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Background Skeletal muscle atrophy is a severe condition that involves loss of muscle mass and quality. Drug intake can also cause muscle atrophy. Biguanide metformin is the first‐line and most widely prescribed anti‐diabetic drug for patients with type 2 diabetes. The molecular mechanism of metformin in muscle is unclear. Methods Myostatin expression was investigated at the protein and transcript levels after metformin administration. To investigate the pathways associated with myostatin signalling, we used real‐time polymerase chain reaction, immunoblotting, luciferase assay, chromatin immunoprecipitation assay, co‐immunoprecipitation, immunofluorescence, primary culture, and confocal microscopy. Serum analysis, physical performance, and immunohistochemistry were performed using our in vivo model. Results Metformin induced the expression of myostatin, a key molecule that regulates muscle volume and triggers the phosphorylation of AMPK. AMPK alpha2 knockdown in the background of metformin treatment reduced the myostatin expression of C2C12 myotubes (−49.86 ± 12.03%, P 3.12‐fold ± 0.13, P 3.3‐fold, P

Methamphetamine (MA) is one of the most commonly abused drugs in the world by illegal drug users. Addiction to MA is a serious public health problem and effective therapies do not exist to date. It has also been reported that behavior induced by psychostimulants such as MA is related to histone deacetylase (HDAC). MeBib is an HDAC6 inhibitor derived from a benzimidazole scaffold. Many benzimidazole-containing compounds exhibit a wide range of pharmacological activity. In this study, we investigated whether HDAC6 inhibitor MeBib modulates the behavioral response in MA self-administered rats. Our results demonstrated that the number of active lever presses in MA self-administered rats was reduced by pretreatment with MeBib. In the hippocampus of rats, we also found MA administration promotes GluN2B, an NMDA receptor subunit, expression, which results in sequential activation of ERK/CREB/BDNF pathway, however, MeBib abrogated it. Collectively, we suggest that MeBib prevents the MA seeking response induced by MA administration and therefore, represents a potent candidate as an MA addiction inhibitor.

Trillions of microorganisms reside in the hosts' gut. Since diverse activities of gut microbiota affect the hosts' health status, maintenance of gut microbiota is important for maintaining human health. Green tea (GT) has multiple beneficial effects on energy metabolism with antiobesity, antidiabetic, and hypolipidemic properties. As GT contains a large amount of bioactive ingredients (e.g., catechins), which can be metabolized by microorganisms, it would be feasible that consumption of GT may cause compositional changes in gut microbiota, and that the changes in gut microbiota would be associated with the beneficial effects of GT. In this study, we demonstrated that consumption of GT extract relieves high-fat diet-induced metabolic abnormalities. Interestingly, GT administration significantly encouraged the growth of Akkermansia muciniphila (Akkermansia), a beneficial microorganism to relieve obesity and related metabolic disorders. Finally, we found that epigallocatechin gallate is the component of GT that stimulates the growth of Akkermansia. According to these data, we propose that GT could be a prebiotic agent for Akkermansia to treat metabolic syndromes.

A simple and reliable multiplex polymerase chain reaction (PCR) method was developed using sequence-characterized amplified region (SCAR) markers to identify kiwifruit (Actinidia spp.) cultivars. We observed 183 different polymorphic random amplified polymorphic DNA (RAPD) markers using 23 arbitrary primers in 30 kiwifruit cultivars. The number of polymorphic bands per primer varied from five (OPM-15, OPN-05, and OPN-19) to 12 (OPG-01), with an average of eight. Fifty-eight cultivar-specific polymorphic RAPD fragments were converted into 16 SCAR markers after validating their specificity. Among them, 15 SCAR markers retained the dominant behavior of the original RAPD fragment, whereas the KA11_774 SCAR marker showed a co-dominant polymorphism. Based on RAPD and SCAR data sets, cluster analyses were performed with the unweighted pair-group method of arithmetic averages. The mean genetic similarities were estimated as 0.703 and 0.677 using the RAPD and SCAR markers, respectively. Thirty kiwifruit cultivars were divided into three clusters for RAPD analysis and into two clusters for SCAR analysis, based on a similarity value of 0.67. Overall, the kiwifruit cultivars with a similar breeding background tended to cluster together. Therefore, RAPD and SCAR markers could be successfully applied to assay genetic relationships for kiwifruit cultivar identification. Cluster analysis of combined RAPD and SCAR markers showed very similar results when compared to the RAPD analysis in dendrogram form. The kiwifruit cultivars tested in this study could be identified using only 10 different SCAR markers (KB08_182, KK08_287, KG19_295, KN15_316, KG01_411, KF07_433, KG09_468, KG09_477, KL01_764, and KA11_774). Furthermore, we optimized SCAR markers for simultaneous identification of the 30 kiwifruit cultivars by multiplex PCR, using two sets of five SCAR primer pairs. The developed SCAR markers and multiplex PCR approach facilitate efficient identification of kiwifruit cultivars.

Metaplasia is a well documented and deleterious effect of crude oil components on bivalved molluscs, including oysters. This reversible transformation of one cell type to another, is a common response to petroleum-product exposure in molluscs. It has been shown experimentally in previous work that eastern oysters (Crassostrea virginica) exposed to petroleum products will exhibit metaplasia of digestive tissues. Here we document for the first time that wild adult oysters inhabiting coastal waters in the northern Gulf of Mexico during and in the aftermath of the Deepwater Horizon oil spill (2010) exhibited metaplasia in both ctenidia and digestive epithelia at significantly higher levels than geographic controls of C. virginica from Chesapeake Bay. Both ctenidial (respiratory and suspension feeding) and digestive tract tissues exhibited significantly higher frequencies of metaplasia in specimens from the Gulf of Mexico compared to those from Chesapeake Bay. Metaplasia included the loss of epithelial cilia, transformations of columnar epithelia, hyperplasia and reduction of ctenidial branches, and vacuolization of digestive tissues. Evidence for a reduction of metaplasia following the oil spill (2010-2013) is suggestive but equivocal.

Hepatocellular carcinoma (HCC) undergoes a stepwise progression from liver cirrhosis to low-grade dysplastic nodule (LGDN), high-grade dysplastic nodule (HGDN), early HCC (eHCC), and progressed HCC (pHCC). Here, we profiled multilayered genomic, epigenomic, and transcriptomic aberrations in the stepwise hepatocarcinogenesis. Initial DNA methylation was observed in eHCC (e.g., DKK3, SALL3, and SOX1) while more extensive methylation was observed in pHCC. In addition, eHCCs showed an initial loss of DNA copy numbers of tumor suppressor genes in the 4q and 13q regions, thereby conferring survival benefits to cancer cells. Transcriptome analysis revealed that HGDNs expressed endoplasmic reticulum (ER) stress–related genes, while eHCC started to express oncogenes. Furthermore, integrative analysis indicated that expression of the serine peptidase inhibitor, Kazal type 1 (SPINK1), played a pivotal role in eHCC development. Significant demethylation of SPINK1 was observed in eHCC compared to HGDN. The study also demonstrated that ER stress may induce SPINK1 demethylation and expression in liver cancer cells. In conclusion, these results reveal the dynamics of multiomic aberrations during malignant conversion of liver cancer, thus providing novel pathobiological insights into hepatocarcinogenesis. Significance: Multiomics profiling and integrative analyses of stepwise hepatocarcinogenesis reveal novel mechanistic and clinical insights into hepatocarcinogenesis.

β-Glucosylglycerol (β-GG) and their derivatives have potential applications in food, cosmetics and the healthcare industry, including antitumor medications. In this study, β-GG and its unnatural glycosides were synthesized through the transglycosylation of two enzymes, Sulfolobus shibatae β-glycosidase (SSG) and Deinococcus geothermalis amylosucrase (DGAS). SSG catalyzed a transglycosylation reaction with glycerol as an acceptor and cellobiose as a donor to produce 56% of β-GGs [β-D-glucopyranosyl-(1→1/3)-D-glycerol and β-D-glucopyranosyl- (1→2)-D-glycerol]. In the second transglycosylation reaction, β-D-glucopyranosyl-(1 → 1/3)-Dglycerol was used as acceptor molecules of the DGAS reaction. As a result, 61% of α-Dglucopyranosyl-( 1→4)-β-D-glucopyranosyl-(1→1/3)-D-glycerol and 28% of α-D-maltopyranosyl- (1→4)-β-D-glucopyranosyl-(1→1/3)-D-glycerol were synthesized as unnatural glucosylglycerols. In conclusion, the combined enzymatic synthesis of the unnatural glycosides of β-GG was established. The synthesis of these unnatural glycosides may provide an opportunity to discover new applications in the biotechnological industry.

On the basis of deguelin, a series of the B,C-ring truncated surrogates with N-substituted amide linkers were investigated as HSP90 inhibitors. The structure activity relationship of the template was studied by incorporating various substitutions on the nitrogen of the amide linker and examining their HIF-1α inhibition. Among them, compound 57 showed potent HIF-1α inhibition and cytotoxicity in triple-negative breast cancer lines in a dose-dependent manner. Compound 57 downregulated expression and phosphorylation of major client proteins of HSP90 including AKT, ERK and STAT3, indicating that its antitumor activity was derived from the inhibition of HSP90 function. The molecular modeling of 57 demonstrated that 57 bound well to the C-terminal ATP-binding pocket in the open conformation of the hHSP90 homodimer with hydrogen bonding and pi-cation interactions. Overall, compound 57 is a potential antitumor agent for triple-negative breast cancer as a HSP90 C-terminal inhibitor.

Cathepsin K is highly expressed in various types of cancers. However, the effect of cathepsin K inhibition in cancer cells is not well characterized. Here, cathepsin K inhibitor (odanacatib; ODN) and knockdown of cathepsin K (siRNA) enhanced oxaliplatin-induced apoptosis in multiple cancer cells through Bax upregulation. Bax knockdown significantly inhibited the combined ODN and oxaliplatin treatment-induced apoptotic cell death. Stabilization of p53 by ODN played a critical role in upregulating Bax expression at the transcriptional level. Casein kinase 2 (CK2)-dependent phosphorylation of OTUB1 at Ser16 played a critical role in ODN- and cathepsin K siRNA-mediated p53 stabilization. Interestingly, ODN-induced p53 and Bax upregulation were modulated by the production of mitochondrial reactive oxygen species (ROS). Mitochondrial ROS scavengers prevented OTUB1-mediated p53 stabilization and Bax upregulation by ODN. These in vitro results were confirmed by in mouse xenograft model, combined treatment with ODN and oxaliplatin significantly reduced tumor size and induced Bax upregulation. Furthermore, human renal clear carcinoma (RCC) tissues revealed a strong correlation between phosphorylation of OTUB1(Ser16) and p53/Bax expression. Our results demonstrate that cathepsin K inhibition enhances oxaliplatin-induced apoptosis by increasing OTUB1 phosphorylation via CK2 activation, thereby promoting p53 stabilization, and hence upregulating Bax.

Methods that link genetic variation to steady-state gene expression levels, such as expression quantitative trait loci (eQTLs), are widely used to functionally annotate trait-associated variants, but they are limited in identifying context-dependent effects on transcription. To address this challenge, we developed the cistrome-wide association study (CWAS), a framework for nominating variants that impact traits through their effects on chromatin state. CWAS associates the genetic determinants of cistromes (e.g., the genome-wide profiles of transcription factor binding sites or histone modifications) with traits using summary statistics from genome-wide association studies (GWAS). We performed CWASs of prostate cancer and androgen-related traits, using a reference panel of 307 prostate cistromes from 165 individuals. CWAS nominated susceptibility regulatory elements or androgen receptor (AR) binding sites at 52 out of 98 known prostate cancer GWAS loci and implicated an additional 17 novel loci. We functionally validated a subset of our results using CRISPRi and in vitro reporter assays. At 28 of the 52 risk loci, CWAS identified regulatory mechanisms that are not observable via eQTLs, implicating genes with complex or context-specific regulation that are overlooked by current approaches that relying on steady-state transcript measurements. CWAS genes include transcription factors that govern prostate development such as NKX3-1, HOXB13, GATA2, and KLF5. Moreover, CWAS boosts discovery power in modestly sized GWAS, identifying novel genetic associations mediated through AR binding for androgen-related phenotypes, including resistance to prostate cancer therapy. CWAS is a powerful and biologically interpretable paradigm for studying variants that influence traits by affecting context-dependent transcriptional regulation.

Simple Summary Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive tumors with a complex genetic landscape. Patients with neurofibromatosis type 1 syndrome (NF1) are at a high risk of MPNSTs, which usually develop from pre-existing benign Schwann cell tumors called plexiform neurofibromas. In this study, we aimed to find genes that, when altered, resulted in MPNST development. Our results suggest that the functional genetic landscape of human MPNST is complex and implicates the hippo/Yes Activated Protein (YAP) pathway in the transformation of neurofibromas. Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive, genomically complex, have soft tissue sarcomas, and are derived from the Schwann cell lineage. Patients with neurofibromatosis type 1 syndrome (NF1), an autosomal dominant tumor predisposition syndrome, are at a high risk for MPNSTs, which usually develop from pre-existing benign Schwann cell tumors called plexiform neurofibromas. NF1 is characterized by loss-of-function mutations in the NF1 gene, which encode neurofibromin, a Ras GTPase activating protein (GAP) and negative regulator of RasGTP-dependent signaling. In addition to bi-allelic loss of NF1, other known tumor suppressor genes include TP53, CDKN2A, SUZ12, and EED, all of which are often inactivated in the process of MPNST growth. A sleeping beauty (SB) transposon-based genetic screen for high-grade Schwann cell tumors in mice, and comparative genomics, implicated Wnt/β-catenin, PI3K-AKT-mTOR, and other pathways in MPNST development and progression. We endeavored to more systematically test genes and pathways implicated by our SB screen in mice, i.e., in a human immortalized Schwann cell-based model and a human MPNST cell line, using CRISPR/Cas9 technology. We individually induced loss-of-function mutations in 103 tumor suppressor genes (TSG) and oncogene candidates. We assessed anchorage-independent growth, transwell migration, and for a subset of genes, tumor formation in vivo. When tested in a loss-of-function fashion, about 60% of all TSG candidates resulted in the transformation of immortalized human Schwann cells, whereas 30% of oncogene candidates resulted in growth arrest in a MPNST cell line. Individual loss-of-function mutations in the TAOK1, GDI2, NF1, and APC genes resulted in transformation of immortalized human Schwann cells and tumor formation in a xenograft model. Moreover, the loss of all four of these genes resulted in activation of Hippo/Yes Activated Protein (YAP) signaling. By combining SB transposon mutagenesis and CRISPR/Cas9 screening, we established a useful pipeline for the validation of MPNST pathways and genes. Our results suggest that the functional genetic landscape of human MPNST is complex and implicate the Hippo/YAP pathway in the transformation of neurofibromas. It is thus imperative to functionally validate individual cancer genes and pathways using human cell-based models, to determinate their role in different stages of MPNST development, growth, and/or metastasis.

We examined changes in hepcidin (closely associated with anemia of chronic inflammation (ACI)) and upstream regulatory pathways after intravenous (IV) iron supplementation in an ACI animal model. ACI was induced in male Sprague-Dawley rats by intraperitoneally administering complete Freund’s adjuvant (CFA). Two weeks after starting CFA treatment, ACI rats received IV iron (CFA-iron) or vehicle (CFA-saline). Three days after IV iron treatment, iron profiles, hepcidin levels, and expression of proteins involved in the signaling pathways upstream of hepcidin transcription in the liver were measured. In CFA-treated rats, anemia with a concomitant increase in the levels of serum inflammatory cytokines and reactive oxygen species occurred. In CFA-iron rats, hemoglobin (Hb) concentration was still lower than that in control rats. In CFA-saline rats, hepatic hepcidin and ferritin levels increased compared with those in control rats and were further increased in CFA-iron rats. In CFA-saline rats, NADPH oxidase- (NOX-) 2, NOX-4, and superoxide dismutase levels in the liver were upregulated compared with those in control rats and their levels were further increased in CFA-iron rats. In CFA-saline rats, activities of the IL-6/STAT and BMP/SMAD pathways were enhanced in the liver compared with those in control rats and their levels were further increased in CFA-iron rats, whereas IL-6 expression remained unaffected after IV iron administration. In HepG2 cells, iron caused phosphorylation of STAT-3 and SMAD1/5 and knockdown of STAT-3 and SMAD1/5 using siRNAs reduced iron-induced hepcidin upregulation to levels similar to those in corresponding control cells. Renal erythropoietin expression and serum erythroferrone concentration were lower in CFA-iron rats than those in control rats. In ACI rats, IV iron supplementation did not recover Hb within three days despite an increase in hepatic ferritin levels, which might be attributable to an additional increase in hepcidin levels that was already upregulated under ACI conditions. Both STAT-3 phosphorylation and SMAD1/5 phosphorylation were associated with hepcidin upregulation after IV iron treatment, and this seems to be linked to iron-induced oxidative stress.

Moon et al. report that antigen presentation via MHC II with coreceptor PD-L1 expressed on intestinal epithelial cells is an important contributing factor for the generation of CD4+CD8αα+ intraepithelial lymphocytes., Conventional CD4+ T cells are differentiated into CD4+CD8αα+ intraepithelial lymphocytes (IELs) in the intestine; however, the roles of intestinal epithelial cells (IECs) are poorly understood. Here, we showed that IECs expressed MHC class II (MHC II) and programmed death–ligand 1 (PD-L1) induced by the microbiota and IFN-γ in the distal part of the small intestine, where CD4+ T cells were transformed into CD4+CD8αα+ IELs. Therefore, IEC-specific deletion of MHC II and PD-L1 hindered the development of CD4+CD8αα+ IELs. Intracellularly, PD-1 signals supported the acquisition of CD8αα by down-regulating the CD4-lineage transcription factor, T helper–inducing POZ/Krüppel-like factor (ThPOK), via the Src homology 2 domain–containing tyrosine phosphatase (SHP) pathway. Our results demonstrate that noncanonical antigen presentation with cosignals from IECs constitutes niche adaptation signals to develop tissue-resident CD4+CD8αα+ IELs., Graphical Abstract

Introduction This study sought to assess preoperative concurrent chemoradiotherapy (CRT) magnetic resonance imaging (MRI)-based findings according to a structured MRI report template for primary staging of rectal cancer, and to evaluate the prognostic relevance of the pre-CRT MRI-based findings in patients with rectal cancer after CRT. Methods We retrospectively evaluated pre- and post-CRT MRI data of patients with pathologically proven rectal adenocarcinoma, between January 2008 and October 2019. Image interpretation was performed independently by two radiologists and each reviewer assessed the cancer characteristics on MRI, based on the structured MRI report for primary staging. MRI-based findings associated with pathologic complete tumour regression grade (TRG) after CRT were analysed by univariate and multivariate analysis. Significant factors from pre-CRT MRI were weighted to score mrTRG in post-CRT MRI. Results On univariate analysis, MR T-stage, tumour infiltration, mesorectal fascia involvement, extramural vascular invasion and serum carcinoembryonic antigen level correlated significantly with pathologic complete response (pCR). Multivariate analysis identified that only MR T-stage was independently associated with pCR (odds ratio, 3.89, 95% confidence interval, 1.18-12.84; P = 0.0278). Adding MRI-based T2-stage as an ancillary finding to mrTRG statistically significantly improved the sensitivity as compared to using only mrTRG for considering a CR. T2_mrTRG was significantly different in terms of the time to tumour progression between the CR and non-CR group. Conclusions The MR T2-stage was independently associated with pCR after CRT in patients with rectal cancer and was helpful as ancillary predictive factor, adding to mrTRG for prediction of pCR.

Ketamine’s inhibitory action on the N-methyl-d-aspartate receptor and anti-inflammatory effects may provide beneficial immunomodulation in cancer surgery. We investigated the effect of subanesthetic-dose ketamine as an adjunct to desflurane anesthesia on natural killer (NK) cell activity and inflammation in patients undergoing colorectal cancer surgery. A total of 100 patients were randomly assigned to a control or ketamine group. The ketamine group received a bolus of 0.25 mg·kg−1 ketamine five minutes before the start of surgery, followed by an infusion 0.05 mg·kg−1·hr−1 until the end of surgery; the control group received a similar amount of normal saline. We measured NK cell activity and proinflammatory cytokines (interleukin-6 [IL-6] and tumour necrosis factor-α [TNF-α]) before surgery and one, 24, and 48 hr after surgery. C-reactive protein (CRP) was measured before surgery and one, three, and five days after surgery. Carcinoembryonic antigen and cancer recurrence/metastasis were assessed two years after surgery. The NK cell activity was significantly decreased after surgery in both groups, but the change was not different between groups in the linear mixed model analysis (P = 0.47). Changes in IL-6, TNF-α, CRP, and carcinoembryonic antigen levels were not different between groups (P = 0.27, 0.69, 0.99, and 0.97, respectively). Cancer recurrence within 2 years after surgery was similar between groups (10% vs 8%, P = 0.62). Intraoperative low-dose ketamine administration did not convey any favourable impacts on overall postoperative NK cell activity, inflammatory responses, and prognosis in colorectal cancer surgery patients. www.clinicaltrial.gov (NCT03273231); registered 6 September 2017.

Increased fat mass, which is induced by the storage of excess nutrients, is considered a causal factor for various metabolic disorders, including insulin resistance, type 2 diabetes, hyperlipidemia, hyperglycemia, hypertension, atherosclerosis, and non-alcoholic fatty liver disease. Therefore, it is necessary to prevent hyperadiposity to sustain a healthy life. Recently, uncoupling proteins (UCPs) were suggested to be molecular targets for curing obesity and its complications. In this study, green satsuma mandarin orange (Citrus unshiu) extract (GME) increased UCP3 expression in cultured myocytes. In a diet-induced obese animal model, administration of GME reduced fat mass and average fat cell size. Similar to in vitro experiments, GME restored expression of UCP3 in skeletal muscle. Moreover, GME also induced UCP2 expression in skeletal muscle. In conclusion, GME is suggested to be a novel functional dietary supplement for adiposity control through induction of UCPs.

Keratin (KRT) 19 is a poor prognostic marker for hepatocellular carcinoma (HCC); however, regulatory mechanisms underlying its expression remain unclear. We have previously reported the presence of fibrous tumor stroma in KRT19-positive HCC, suggesting that cross-talk between cancer-associated fibroblasts (CAF) and tumor epithelial cells could regulate KRT19 expression. This was investigated in this study using an in vitro model of paracrine interaction between HCC cell lines (HepG2, SNU423) and hepatic stellate cells (HSC), a major source of hepatic myofibroblasts. HSCs upregulated transcription and translation of KRT19 in HCC cells via paracrine interactions. Mechanistically, hepatocyte growth factor (HGF) from HSCs activated c-MET and the MEK–ERK1/2 pathway, which upregulated KRT19 expression in HCC cells. Furthermore, AP1 (JUN/FOSL1) and SP1, downstream transcriptional activators of ERK1/2, activated KRT19 expression in HCC cells. In clinical specimens of human HCC (n = 339), HGF and KRT19 protein expression correlated with CAF levels. In addition, HGF or MET protein expression was associated with FOSL1 and KRT19 expression and was found to be a poor prognostic factor. Analysis of data from The Cancer Genome Atlas also revealed KRT19 expression was closely associated with CAF and MET-mediated signaling activities. These results provide insights into the molecular background of KRT19-positive HCC that display an aggressive phenotype. Significance: These findings reveal KRT19 expression in hepatocellular carcinoma is regulated by cross-talk between cancer-associated fibroblasts and HCC cells, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(7); 1619–31. ©2018 AACR.

Background Hyperglycemia (HG) exacerbates myocardial ischemia/reperfusion (I/R) injury and renders protective strategies ineffective by amplified inflammatory response via enhanced high-mobility group box-1 (HMGB1) release. This study investigated the role of ethyl pyruvate (EP) against myocardial I/R injury under a clinically relevant HG condition. Methods Sprague-Dawley rats (n=76) were randomly assigned to 6 groups: normoglycemia (NG)-Sham, NG-I/R-control (C, saline), NG-I/R-EP treatment (50mg/kg) upon reperfusion, HG-Sham, HG-I/R-C, and HG-I/R-EP treatment upon reperfusion. HG was induced by 1.2g/kg dextrose. I/R was induced by ligation of the left anterior descending artery for 30min followed by 4h of reperfusion. Results HG resulted in exacerbation of myocardial infarct size by 19% with amplified activation of HMGB1–receptors of advanced glycation end products/toll like receptors–NF-κB pathway compared to NG following I/R, which all could be attenuated by EP. EP treatment was associated with diminished tumor necrosis factor-α, interleukin-1β, and interleukin-6 expressions. It also served to normalize the increase in pro-apoptotic Bax and the decrease in anti-apoptotic Bcl-2 protein levels. These effects were associated with decreased myocardial apoptosis and infarct size (by 30% and 36% in the NG and HG groups, respectively) regardless of the glycemic condition. Conclusion HG exacerbated myocardial I/R injury through amplified inflammatory response via increased HMGB1 level. EP treatment upon reperfusion conveyed significant myocardial protection against the I/R injury under both NG and HG conditions. Common to both glycemic conditions, associated mechanisms involved attenuated increase in HMGB1 level and suppression of its down-stream pathways.

이 연구는 대학 태권도선수의 자기효능감과 취업불안 및 구직준비의 관계를 규명하는데 목적이 있다. 연구대상자는 2017년 서울, 경기, 충청권에 재학중인 대학태권도 선수로 모집단으로 선정하였으며, 무작위로 11개소를 선정한 후 각 학교당 20명씩 할당 표집한 후 응답의 일관성이 결여되는 16부를 제외한 204명을 최종 연구자료로 사용하였다. 조사도구는 선행연구에서 조사된 설문지를 수정 및 보완하여 사용하였으며, 연구자가 직접 연구기관인 학교를 방문하여 연구를 진행하였다. 자료처리는 통계패키지 프로그램인 SPSS 22.0을 이용하여 상관관계 분석과 다중회귀분석 방법을 연구목적에 맞게 사용한 결과 다음과 같은 결론을 얻었다. 첫째, 자기효능감은 취업불안에 부분적인 영향을 미쳤고, 둘째, 자기효능감은 구직준비에 부분적인 영향을 미쳤다. 셋째, 취업불안은 구직준비에 부분적인 영향을 미쳤다. 이 연구는 차후 대학 태권도선수의 취업불안을 해소하고, 구직준비활동에 기초자료 또는 지표로 활용할 수 있을 것이다.

Being self-incompatible, most Asian plums should be planted with a compatible pollinizer to produce fruits. Therefore, the selection of an adequate pollinizer is essential when new plum cultivars are released. To select a suitable pollinizer for ‘Summer Fantasia’ plum, the S genotype, cross-compatibility, pollen viability, and flowering time of five candidate cultivars were evaluated. Using polymerase chain reaction (PCR) analysis, the S genotype of ‘Summer Fantasia’ was determined as SaSc, which was expected to be compatible with other S genotypes. To test cross-compatibility, the trees were covered with caging net to prevent unintended pollination, and pistils were hand pollinated without emasculation. Fruit set percentage was calculated 10 weeks after pollination. The fruit set percentage resulting from the cross between ‘Summer Fantasia’ and ‘Taiyo’ (SbSc) was 13.8%, whereas that resulting from other combinations was less than 5.0%. Pollen germination percentage was investigated to monitor pollen viability; it varied yearly among cultivars, although ‘Formosa’ (10.6%) and ‘Taiyo’ (13.8%) showed the highest pollen germination percentages among the cultivars. When averaged over three years and two locations, ‘Summer Fantasia’ bloomed 2–3 days after ‘Akihime’, ‘Formosa’, ‘Oishiwase’, and ‘Purple Queen’. Blooming period of ‘Summer Fantasia’ and ‘Taiyo’ overlapped almost entirely. Overall, the results indicated that ‘Taiyo’ was the most suitable pollinizer for ‘Summer Fantasia’.

// Tam Thuy Lu Vo 1 , Ji-Hyeon Park 1 , Ji Hae Seo 2 , Eun Ji Lee 1 , Hoon Choi 1 , Sung-Jin Bae 1 , Hoang Le 1 , Sunho An 1 , Hye Shin Lee 1 , Hee-Jun Wee 1 and Kyu-Won Kim 1, 3 1 SNU-Harvard NeuroVascular Protection Research Center, College of Pharmacy and The Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Korea 2 Department of Biochemistry, School of Medicine, Keimyung University, Daegu 42601, Korea 3 Crop Biotechnology Institute, GreenBio Science and Technology, Seoul National University, Pyeongchang 25354, Korea Correspondence to: Kyu-Won Kim, email: qwonkim@snu.ac.kr Keywords: aurora kinase A, ARD1, lysine acetylation, cell proliferation, cell migration Abbreviations: AuA: Aurora kinase A; ARD1: Arrest defective protein 1; D-box: destruction box; DAD: D-box activating domain. Received: May 04, 2017 Accepted: June 30, 2017 Published: July 18, 2017 ABSTRACT Aurora kinase A (AuA) is a prerequisite for centrosome maturation, separation, and mitotic spindle assembly, thus, it is essential for cell cycle regulation. Overexpression of AuA is implicated in poor prognosis of many types of cancer. However, the regulatory mechanisms underlying the functions of AuA are still not fully understood. Here, we report that AuA colocalizes with arrest defective protein 1 (ARD1) acetyltransferase during cell division and cell migration. Additionally, AuA is acetylated by ARD1 at lysine residues at positions 75 and 125. The double mutations at K75/K125 abolished the kinase activity of AuA. Moreover, the double mutant AuA exhibited diminished ability to promote cell proliferation and cell migration. Mechanistic studies revealed that AuA acetylation at K75/K125 promoted cell proliferation via activation of cyclin E/CDK2 and cyclin B1. In addition, AuA acetylation stimulated cell migration by activating the p38/AKT/MMP-2 pathway. Our findings indicate that ARD1-mediated acetylation of AuA enhances cell proliferation and migration, and probably contributes to cancer development.

Policosanols have hypocholesterolemic activity; however, the molecular mechanism of the policosanol effects is currently poorly characterized. We hypothesized that hexacosanol, a policosanol compound derived from barley sprout, may decrease cellular and plasma cholesterol levels; we thus investigated the hypocholesterolemic activity and mechanism of hexacosanol on both hepatocytes and high-fat-induced obese C57BL/6J mice. The reduction of total cholesterol, free cholesterol, and cholesteryl ester concentrations was confirmed in hexacosanol-stimulated hepatocytes (-38%, -33%, and -53%, respectively). Plasma, hepatic cholesterol concentrations, and hepatic steatosis were significantly reduced in high-fat-fed mice orally administered with hexacosanol (0.7 mg/kg body weight a day) for 8 weeks compared with those of vehicle-fed control mice (-15% and -40%, respectively). Hexacosanol in fact bound to the allosteric regulation site of AMP-activated protein kinase (AMPK)-β subunit and thus activated AMPK that inhibited the activity of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase by inhibitory phosphorylation. In addition, activation of AMPK by hexacosanol induced hepatic autophagy activity, which could further reduce hepatic lipid accumulation. Alternatively, hexacosanol suppressed the nuclear translocation and activation of sterol regulatory element-binding protein-2 (SREBP-2), a key transcription factor in cholesterol biosynthesis. These results collectively suggest that hexacosanol is a major hypocholesterolemic compound in barley sprouts with regulation of AMPK activation and SREBP-2 suppression. These suppress 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase at both mRNA expression and protein activity levels. In conclusion, hexacosanol activates AMPK and hepatic autophagy and inhibits SREBP2, resulting in hypocholesterolemic activities and improvement of hepatic steatosis.

Cancer cells are continuously challenged by adverse environmental factors including hypoxia, metabolite restriction, and immune reactions, and must adopt diverse strategies to survive. Heat shock protein (Hsp) 70 plays a central role in protection against stress-induced cell death by maintaining protein homeostasis and interfering with the process of programmed cell death. Recent findings have suggested that Hsp70 acetylation is a key regulatory modification required for its chaperone activity, but its relevance in the process of programmed cell death and the underlying mechanisms involved are not well understood. In this study, we sought to investigate mechanisms mediated by Hsp70 acetylation in relation to apoptotic and autophagic programmed cell death. Upon stress-induced apoptosis, Hsp70 acetylation inhibits apoptotic cell death, mediated by Hsp70 association with apoptotic protease-activating factor (Apaf)-1 and apoptosis-inducing factor (AIF), key modulators of caspase-dependent and -independent apoptotic pathways, respectively. Hsp70 acetylation also attenuated autophagic cell death associated with upregulation of autophagy-related genes and autophagosome induction. Collectively, these results suggest that the acetylation of Hsp70 plays key regulatory roles in cell death pathways as well as in its function as a chaperone, together enabling cellular protection in response to stress.

BackgroundStress is an important cause of skin disease, including hair loss. The hormonal response to stress is due to the HPA axis, which comprises hormones such as corticotropin releasing factor (CRF), adrenocorticotropic hormone (ACTH), and cortisol. Many reports have shown that CRF, a crucial stress hormone, inhibits hair growth and induces hair loss. However, the underlying mechanisms are still unclear. The aim of this study was to examine the effect of CRF on human dermal papilla cells (DPCs) as well as hair follicles and to investigate whether the HPA axis was established in cultured human DPCs.ResultsCRF inhibited hair shaft elongation and induced early catagen transition in human hair follicles. Hair follicle cells, both human DPCs and human ORSCs, expressed CRF and its receptors and responded to CRF. CRF inhibited the proliferation of human DPCs through cell cycle arrest at G2/M phase and induced the accumulation of reactive oxygen species (ROS). Anagen-related cytokine levels were downregulated in CRF-treated human DPCs. Interestingly, increases in proopiomelanocortin (POMC), ACTH, and cortisol were induced by CRF in human DPCs, and antagonists for the CRF receptor blocked the effects of this hormone.ConclusionThe results of this study showed that stress can cause hair loss by acting through stress hormones. Additionally, these results suggested that a fully functional HPA axis exists in human DPCs and that CRF directly affects human DPCs as well as human hair follicles under stress conditions.

PURPOSE Forkhead box C1 (FOXC1) is critical for maintaining bone marrow microenvironments during hematopoiesis, but its role in hematological malignancies remains obscure. Here, we investigated whether FOXC1 regulates tumor dormancy and activation in the microenvironments of T and natural killer (NK) cell lymphomas. Materials and Methods One hundred and twenty cases of T and NK cell lymphomas were included; the immunohistochemical expression of FOXC1 was investigated in stromal cells, and numbers of FOXC1+ stromal cells were counted. Furthermore, the expression of phosphorylated p38 (p-p38) and phosphorylated ERK1/2 (p-ERK1/2) in tumor cells was investigated using immunohistochemistry. RESULTS FOXC1 was variably expressed in C-X-C motif chemokine 12-associated reticular stromal cells, histiocytes, (myo)fibroblasts, and endothelial cells. The phenotypes of cases were categorized as dormant (high p-p38/low p-ERK1/2; n=30, 25.0%), active (high p-ERK1/2/low p-p38; n=25, 20.8%), or intermediate (others; n=65, 54.2%). Lower FOXC1+ stromal cell infiltration was associated with the dormant phenotype, the precursor T lymphoblastic leukemia/lymphoma subtype, and inferior overall survival rates, whereas higher FOXC1+ stromal cell infiltration was associated with the active phenotype and favorable patient prognosis (p < 0.05 for all). CONCLUSION These results suggested that FOXC1+ stromal cells within the microenvironments of T and NK cell lymphomas might be related to tumor phenotypes.

Background Detection of Helicobacter pylori in gastric biopsy is important for appropriate treatment and prevention of gastric carcinoma and lymphoma. A novel peptide nucleic acid probe (PNA)-based real-time polymerase chain reaction (PCR) method was developed for detection of H pylori and A2142G/A2143G mutation of the 23S rRNA gene, which is associated with clarithromycin resistance. Methods To evaluate the performance of the PNA probe-based PCR method, a total of 409 gastric biopsy samples were analyzed by PNA probe-based PCR and compared with other H pylori detection methods, including hematoxylin and eosin (HE) and Warthin-Starry (WS) staining, immunohistochemistry (IHC). A2142G/A2143G mutation of the 23S rRNA gene was tested by dual priming oligonucleotide (DPO)-based PCR and Sanger sequencing to evaluate PNA probe-based PCR. Results Among 271 cases that were positive for H pylori on IHC which was considered as a standard method, 264 cases (97.4%) and 259 cases (95.6%) were positively detected by HE/WS and PNA probe-based qPCR, respectively. Of 100 H pylori-positive patients tested by IHC, H pylori was detected in 93 cases (93.0%) by PNA probe-based PCR, 86 cases (86.0%) by DPO-based PCR, and 93 cases (93.0%) by conventional PCR. The sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of PNA probe-based qPCR were 93.0%, 94.9%, 93.9%, 94.9%, and 93.0%, respectively, which were all higher than those of DPO-based PCR. When Sanger sequencing was determined as a standard method to detect A2142G/A2143G mutations, the sensitivity of the PNA- and DPO-based methods was 100% and 94.4%, respectively, and the specificity was 100% for both methods. Conclusion PNA probe-based qPCR is an appropriate method for detecting H pylori and the clarithromycin resistance-associated mutation type.

Hepatocellular carcinoma (HCC) is subclassified into five gross types, namely, vaguely nodular (VN), single nodular (SN), single nodular with extranodular growth (SNEG), confluent multinodular (CM), and infiltrative (INF) type. However, the pathological background underlying differences in biological behavior of different gross types of HCC remains unclear. The histopathological features, clinical outcomes of HCC gross types, and their relationships with stemness-related marker status and fibrotic/hypoxic tumor microenvironment (TME) were evaluated in 266 resected HCCs. The stemness-related markers (CD24, CD44, CD133, SALL4, YAP1, K19 and EpCAM), fibrous tumor stroma (αSMA), and hypoxia (CAIX) were evaluated with immunohistochemistry. Poorer differentiation, reduced capsule formation, higher microvascular invasion, larger tumor size and larger area of necrosis were observed in order of VN-SN-SNEG-CM-INF type (p = 0.005 for all, linear-by-linear association). The expression of summed stemness-related markers and hypoxic/fibrotic TME showed an increasing trend in order of VN-SN-SNEG-CM-INF type (p

Tumor necrosis factor‑associated apoptosis‑inducing ligand (TRAIL) is considered to be a potential therapeutic target for various types of cancer. However, colon cancer is difficult to treat due to its resistance to TRAIL. Therefore, various trials have been conducted to overcome TRAIL resistance in colon cancer. The present study aimed to determine whether icariin (ICA) may sensitize human colon cancer cells to TRAIL‑induced apoptosis in vitro and in vivo. In the investigation of the effect of ICA on TRAIL‑induced apoptosis, the LIVE/DEAD assay results demonstrated that TRAIL plus ICA synergistically induced apoptosis in 49% of HCT116 colon cancer cells. These results were confirmed using long‑term colony formation assay. ICA potentiated TRAIL‑induced apoptosis by modulating the expression of apoptotic proteins and the induction of cell surface death receptors (DRs) 4 and 5. Upregulation of DRs by ICA was also observed at the transcriptional level by RT‑PCR. The expression of DR by ICA was increased through the production of reactive oxygen species (ROS). The results also suggested that increased expression of DR by ICA may be due to the activation of ERK and induction of the transcription factor CCAAT enhancer‑binding protein homologous protein (CHOP). NAC, a ROS scavenger, reduced the effect of ICA on ERK activation, DR induction and sensitization of TRAIL‑induced apoptosis. In addition, ICA enhanced the effects of TRAIL to reduce tumor growth in an in vivo xenograft mouse model. Overall, the present study provided evidence that ICA sensitized tumor cells to TRAIL‑induced apoptosis via ROS‑, ERK‑ and CHOP‑mediated upregulation of DR5 and DR4. Based on these results, it is suggested that the antitumor activity of ICA and TRAIL co‑treatment in vitro and in vivo may be used as an effective therapeutic agent in chemotherapy.

Metabolomics is a powerful tool used to understand comprehensive changes in the metabolic response and to study the phenotype of an organism by instrumental analysis. It most commonly involves mass spectrometry followed by data mining and metabolite assignment. For the last few decades, hair has been used as a valuable analytical sample to investigate retrospective xenobiotic exposure as it provides a wider window of detection than other biological samples such as saliva, plasma, and urine. Hair contains functional metabolomes such as amino acids and lipids. Moreover, segmental analysis of hair based on its growth rate can provide information on metabolic changes over time. Therefore, it has great potential as a metabolomics sample to monitor chronic diseases, including drug addiction or abnormal conditions. In the current review, the latest applications of hair metabolomics in animal studies and clinical settings are highlighted. For this purpose, we review and discuss the characteristics of hair as a metabolomics sample, the analytical techniques employed in hair metabolomics and the consequence of hair metabolome alterations in recent studies. Through this, the value of hair as an alternative biological sample in metabolomics is highlighted.

Based on the lead compound L-80 (compound 2), a potent heat shock protein 90 (HSP90) inhibitor, a series of C-ring truncated deguelin analogs were designed, synthesized and evaluated for Hypoxia Inducible Factor-1α (HIF-1α) inhibition as a primary screening method. Their structure-activity relationship was investigated in a systematic manner by varying the A/B ring, linker and D/E ring, respectively. Among the synthesized inhibitors, compound 5 exhibited potent HIF-1α inhibition in a dose-dependent manner and significant antitumor activity in human non-small cell lung carcinoma (H1299), with better activities than L-80. It also inhibited in vitro hypoxia-mediated angiogenic processes in human retinal microvascular endothelial cells (HRMEC). The docking study of 5 showed a similar binding mode as L-80: it occupied the C-terminal ATP-binding pocket of HSP90, indicating that the anticancer and antiangiogenic activities of 5 were derived from HIF-1α destabilization by inhibiting the C-terminal ATP-binding site of hHSP90.

// Yoon Yang Jung 1 , Ji Hae Nahm 2 , Hyun-Soo Kim 3 1 Department of Pathology, Myongji Hospital, Seonam University College of Medicine, Goyang-si, Republic of Korea 2 Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea 3 Department of Pathology, Severance Hospital, Yonsei University College of Medicine, Seoul, Republic of Korea Correspondence to: Hyun-Soo Kim, email: hyunsookim@yuhs.ac Keywords: cervix, glassy cell carcinoma, cytology, human papillomavirus Received: August 02, 2016 Accepted: September 17, 2016 Published: September 30, 2016 ABSTRACT A retrospective analysis was performed to describe the cytomorphological and histopathological findings and human papillomavirus (HPV) genotypes for glassy cell carcinoma (GCC) of the uterine cervix. Five cases of cervical GCC, in which the glassy cell features constituted at least 95% of the specimen, were included. Four patients had stage IIB GCCs and one had stage IIIB GCC. All patients underwent concurrent chemoradiation therapy. Based on pretreatment cytology, only 1 of the 5 cases was correctly diagnosed as GCC. The remaining cases were diagnosed as carcinoma of undetermined type, adenocarcinoma, poorly differentiated carcinoma, or unsatisfactory for evaluation. Cytological specimens had moderate cellularity and contained small clusters of tumor cells admixed with amphophilic, granular tumor diathesis. The tumor cells possessed large, round to oval nuclei and abundant, granular, ground-glass cytoplasm. The nuclei exhibited prominent eosinophilic nucleoli. The cytoplasm displayed sharp margins and molding, resulting in “intercellular windows” between neighboring attached cells. HPV genotyping revealed that high-risk HPV types 18, 16, and 31 were detected in 3, 1, and 1 cases, respectively. Consistent with this finding, all cases exhibited block p16 positivity, confirming the association of HPV infection with GCC. In conclusion, a distinct cytoplasmic margin, the characteristic histopathological feature of GCC, was observed in liquid-based cytological preparations. We suggest that sharp cytoplasmic outlines with molding and intercellular windows are characteristic cytomorphological features of GCC. Detection of high-risk HPV in all cases strongly supported the notion that high-risk HPV is involved in the pathogenesis of GCC.

Most of the commercial devices for vitrification are directly immersed into the warming solution (WS) for increasing of warming rate. However, the previous modified cut standard straw (MCS) which has reported is difficult to immerse into the WS. The aim of this study was to investigate whether the long cut straw (LCS) could be useful as a stable tool for vitrified-warmed human blastocysts. A total of 138 vitrified-warmed cycles were performed between November 2013 and November 2014 (exclusion criteria: women ≥38 years old, poor responder, surgical retrieval sperm, and severe male factor). The artificial shrinkage was conducted using 29-gauge needles. Ethylene glycol and dimethyl sulfoxide (7.5% and 15% (v/v)) were used as cryoprotectants. Freezing and warming were conducted using the LCS tool. The cap of LCS was removed using the forceps in the liquid nitrogen (LN2) and then directly immersed into the first WS for 1 min at 37℃ (1 M sucrose). Only re-expanded blastocysts were transferred after it was cultured in sequential media for 18-20 h. A total of 294 blastocysts were warmed, and all were recovered (100%). Two hundred eighty-five embryos were survived (96.9%). The vitrifiedwarmed blastocysts of all patients were transferred without any cancellation. We were able to achieve a reasonable implantation (24.2%), following by clinical pregnancy (36.2%), which then continued to ongoing pregnancy (36.2%), and live birth (31.2%). Using LCS is achieved the acceptable rates of survival, pregnancy and live birth. Therefore, the LCS could be considered as a stable and simple tool for human embryo vitrificaton.

Cirrhosis, the end-stage of hepatic fibrosis, is not only life-threatening by itself, but also a causative factor of liver cancer. Despite efforts to develop treatment for liver fibrosis, there are no approved agents as anti-fibrotic drugs to date. In the present study, we aimed to investigate the anti-fibrotic effect of the AMP-activated protein kinase (AMPK) activator, HL156A. A mouse model of thioacetamide (TAA)-induced liver fibrosis was used to examine the effect of HL156A in vivo. Mice received either TAA alone or a combination of TAA and HL156A intraperitoneally for a total duration of 6 weeks. Including HL156A during exposure to TAA significantly reduced extracellular matrix (ECM) deposition and production of the hepatic transforming growth factor-β1 (TGF-β1). Immunohistochemical analysis revealed that the activation of hepatic stellate cells and the capillarization of liver sinusoids were also diminished significantly by HL156A co-treatment. The anti-fibrotic effect of HL156A was further studied in vitro by using a rat hepatic stellate cell line, HSC-T6 cells. The induction of α-smooth muscle actin (α-SMA) by TGF-β1 treatment was reversed by HL156A, which was likely via the activation of AMPK. Moreover, HL156A showed anti-inflammatory effects on macrophages. Treatment with HL156A diminished LPS-induced activation of both Raw264.7 macrophage cells and primary cultured mouse macrophages. Taken together, these results imply that the AMPK activator HL156A inhibits hepatic fibrosis via multiple mechanisms and could be a potentially effective agent for fibrosis treatment.

The therapeutic efficacy of most anti-cancer drugs depends on their apoptosis-inducing abilities. Previously, we showed that a peptide containing the mitochondrial targeting domain (MTD) found in Noxa, a BH-3 only protein of Bcl-2 family, induces necrosis. Here, a fusion peptide of neuropilin-1 (NRP-1) targeting peptide and MTD peptide, designated tumor homing motif 17:MTD (TU17:MTD), was found to induce necrosis in cancer cells in vitro and to cause the regression of tumors when intravenously injected into mice bearing subcutaneous CT26 colorectal carcinoma tumors. The necrosis within tumor tissues was evident upon administering TU17:MTD. TU17:MTD penetrated into tumor cells by targeting to Neuropilin-1, which could be blocked by anti-NRP-1 antibody. The efficacy of TU17:MTD on tumor regression was higher than that of TU17:D(KLAKLAK)2, a fusion peptide of NRP-1 targeting peptide and a pro-apoptotic peptide. The necrotic cell death within tumor tissues was evident at day 1 after administering TU17:MTD systemically. Transplanted subcutaneous substantially reduced in size within two weeks and 5 days, respectively, with no apparent side effects. Together, these results propose that the pro-necrotic peptide MTD may present an alternative approach for development of targeted anti-cancer agents.

Proteomic identification of differentially expressed proteins after treatment with berberine, a natural alkaloid, was investigated in this study. The 23 differentially expressed proteins, 3 of which were enhanced and 20 were reduced, were associated with diverse biological functions including cell death and molecular chaperone. The proteomic result was also validated by western blot analysis of ezrin, septin, UQCRC1 and elongation factor Tu. Moreover, changes in total activity and isozyme pattern of antioxidant enzymes were examined via activity assay and in-situ activity staining, respectively. Total activities of catalase and superoxide dismutase were enhanced, while those of glutathione peroxidase and glutathione reductase were unchanged by berberine. Taken together, the results showed that berberine elicited alteration of protein expression patterns and signaled the induction of isozyme of catalase and superoxide dismutase in melanoma cells.

Nuclear factor E2-related factor2 (Nrf2) activation is associated with both cytoprotective effects and malignant behavior of cancer cells. This study aimed to evaluate the clinicopathological implications of the expression of Nrf2, pNrf2, and its regulator Keap1 in human hepatocellular carcinomas (HCCs). Tissue microarrays consisting of 285 surgically resected HCCs were immunohistochemically stained with pNrf2, Nrf2, Keap1, stemness-related markers (keratin 19 (K19), epithelial cell adhesion molecule (EpCAM)), carbonic anhydrase IX (CAIX), epithelial&ndash, mesenchymal transition (EMT)-related markers (ezrin, uPAR, E-cadherin), and p53, and the results were correlated with the clinicopathological features. pNrf2 expression was significantly associated with increased proliferative activity, as well as EpCAM, ezrin, p53, and CAIX expression and E-cadherin loss (p &lt, 0.05, all). Strong cytoplasmic Nrf2 expression was associated with CAIX and ezrin expression (p &lt, 0.05, both). Keap1 was associated with increased proliferative activity, portal vein invasion, EMT-related markers, and p53 expression in CAIX-negative HCCs (p &lt, 0.05, all). Both pNrf2 and cytoplasmic Nrf2 expression were associated with decreased overall survival (p &lt, 0.05, both), and cytoplasmic Nrf2 expression was an independent predictor of decreased overall survival on multivariate analysis (hazard ratio 4.15, p &lt, 0.001). Both pNrf2 and cytoplasmic Nrf2 expression were associated with poor survival and aggressive behavior of HCC. In addition, Keap1 expression was also associated with aggressive HCC behavior in CAIX-negative HCCs, suggesting that Keap1 expression should be interpreted in the context of hypoxia status.

The CCAAT/enhancer-binding protein &beta, (C/EBP&beta, ) is a transcription factor that regulates cellular proliferation, differentiation, apoptosis and tumorigenesis. Although the pro-oncogenic roles of C/EBP&beta, have been implicated in various human cancers, how it contributes to tumorigenesis or tumor progression has not been determined. Immunohistochemistry with human non-small cell lung cancer (NSCLC) tissues revealed that higher levels of C/EBP&beta, protein were expressed compared to normal lung tissues. Knockdown of C/EBP&beta, by siRNA reduced the proliferative capacity of NSCLC cells by delaying the G2/M transition in the cell cycle. In C/EBP&beta, knockdown cells, a prolonged increase in phosphorylation of cyclin dependent kinase 1 at tyrosine 15 (Y15-pCDK1) was displayed with simultaneously increased Wee1 and decreased Cdc25B expression. Chromatin immunoprecipitation (ChIP) analysis showed that C/EBP&beta, bound to distal promoter regions of WEE1 and repressed WEE1 transcription through its interaction with histone deacetylase 2. Treatment of C/EBP&beta, knockdown cells with a Wee1 inhibitor induced a decrease in Y15-pCDK1 and recovered cells from G2/M arrest. In the xenograft tumors, the depletion of C/EBP&beta, significantly reduced tumor growth. Taken together, these results indicate that Wee1 is a novel transcription target of C/EBP&beta, that is required for the G2/M phase of cell cycle progression, ultimately regulating proliferation of NSCLC cells.

Transcriptional regulation is highly disease and cell-type specific. We performed H3K27ac chromatin immunoprecipitation and transcriptomic sequencing in primary tumors for the four different subtypes of invasive epithelial ovarian cancer (OC). Histotype-specific regulatory elements (REs) were enriched in enhancers (PIn silicoprediction of putative target genes for histotype-specific REs identified genes (WFDC2, P=5.5×10-5) and pathways (PI3K-Akt signaling, PPAX8andCA125) are associated with super-enhancers (SEs) in all OCs, while others are histotype-specific, includingPPP1R3Bwhich is associated with SEs specific to clear cell OC. Integrated analysis of active chromatin landscapes with somatic single nucleotide variants (SNVs) from whole genome sequencing (WGS) of 232 primary OCs identified frequently mutated REs, including theKLF6promoter (P=8.2×10-8) and a putative enhancer at chromosome 6p22.1 (P-11), while the collection ofcisregulatory elements associated withPAX8was the most frequently mutated set of enhancers in OC (P=0.003). Functional analyses supported our findings: Knockdown ofPPP1R3Bin clear cell OC cells significantly reduced intracellular glycogen content, a signature feature of this histotype; and stable knockout of a 635 bp region in the 6p22.1 enhancer induced downregulation of two predicted target genes,ZSCAN16andZSCAN12(P=6.6 x 10-4and P=0.02). In summary, we have characterized histotype-specific epigenomic and transcriptomic landscapes in OC and defined likely functional REs based on somatic mutation analysis of ovarian tumors.

The CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that regulates cellular proliferation, differentiation, apoptosis and tumorigenesis. Although the pro-oncogenic roles of C/EBPβ have been implicated in various human cancers, how it contributes to tumorigenesis or tumor progression has not been determined. Immunohistochemistry with human non-small cell lung cancer (NSCLC) tissues revealed that higher levels of C/EBPβ protein are expressed compared to normal lung tissues. Knockdown of C/EBPβ by siRNA reduced the proliferative capacity of NSCLC cells by delaying G2/M transition of the cell cycle. In C/EBPβ-knockdown cells, a prolonged increase in phosphorylation of cyclin dependent kinase 1 at tyrosine 15 (Y15-pCDK1) was displayed with increased Wee1 and decreased Cdc25B expression, simultaneously. ChIP analysis showed that C/EBPβ bound to distal promoter regions of WEE1 and repressed WEE1 transcription through the interaction with histone deacetylase 2. Treatment of C/EBPβ-knockdown cells with a Wee1 inhibitor induced a decrease in Y15-pCDK1 and recovered cells from G2/M arrest. In the xenograft tumors, the depletion of C/EBPβ significantly reduced tumor growth. Taken together, these results indicate that Wee1 is a novel transcription target of C/EBPβ that is required for the G2/M phase of cell cycle progression, ultimately regulating proliferation of NSCLC cells.

Mitochondrial dysfunction and subsequent enhanced oxidative stress is implicated in the pathogenesis of autism spectrum disorder (ASD). Mitochondrial transcription factor B2 (TFB2M) is an essential protein in mitochondrial gene expression. No reports have described TFB2M mutations and variations involved in any human diseases. We identified a rare homozygous c.790C>T (His264Tyr) variation in TFB2M gene in two Korean siblings with ASD by whole-exome sequencing. The roles of the TFB2M variation in the pathogenesis of ASD were investigated. Patient fibroblasts revealed increased transcription of mitochondrial genes and mitochondrial function in terms of ATP, membrane potential, oxygen consumption, and reactive oxygen species (ROS). Overexpression of the TFB2M variant in primary-cultured fibroblasts demonstrated significantly increased transcription of mitochondrial genes and mitochondrial function compared with overexpression of wild-type TFB2M. Molecular dynamics simulation of the TFB2M variant protein suggested an increase in the rigidity of the hinge region, which may cause alterations in loading and/or unloading of TFB2M on target DNA. Our results suggest that augmentation of mitochondrial gene expression and subsequent enhancement of mitochondrial function may be associated with the pathogenesis of ASD in Korean patients.

Apoptotic cell clearance by phagocytes is essential in tissue homeostasis. We demonstrated that conditioned medium (CM) from macrophages exposed to apoptotic cancer cells inhibits the TGFβ1-induced epithelial–mesenchymal transition (EMT), migration, and invasion of cancer cells. Apoptotic 344SQ (ApoSQ) cell-induced PPARγ activity in macrophages increased the levels of PTEN, which was secreted in exosomes. Exosomal PTEN was taken up by recipient lung cancer cells. ApoSQ-exposed CM from PTEN knockdown cells failed to enhance PTEN in 344SQ cells, restore cellular polarity, or exert anti-EMT and anti-invasive effects. The CM that was deficient in PPARγ ligands, including 15-HETE, lipoxin A4, and 15d-PGJ2, could not reverse the suppression of PPARγ activity or the PTEN increase in 344SQ cells and consequently failed to prevent the EMT process. Moreover, a single injection of ApoSQ cells inhibited lung metastasis in syngeneic immunocompetent mice with enhanced PPARγ/PTEN signaling both in tumor-associated macrophages and in tumor cells. PPARγ antagonist GW9662 reversed the signaling by PPARγ/PTEN; the reduction in EMT-activating transcription factors, such as Snai1 and Zeb1; and the antimetastatic effect of the ApoSQ injection. Thus, the injection of apoptotic lung cancer cells may offer a new strategy for the prevention of lung metastasis.

The phenolic compounds and radical scavenging activity of ethanolic extracts of five adzuki beans were evaluate according to cultivar. The predominant phenolic acids in five cultivars of adzuki bean were (+)-catechin and gallic acid. Antioxidant capacities were determined using 2,2-diphenyl-1-picrylhydrazyl and 2,2-azinobis (3-ethylbenothiazoline-6-sulphonic acid) diammonium salt, and reducing power was positively enhanced according to the total polyphenolic content. In a cell-based assay, adzuki beans showed cytoprotective effects against oxidative stress induced by tert-butyl hydroperoxide and inhibitory effects on the production of reactive oxygen species, except for the cultivar Vigna angularis var. nipponensis cv. Whinnarae. In conclusion, (+)-catechin was the predominant phenolic compound found in adzuki beans, but there were differences according to the cultivar. Overall, the adzuki bean cultivars showed different antioxidant activities and cytoprotective effects according to the concentration and composition of phenolic compounds.