TRAIP. In addition, an epistatic analysis of Zfp212, the mouse homolog of human ZNF212, in mouse embryonic stem cells (mESCs), shows that it appears to act upstream of both the Neil3 and Fanconi anemia (FA) pathways of ICLs repair. We find that human ZNF212 interacted directly with NEIL3 and promotes its recruitment to ICL lesions. Collectively, our findings identify ZNF212 as a new factor involved in the DDR, HR-mediated repair and ICL repair though direct interaction with TRAIP.

Preclinical activities of Cassia tora Linn against aging-related diseases.

Globally, an aging population is increasing, and aging is a natural physiological process and a major risk factor for all age-related diseases. It seriously threatens personal health and imposes a great economic burden. Therefore, there is a growing scientific interest in strategies for well-aging with prevention and treatment of age-related diseases. The seed, root, stem or leaves of Cassia tora Linn. are useful for anti-bacteria, anti-hyperlipidemia and anti-obesity due to its pharmacological activities such as anti-inflammation and anti-oxidant both in vitro and in vivo. Nevertheless, no clinical trials have been attempted so far, therefore here we would like to understand the current preclinical activities for aging-related disease models including cataract, metabolic dysfunction and neurodegeneration, then discuss their preparation for clinical trials and perspectives.

De novo sphingolipid biosynthesis necessitates detoxification in cancer cells.

Sphingolipids play important signaling and structural roles in cells. Here, we find that during de novo sphingolipid biosynthesis, a toxic metabolite is formed with critical implications for cancer cell survival. The enzyme catalyzing the first step in this pathway, serine palmitoyltransferase complex (SPT), is upregulated in breast and other cancers. SPT is dispensable for cancer cell proliferation, as sphingolipids can be salvaged from the environment. However, SPT activity introduces a liability as its product, 3-ketodihydrosphingosine (3KDS), is toxic and requires clearance via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). In cancer cells, but not normal cells, targeting KDSR induces toxic 3KDS accumulation leading to endoplasmic reticulum (ER) dysfunction and loss of proteostasis. Furthermore, the antitumor effect of KDSR disruption can be enhanced by increasing metabolic input (via high-fat diet) to allow greater 3KDS production. Thus, de novo sphingolipid biosynthesis entails a detoxification requirement in cancer cells that can be therapeutically exploited.

Bee Venom Activates the Nrf2/HO-1 and TrkB/CREB/BDNF Pathways in Neuronal Cell Responses against Oxidative Stress Induced by Aß1-42.

Honeybee venom has recently been considered an anti-neurodegenerative agent, primarily due to its anti-inflammatory effects. The natural accumulation of amyloid-beta (Aß) in the brain is reported to be the natural cause of aging neural ability downfall, and oxidative stress is the main route by which Aß ignites its neural toxicity. Anti-neural oxidative stress is considered an effective approach for neurodegenerative therapy. To date, it is unclear how bee venom ameliorates neuronal cells in oxidative stress induced by Aß. Here, we evaluated the neuroprotective effect of bee venom on Aß-induced neural oxidative stress in both HT22 cells and an animal model. Our results indicate that bee venom protected HT22 cells against apoptosis induced by Aß1-42. This protective effect was explained by the increased nuclear translocation of nuclear factor erythroid 2-like 2 (Nrf2), consequently upregulating the production of heme oxygenase-1 (HO-1), a critical cellular instinct antioxidant enzyme that neutralizes excessive oxidative stress. Furthermore, bee venom treatment activated the tropomyosin-related kinase receptor B (TrkB)/cAMP response element-binding (CREB)/brain-derived neurotrophic factor (BDNF), which is closely related to the promotion of cellular antioxidant defense and neuronal functions. A mouse model with cognitive deficits induced by Aß1-42 intracerebroventricular (ICV) injections was also used. Bee venom enhanced animal cognitive ability and enhanced neural cell genesis in the hippocampal dentate gyrus region in a dose-dependent manner. Further analysis of animal brain tissue and serum confirmed that bee venom reduced oxidative stress, cholinergic system activity, and intercellular neurotrophic factor regulation, which were all adversely affected by Aß1-42. Our study demonstrates that bee venom exerts antioxidant and neuroprotective actions against neural oxidative stress caused by Aß1-42, thereby promoting its use as a therapeutic agent for neurodegenerative disorders.

CD4+/CD8+ Ratio and Growth Differentiation Factor 8 Levels in Peripheral Blood of Large Canine Males Are Useful Parameters to Build an Age Prediction Model.

PURPOSE: To build an age prediction model, we measured CD4+ and CD8+ cells, and humoral components in canine peripheral blood. MATERIALS AND METHODS: Large Belgian Malinois (BGM) and German Shepherd Dog (GSD) breeds (n=27), aged from 1 to 12 years, were used for this study. Peripheral bloods were obtained by venepuncture, then plasma and peripheral blood mononuclear cells (PBMCs) were separated immediately. Six myokines, including interleukin (IL)-6, IL-8, IL-15, leukemia inhibitory factor (LIF), growth differentiation factor 8 (GDF8), and GDF11 were measured from plasma and CD4+/CD8+ T-lymphocytes ratio were measured from PBMC. These parameters were then tested with age prediction models to find the best fit model. RESULTS: We found that the T-lymphocyte ratio (CD4+/CD8+) was significantly correlated with age (r=0.46, p=0.016). Among the six myokines, only GDF8 showed a significant correlation with age (r=0.52, p=0.005). Interestingly, these two markers showed better correlations in male dogs than females, and BGM breed than GSD. Using these two age biomarkers, we could obtain the best fit in a quadratic linear mixed model (r=0.77, p=3×10-6). CONCLUSIONS: Age prediction is a challenging task because of complication with biological age. Our quadratic linear mixed model using CD4+/CD8+ ratio and GDF8 level showed a meaningful age prediction.

CosR Regulation of perR Transcription for the Control of Oxidative Stress Defense in Campylobacter jejuni.

Oxidative stress resistance is an important mechanism to sustain the viability of oxygen-sensitive microaerophilic Campylobacter jejuni. In C. jejuni, gene expression associated with oxidative stress defense is modulated by PerR (peroxide response regulator) and CosR (Campylobacter oxidative stress regulator). Iron also plays an important role in the regulation of oxidative stress, as high iron concentrations reduce the transcription of perR. However, little is known about how iron affects the transcription of cosR. The level of cosR transcription was increased when the defined media MEMα (Minimum Essential Medium) was supplemented with ferrous (Fe2+) and ferric (Fe3+) iron and the Mueller-Hinton (MH) media was treated with an iron chelator, indicating that iron upregulates cosR transcription. However, other divalent cationic ions, such as Zn2+, Cu2+, Co2+, and Mn2+, did not affect cosR transcription, suggesting that cosR transcription is regulated specifically by iron. Interestingly, the level of perR transcription was increased when CosR was overexpressed. The positive regulation of perR transcription by CosR was observed both in the presence or in the absence of iron. The results of the electrophoretic mobility shift assay showed that CosR directly binds to the perR promoter. DNase I footprinting assays revealed that the CosR binding site in the perR promoter overlaps with the PerR box. In the study, we demonstrated that cosR transcription is increased in iron-rich conditions, and CosR positively regulates the transcription of PerR, another important regulator of oxidative stress defense in C. jejuni. These results provide new insight into how C. jejuni regulates oxidative stress defense by coordinating the transcription of perR and cosR in response to iron.

Consequences of aneuploidy in human fibroblasts with trisomy 21.

An extra copy of chromosome 21 causes Down syndrome, the most common genetic disease in humans. The mechanisms contributing to aneuploidy-related pathologies in this syndrome, independent of the identity of the triplicated genes, are not well defined. To characterize aneuploidy-driven phenotypes in trisomy 21 cells, we performed global transcriptome, proteome, and phenotypic analyses of primary human fibroblasts from individuals with Patau (trisomy 13), Edwards (trisomy 18), or Down syndromes. On average, mRNA and protein levels were increased by 1.5-fold in all trisomies, with a subset of proteins enriched for subunits of macromolecular complexes showing signs of posttranscriptional regulation. These results support the lack of evidence for widespread dosage compensation or dysregulation of chromosomal domains in human autosomes. Furthermore, we show that several aneuploidy-associated phenotypes are present in trisomy 21 cells, including lower viability and increased dependency on serine-driven lipid synthesis. Our studies establish a critical role of aneuploidy, independent of triplicated gene identity, in driving cellular defects associated with trisomy 21.

SDE2 integrates into the TIMELESS-TIPIN complex to protect stalled replication forks.

Protecting replication fork integrity during DNA replication is essential for maintaining genome stability. Here, we report that SDE2, a PCNA-associated protein, plays a key role in maintaining active replication and counteracting replication stress by regulating the replication fork protection complex (FPC). SDE2 directly interacts with the FPC component TIMELESS (TIM) and enhances its stability, thereby aiding TIM localization to replication forks and the coordination of replisome progression. Like TIM deficiency, knockdown of SDE2 leads to impaired fork progression and stalled fork recovery, along with a failure to activate CHK1 phosphorylation. Moreover, loss of SDE2 or TIM results in an excessive MRE11-dependent degradation of reversed forks. Together, our study uncovers an essential role for SDE2 in maintaining genomic integrity by stabilizing the FPC and describes a new role for TIM in protecting stalled replication forks. We propose that TIM-mediated fork protection may represent a way to cooperate with BRCA-dependent fork stabilization.

Alternative Options for Skin Cancer Therapy via Regulation of AKT and Related Signaling Pathways.

Global environmental pollution has led to human exposure to ultraviolet (UV) radiation due to the damaged ozone layer, thereby increasing the incidence and death rate of skin cancer including both melanoma and non-melanoma. Overexpression and activation of V-akt murine thymoma viral oncogene homolog (AKT, also known as protein kinase B) and related signaling pathways are major factors contributing to many cancers including lung cancer, esophageal squamous cell carcinoma and skin cancer. Although BRAF inhibitors are used to treat melanoma, further options are needed due to treatment resistance and poor efficacy. Depletion of AKT expression and activation, and related signaling cascades by its inhibitors, decreases the growth of skin cancer and metastasis. Here we have focused the effects of AKT and related signaling (PI3K/AKT/mTOR) pathways by regulators derived from plants and suggest the need for efficient treatment in skin cancer therapy.

[Development and Evaluation of the Life Respect Enhancement Program for Nursing Officers].

PURPOSE: The present study was conducted to develop the Life Respect Enhancement Program and test its effectiveness on suicide knowledge, suicide intervention skills, self-efficacy in suicide prevention, and gatekeeper behaviors among nursing officers. METHODS: The program was developed according to the ADDIE model and finalized after practical need analysis, expert verification, and a preliminary study. The present study used a concurrent embedded mixed-method research design. To compare the effects of the Program and the control group on the outcome variables, a quantitative study was conducted using a nonequivalent control group with a pretest-posttest design. Quantitative data were analyzed using χ²-tests, t-tests, and RM-ANOVA. A qualitative study was conducted using FGI with six members of the experimental group 3 months after intervention completion. RESULTS: In total, 56 nursing officers participated in the 3-month follow-up test. Compared to the control group, the experimental group showed significant improvements in suicide knowledge, suicide intervention skills, and self-efficacy in suicide prevention scores at post-test. The significant group differences in suicide knowledge and suicide intervention skills were maintained at 3 months. The gatekeeper behavior scores of the experimental group were also significantly higher than those of the control group at 3 months. Qualitative analysis of the participants' experiences with the Program yielded two themes and seven sub-themes. CONCLUSION: The newly developed Life Respect Enhancement Program demonstrates significant effects on suicide knowledge, suicide intervention skills, and gatekeeper behaviors. Thus, this program is recommended to promote suicide prevention competencies among nursing officers in military clinical settings.

Intracellular delivery of Parkin rescues neurons from accumulation of damaged mitochondria and pathological α-synuclein.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by mitochondrial dysfunction, Lewy body formation, and loss of dopaminergic neurons. Parkin, an E3 ubiquitin ligase, is thought to inhibit PD progression by removing damaged mitochondria and suppressing the accumulation of α-synuclein and other protein aggregates. The present study describes a protein-based therapy for PD enabled by the development of a cell-permeable Parkin protein (iCP-Parkin) with enhanced solubility and optimized intracellular delivery. iCP-Parkin recovered damaged mitochondria by promoting mitophagy and mitochondrial biogenesis and suppressed toxic accumulations of α-synuclein in cells and animals. Last, iCP-Parkin prevented and reversed declines in tyrosine hydroxylase and dopamine expression concomitant with improved motor function induced by mitochondrial poisons or enforced α-synuclein expression. These results point to common, therapeutically tractable features in PD pathophysiology, and suggest that motor deficits in PD may be reversed, thus providing opportunities for therapeutic intervention after the onset of motor symptoms.

ATAD5 restricts R-loop formation through PCNA unloading and RNA helicase maintenance at the replication fork.

R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflict (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or ATAD5-interacting RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.

Inhibition of the oligosaccharyl transferase in Caenorhabditis elegans that compromises ER proteostasis suppresses p38-dependent protection against pathogenic bacteria.

The oligosaccharyl transferase (OST) protein complex mediates the N-linked glycosylation of substrate proteins in the endoplasmic reticulum (ER), which regulates stability, activity, and localization of its substrates. Although many OST substrate proteins have been identified, the physiological role of the OST complex remains incompletely understood. Here we show that the OST complex in C. elegans is crucial for ER protein homeostasis and defense against infection with pathogenic bacteria Pseudomonas aeruginosa (PA14), via immune-regulatory PMK-1/p38 MAP kinase. We found that genetic inhibition of the OST complex impaired protein processing in the ER, which in turn up-regulated ER unfolded protein response (UPRER). We identified vitellogenin VIT-6 as an OST-dependent glycosylated protein, critical for maintaining survival on PA14. We also showed that the OST complex was required for up-regulation of PMK-1 signaling upon infection with PA14. Our study demonstrates that an evolutionarily conserved OST complex, crucial for ER homeostasis, regulates host defense mechanisms against pathogenic bacteria.

PCNA Unloading Is Negatively Regulated by BET Proteins.

Proliferating cell nuclear antigen (PCNA) is a DNA clamp essential for DNA replication. During DNA synthesis, PCNA is continuously loaded onto and unloaded from DNA. PCNA recruits various proteins to nascent DNA to facilitate chromosome duplication. Therefore, timely PCNA unloading is crucial for high-fidelity DNA replication. The ATAD5-RFC-like complex (ATAD5-RLC) unloads PCNA from replicated DNA. It is unclear how ATAD5-RLC activity is regulated to prevent premature PCNA unloading. Here, we find that BRD4, an acetyl-histone-binding chromatin reader, inhibits the PCNA-unloading activity of ATAD5-RLC. The BRD4 ET domain interacts with a region upstream of the ATAD5 PCNA-unloading domain. BRD4-ATAD5 binds to acetyl-histones in nascent chromatin. BRD4 release from chromatin correlates with PCNA unloading. Disruption of the interaction between BRD4 and acetyl-histones or between BRD4 and ATAD5 reduces the PCNA amount on chromatin. In contrast, the overexpression of BRD4 increases the amount of chromatin-bound PCNA. Thus, acetyl-histone-bound BRD4 fine-tunes PCNA unloading from nascent DNA.

ATAD5 promotes replication restart by regulating RAD51 and PCNA in response to replication stress.

Maintaining stability of replication forks is important for genomic integrity. However, it is not clear how replisome proteins contribute to fork stability under replication stress. Here, we report that ATAD5, a PCNA unloader, plays multiple functions at stalled forks including promoting its restart. ATAD5 depletion increases genomic instability upon hydroxyurea treatment in cultured cells and mice. ATAD5 recruits RAD51 to stalled forks in an ATR kinase-dependent manner by hydroxyurea-enhanced protein-protein interactions and timely removes PCNA from stalled forks for RAD51 recruitment. Consistent with the role of RAD51 in fork regression, ATAD5 depletion inhibits slowdown of fork progression and native 5-bromo-2'-deoxyuridine signal induced by hydroxyurea. Single-molecule FRET showed that PCNA itself acts as a mechanical barrier to fork regression. Consequently, DNA breaks required for fork restart are reduced by ATAD5 depletion. Collectively, our results suggest an important role of ATAD5 in maintaining genome integrity during replication stress.