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Canine mammary gland tumour (cMGT) is the most common tumour in intact female dogs. Surgery is the only effective treatment for cMGT, and dogs with metastasis at the time of diagnosis or those diagnosed at an advanced stage have poorer prognosis. Thus, novel diagnostic biomarkers and therapeutic targets are needed. Neurokinin-1 receptor (NK-1 receptor) is involved in cancer progression and has been detected in various malignant tumours including breast cancer in humans. Furthermore, NK-1 receptor antagonists inhibit cancer progression. We evaluated NK-1 receptor expression in malignant and benign cMGT compared with that in normal mammary gland tissues and analysed the relationship between the expression of NK-1 receptor and histopathological type or malignancy grade. Specimens from 34 malignant MGT and 35 benign MGT cases were used for immunohistochemistry and scored according to intensity and percentage. Healthy margins from each tumour were used as internal controls. The scores for NK-1 receptor intensity, percentage of positive cells and overall immunohistochemistry were higher in malignant MGT than in benign MGT and normal tissue (p < .000). NK-1 receptor expression was not correlated with either malignancy grade or histopathological type. Expression of the NK-1 receptor in malignant MGT was higher than that in benign MGT and normal tissues. Thus, NK-1 receptor could be considered a novel therapeutic target for cMGT. Further studies using other quantitative tests such as western blotting or PCR and the evaluation of substance P in patient tumour tissue or serum are needed., (© 2020 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Objective: We investigated the expression pattern of long noncoding RNAs (lncRNA) and messenger RNAs (mRNA) from two different intracerebral hemorrhage (ICH) rat models, and performed gene ontology and gene/protein interaction analyses., Methods: We harvested hemorrhagic brain 1, 3, and 7 days after ICH induction by stereotactic collagenase injection. We performed microarray analyses with Agilent array platform to compare the expression of lncRNA and mRNAs from hemorrhagic and normal brains. The RNA expression patterns were also examined from the autologous blood injection ICH model at days 1 and 3, and significantly altered lncRNAs from two ICH models were validated by quantitative reverse transcriptase-polymerase chain reaction. Gene ontology analysis and pathway analysis were performed with differentially expressed mRNAs after ICH. Gene and protein interaction analysis was performed to elucidate the functional role of upregulated lncRNA in neuronal damage., Results: Among the 13,661 lncRNAs studied, 83, 289, and 401 lncRNAs were significantly elevated after 1, 3, and 7 days after collagenase-induced ICH, respectively. NR&#95;027324, or H19, was the most upregulated lncRNA after 1 day from the two ICH models and its elevation persisted until the 7th day. Gene ontology analysis revealed that immune-related biological processes such as immune response, immune system process, and defense response were upregulated from both ICH models. Gene and protein interaction study demonstrated that NR&#95;027324 was closely related to the type I interferon signaling pathway., Interpretation: This study illustrates the dynamic expression pattern of the lncRNA profile following ICH, and that H19 is the most consistently upregulated lncRNA after ICH., (© 2019 The Authors. Annals of Clinical and Translational Neurology published by Wiley Periodicals, Inc on behalf of American Neurological Association.)

Background: To initiate mucosal immune responses, antigens in the intestinal lumen must be transported into gut-associated lymphoid tissue through M cells. Recently, it has been increasingly recognized that receptor activator of NF-kB ligand (RANKL) controls M cell differentiation by interacting with RANK expressed on the sub-epithelium of Peyer's patches. In this study, we increased the number of M cells using soluble RANKL (sRANKL) as a potent mucosal adjuvant., Results: For efficient oral delivery of sRANKL, we constructed recombinant Lactococcus lactis (L. lactis) IL1403 secreting sRANKL (sRANKL-LAB). The biological activity of recombinant sRANKL was confirmed by observing RANK-RANKL signaling in vitro. M cell development in response to oral administration of recombinant L. lactis was determined by 1.51-fold higher immunohistochemical expression of M cell marker GP-2, compared to that of non-treatment group. In addition, an adjuvant effect of sRANKL was examined by immunization of mice with M-BmpB as a model antigen after treatment with sRANKL-LAB. Compared with the wild-type L. lactis group, the sRANKL-LAB group showed significantly increased systemic and mucosal immune responses specific to M-BmpB., Conclusions: Our results show that the M cell development by sRANKL-LAB can increase the antigen transcytotic capability of follicle-associated epithelium, and thereby enhance the mucosal immune response, which implies that oral administration of sRANKL is a promising adjuvant strategy for efficient oral vaccination.

Objectives: Soldiers lose muscle and bone density during sustained operations. We investigated the impact of β-hydroxy-β-methylbutyrate (HMB) on bone properties, muscle mass, and markers of skeletal muscle regeneration under simulated military sustained operations., Methods: Male mice were divided into four groups (10/group): (1) ALT = ad libitum + trained (1h/d for 3 d/wk); (2) ALTH = ALT + HMB (0.5 g/kg BW/d); (3) C = caloric restricted (-30%) + trained (6h/d, 6d/wk); and (4) CH = C + HMB. Assessments included bone mineral density/content by dual-energy X-ray absorptiometry, muscle wet weight (quadriceps) and expression of selected genes regulating muscle mass and protein turnover. Analysis of variances were used with significance set at p < 0.05., Results: Bone mineral content increased in the ALT group (+16%) and decreased in the C group (-32%). Quadriceps muscle mass was lower in C (-27%) and CH (-19%) compared to ALT and ALTH. Myogenin mRNA expression was higher in C than ALT, ALTH and CH. Protein kinase B (Akt) mRNA expression was higher in both C and CH than ALT and ALTH. Mammalian target of rapamycin expression was higher in CH than ALT and ALTH. Muscle RING-finger protein-1 expression was higher in both C and CH than ALT and ALTH., Conclusion: HMB intake improved bone properties and attenuated the depression of protein synthesis during a simulated sustained military operation., (Reprint & Copyright © 2014 Association of Military Surgeons of the U.S.)

Caspases are a family of cysteine proteases that play an important role in the apoptotic pathway. Caspase-6 is an apoptosis effector that cleaves a variety of cellular substrates. The active form of the enzyme is required for use in research. However, it has been difficult to obtain sufficient quantities of active caspase-6 from Escherichia coli. In the present study, we constructed a caspase-6 with a 23-amino-acid deletion in the pro-domain. This engineered enzyme was expressed as a soluble protein in E. coli and was purified using affinity resin. In vitro enzyme assay and cleavage analysis revealed that the engineered active caspase-6 protein had characteristics similar to those of wild-type caspase-6. This novel method can be a valuable tool for obtaining active caspase-6 that can be used for screening caspase-6-specific substrates, which in turn can be used to elucidate the function of caspase-6 in apoptosis.

Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion. Nearly all TAP-tagged Xo2276 existed in an insoluble form; addition of rice leaf extracts from a Xoo-susceptible rice cultivar, Milyang23, significantly stimulated secretion of TAP-tagged Xo2276 into the medium. In a T3SS-defective Xoo mutant strain, secretion of TAP-tagged Xo2276 was blocked. Xo2276 is a Xoo ortholog of Xanthomonas campestris pv. vesicatoria (Xcv) AvrBs3 and contains a conserved DNA-binding domain (DBD), which includes 19.5 tandem repeats of 34 amino acids. Xo2276- DBD was expressed in E. coli and purified. Direct in vitro recognition of Xo2276-DBD on a putative target DNA sequence was confirmed using an electrophoretic mobility shift assay. This is the first study measuring the homologous expression and secretion of Xo2276 in vitro using rice leaf extract and its direct in vitro binding to the specific target DNA sequence.

Conclusion: Genetic dysregulation may be associated with development of obstructive sleep apnea (OSA)., Objectives: Genetic expression was investigated in the soft palate of patients with OSA to evaluate the biomolecular characteristics of the tissue where airway obstruction actually occurs., Methods: Soft palate mucosa was obtained during uvulopalatal flap surgery. Three patients with apnea/hypopnea index (AHI) >30/h and three simple snoring patients with AHI < 5/h were enrolled. After total RNA was extracted and amplified, gene expression levels were determined by microarray analysis, and relative signal intensities for each gene were evaluated., Results: Of the 45,034 genes analyzed, 137 were up-regulated in OSA patients compared with simple snorers and 95 were down-regulated. Gene network analysis showed that genes related to RNA interference and transcription were most commonly involved in the functional pathophysiology of OSA. In particular, genes encoding proliferating cell nuclear antigen and proteasome 26S subunit ATPase 6 were functionally connected with other genes.

Translationally controlled tumor protein (TCTP), also termed P23 in human, belongs to a family of calcium- and tubulin-binding proteins, and it is generally regarded as a growth-regulating protein. Recently, Arabidopsis TCTP (AtTCTP) has been reported to function as an important growth regulator in plants. On the other hand, plant TCTP has been suggested to be involved in abiotic stress signaling such as aluminum, salt, and water deficit by a number of microarray or proteomic analyses. In this study, the biological functions of AtTCTP were investigated by using transgenic Arabidopsis plants overexpressing AtTCTP. Interestingly, AtTCTP overexpression enhanced drought tolerance in plants. The expression analysis showed that AtTCTP was expressed in guard cells as well as in actively growing tissues. Physiological studies of the overexpression lines showed increased ABA- and calcium-induced stomatal closure ratios and faster stomatal closing responses to ABA. Furthermore, in vitro protein-protein interaction analysis confirmed the interaction between AtTCTP and microtubules, and microtubule cosedimentation assays revealed that the microtubule binding of AtTCTP increased after calcium treatment. These results demonstrate that the overexpression of AtTCTP confers drought tolerance to plants by rapid ABA-mediated stomatal closure via the interaction with microtubules in which calcium binding enhances the interaction. Collectively, the present results suggest that the plant TCTP has molecular properties similar to animal TCTPs, such as tubulin- and calcium-binding, and that it functions in ABA-mediated stomatal movement, in addition to regulating the growth of plants.

Objective: The associations between arrest-defective protein 1 (ARD1) gene expression and the clinicopathological characteristics and clinical outcomes of 94 patients undergoing hepatectomy for hepatocellular carcinoma (HCC) were investigated., Methods: ARD1 mRNA levels in HCC and corresponding non-cancerous tissues were quantified by real-time PCR. The gene expression of the tumor relative to that in the non-tumor tissues was calculated using the 2(-)(ΔΔ)(CT) method. The subjects were classified into high expression (2(-)(ΔΔ)(CT) > 1, n = 38) and low expression (2(-)(ΔΔ)(CT) ≤ 1, n = 56) groups., Results: The HCCs did not differ from matched liver tissues in terms of ARD1 mRNA levels. The high expression group had more often microvascular invasion than the low expression group (32 vs. 14%; p = 0.045). The two groups did not differ significantly in terms of other patient or tumor variables. The median follow-up period was 92.1 months. The 5-year recurrence-free and overall survival rates were 34 and 76% for the high expression group, respectively, which were similar to the rates of the low expression group (46 vs. 73%, p = 0.98 and p = 0.52, respectively)., Conclusions: Intratumoral ARD1 mRNA overexpression was involved in the microvascular invasion process in patients with HCC, although it did not associate strongly with postresectional outcomes., (Copyright © 2012 S. Karger AG, Basel.)

Four membrane calcium (Ca²⁺) channels are known to play an important role in calcium transport: transient receptor potential cation channel subfamily V members 5 and 6 (Trpv5/6), Na⁺/Ca²⁺ exchanger 1 (Ncx1) and plasma membrane Ca²⁺-transporting ATPase1 (Pmca1b). These channels have been shown to mediate the transport of Ca²⁺ between the cytosol and bloodstream in multiple tissues, including the duodenum, kidney, and uterus. Trpv5 and Trpv6 are known to selectively import Ca²⁺ into cells, while Ncx1 and Pmca1b function to extrude Ca²⁺. The present study examined and compared expression patterns of these genes in the canine duodenum, kidney and uterus. We confirmed expression of all four genes in this canine model. To collect tissues for analysis, two adult (2.5-year-old) female dogs were euthanized and tissue samples were collected from an abdominal incision. Expression of Trpv5/6, Ncx1 and Pmca1b was analyzed by reverse transcription polymerase chain reaction (RT-PCR), Western blot analysis, and immunohistochemistry. Trpv5, Trpv6 and Ncx1 mRNA were highly expressed in the kidney, while Pmca1b mRNA was primarily detected in the uterus. Protein expression was verified by Western blot analysis and localized by immunohistochemistry. Protein expression showed patterns similar to those of mRNA expression. Taken together, these results indicate that transcriptional and transitional expression of Tprv5, Trpv6, Ncx1 and Pmca1b may play a critical role in calcium transport in the canine duodenum, kidney and uterus.

A gene encoding bacillopeptidase F, bpr86-1, was cloned from B. amyloliquefaciens CH86-1 isolated from cheonggukjang. This gene could encode a preproenzyme of 1,431 amino acids. When bpr86-1 was introduced into B. subtilis WB600 via pHY300PLK, an E. coli-Bacillus shuttle vector, the transformant showed fibrinolytic activity. During growth on LB, the fibrinolytic activity of cells increased sharply when they entered the stationary phase. The highest activity (761.4 mU/mg protein) was observed at 96 h of cultivation.

Marie Unna hereditary hypotrichosis (MUHH) is a rare autosomal dominant hair disorder. Through the study of a mouse model, we identified a mutation in the 5'-untranslated region of the hairless (HR) gene in patients with MUHH in a Caucasian family. The corresponding mutation, named 'hairpoor', was found in mutant mice that were generated through N-ethyl-N-nitrosourea mutagenesis. Hairpoor mouse mutants display partial hair loss at an early age and progress to near alopecia, which resembles the MUHH phenotype. This mutation conferred overexpression of HR through translational derepression and, in turn, decreased the expression of Sfrp2, an inhibitor of the Wnt signaling pathway. This study indicates that the gain in function of HR also results in alopecia, as seen with the loss of function of HR, via abnormal upregulation of the Wnt signaling pathway.

The bacterial beta-ketoacyl-ACP synthase III (KASIII) encoded by the gene fabH (Xoo4209) from Xanthomonas oryzae pv. oryzae, a plant pathogen, is an important enzyme in the elongation steps of fatty-acid biosynthesis. It is expected to be one of the enzymes responsible for bacterial blight (BB), a serious disease that results in huge production losses of rice. As it represents an important target for the development of new antibacterial drugs against BB, determination of the crystal structure of the KAS III enzyme is essential in order to understand its reaction mechanism. In order to analyze the structure and function of KAS III, the fabH (Xoo4209) gene was cloned and the enzyme was expressed and purified. The KASIII crystal diffracted to 2.05 A resolution and belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 69.8, b = 79.5, c = 62.3 A. The unit-cell volume of the crystal is compatible with the presence of a single monomer in the asymmetric unit, with a corresponding Matthews coefficient V(M) of 2.27 A(3) Da(-1) and a solvent content of 45.8%.

Objectives/hypothesis: This study aimed to evaluate the effect of dexamethasone on the expression of transforming growth factor (TGF)-beta in the mouse model of allergic rhinitis., Study Design: Female BALB/c mice were randomly assigned to four groups, including two control groups and two treatment groups., Methods: General sensitization and local challenge were performed with ovalbumin (OVA). In the treatment groups, dexamethasone was injected intraperitoneally 3 hours before general sensitization or local challenge. Symptom score, eosinophil infiltration, and immunostaining for TGF-beta1 and CD4 in nasal mucosa, and TGF-beta1 and OVA-specific immunoglobulin E (IgE) in sera were analyzed., Results: Dexamethasone administration before general sensitization reduced the symptom score, OVA-specific IgE, and eosinophil infiltration and increased the serum level of TGF-beta1 significantly. Dexamethasone administration before local challenge reduced only the eosinophil infiltration significantly. Immunoreactivity of TGF-beta1 and CD4 was lower in both treatment groups., Conclusion: These results suggest that dexamethasone may play an important role in the regulation of allergic reactions by at least two mechanisms; one by suppressing allergic sensitization through decrease of CD4+ T cells and increase of TGF-beta, and the other by suppressing late allergic reactions through the inhibition of proliferation and chemotaxis of inflammatory cells such as eosinophils.

The IL-4 receptor (IL-4R) and IL-13 genes are candidate genes in atopic diseases. The IL-4Ralpha chain and IL-13 promoter polymorphisms are gain-of-function mutations associated with atopy. We tested whether polymorphisms in the IL-4Ralpha chain and the coding region of the IL-13 gene are associated with allergic rhinitis in a Korean population. Polymerase chain reaction-based assays for IL-4Ralpha Gln551Arg and IL-13 exon 4 G2044A were used for genotyping. There were no differences in the frequencies of the genotypes and alleles of IL-4Ralpha between the controls and patients. The frequency of the IL-13 exon 4 2044A allele was statistically different between the controls and patients. Our results suggest that the IL-13 exon 4 G2044A polymorphism confers susceptibility to the development of allergic rhinitis in Koreans, whereas the IL-4Ralpha Gln551Arg polymorphism is not related to allergic rhinitis.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental and food contaminants with known or suspected carcinogenic properties. In this study, we have evaluated whether PAHs activate the early growth response (EGR-1) gene and bind to peroxisome proliferator-activated receptor alpha (PPAR alpha) and delta (PPAR beta/delta) in cell culture systems. Luciferase reporter systems were employed and several PAHs were evaluated for their ability to activate EGR-1 and PPARs. Some PAHs enhanced EGR-1 expression and activated PPAR alpha and PPAR beta. Among them, benz(a)anthracene was found to act as a relatively potent activator of PPAR alpha and PPAR beta/delta, and to significantly enhance EGR-1 transcription. These in vitro assays were confirmed by Western blot analysis, using cell lysates of tissue samples from mouse trapped at a highly contaminated Superfund site in the Chattanooga Creek floodplain in Chattanooga, Tennessee. We have found that a PPAR target gene, glycogen synthase kinase-3beta (GSK-3beta), was down-regulated and EGR-1 was up-regulated in the mouse samples of Chattanooga Creek. In addition, select PAHs repressed GSK-3beta and induced CYP4A in FaO rat hepatoma cells. In conclusion, PAHs activate PPAR alpha and PPAR beta/delta, and up-regulate EGR-1 expression in vitro as well as in vivo. These data may provide a diversity of PAH activity in several biological pathways.

To efficiently perform collection and functional studies of large-scale cDNAs, we constructed multi-functional cDNA vectors for efficient full-length cDNA cloning, direct sequencing, easy screening, and the expression of cDNA in vitro and in vivo without subcloning the cDNA into other vectors. The constructed vectors, pCNS and pCNS-D2, contain a multi-cloning site for uni-directional full-length cDNA cloning, T7 and Sp6 RNA polymerase promoters for in vitro transcription and translation, and hCMV immediate early promoter and BGH poly(A) to allow expression in mammalian cells. Using these vectors, we constructed full-length enriched cDNA libraries containing 60-75% of the full-length cDNAs using two different oligo-capping methods. The subtracted cDNA libraries could also be constructed by removing of EF1-alpha cDNA, a highly expressed cDNA. In addition, we confirmed the translation of EF1-alpha cDNA in vitro and the expression of luciferase cDNA in mammalian cells. The expression efficiency of luciferase cDNA in different cell lines, such as HeLa, Hep3B, SNU638, and SNU668, showed that pCNS vectors can highly express target genes in different cell types. These results indicated that our multi-purpose vectors, pCNS and pCNS-D2, are useful tools for the construction of full-length cDNA libraries and high-throughput based functional study of cDNAs., (Copyright 2004 Elsevier Inc.)

Suppression subtractive hybridization, a PCR-based method for cDNA subtraction, was used to identify differentially expressed genes in keratinocytes. Differentiation was induced by elevating the calcium level in the cell culture medium. Using HaCaT immortalized keratinocytes cultured in the presence of a high calcium concentration, we isolated 60 clones representing 48 different genes. By reverse Northern analysis, 13 genes were scored as overexpressed in these HaCaT cells. Northern blot analysis was used to confirm differential gene expression. Six genes, keratin 1, plasminogen activator inhibitor type 2 (PAI-2), ferritin H, peroxiredoxin 5 (PRDX5), insulin-like growth factor binding protein-3 (IGFBP-3), and one EST gene, were differentially expressed in HaCaT cells cultured in the presence of a high calcium concentration. Two of these genes, keratin 1 and PAI-2, are differentially expressed during keratinocyte terminal differentiation. IGFBP-3, which has reduced expression during epidermal differentiation, was increased after culture in a high-calcium medium for 2 or 5 days. Overexpression of the ferritin H and PRDX5 genes due to elevated calcium has not been reported in keratinocytes. We demonstrated the expression of IGFBP-3, ferritin H, PRDX5, and one gene of a matching sequence from the EST database during differentiation in primary cultured normal human keratinocytes. The EST gene expressed two transcripts of 1.8 kb and 2.5 kb in HaCaT cells, and the transcripts were confirmed to increase in keratinocytes cultured in a high-calcium medium.

Peritoneal metastases (PM) in colorectal cancer (CRC) is associated with a dismal prognosis. Identifying and exploiting new biomarkers, signatures, and molecular targets for personalised interventions in the treatment of PM in CRC is imperative. We conducted transcriptomic profiling using RNA-seq data generated from the primary tissues of 19 CRC patients with PM. Using our dataset established in a previous study, we identified 1422 differentially expressed genes compared to non-metastatic CRC. The profiling demonstrated no differential expression in liver and lung metastatic CRC. We selected 12 genes based on stringent criteria and evaluated their expression patterns in a validation cohort of 32 PM patients and 84 without PM using real-time reverse transcription-polymerase chain reaction. We selected cartilage intermediate layer protein 2 (CILP2) because of high mRNA expression in PM patients in our validation cohort and its association with a poor prognosis in The Cancer Genome Atlas. Kaplan–Meier survival analysis in our validation cohort demonstrated that CRC patients with high CILP2 expression had significantly poor survival outcomes. Knockdown of CILP2 significantly reduced the proliferation, colony-forming ability, invasiveness, and migratory capacity and downregulated the expression of molecules related to epithelial-mesenchymal transition in HCT116 cells. In an in vivo peritoneal dissemination mouse knockdown of CILP2 also inhibited CRC growth. Therefore, CILP2 is a promising biomarker for the prediction and treatment of PM in CRC. [ABSTRACT FROM AUTHOR]

Abnormal activation of receptor tyrosine kinases (RTKs) contributes to tumorigenesis, while protein tyrosine phosphatases (PTPs) contribute to tumor control. One of the most representative PTPs is Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1), which is associated with either an increased or decreased survival rate depending on the cancer type. Hypermethylation in the promoter region of PTPN6, the gene for the SHP-1 protein, is a representative epigenetic regulation mechanism that suppresses the expression of SHP-1 in tumor cells. SHP-1 comprises two SH2 domains (N-SH2 and C-SH2) and a catalytic PTP domain. Intramolecular interactions between the N-SH2 and PTP domains inhibit SHP-1 activity. Opening of the PTP domain by a conformational change in SHP-1 increases enzymatic activity and contributes to a tumor control phenotype by inhibiting the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathway. Although various compounds that increase SHP-1 activation or expression have been proposed as tumor therapeutics, except sorafenib and its derivatives, few candidates have demonstrated clinical significance. In some cancers, SHP-1 expression and activation contribute to a tumorigenic phenotype by inducing a tumor-friendly microenvironment. Therefore, developing anticancer drugs targeting SHP-1 must consider the effect of SHP-1 on both cell biological mechanisms of SHP-1 in tumor cells and the tumor microenvironment according to the target cancer type. Furthermore, the use of combination therapies should be considered. [ABSTRACT FROM AUTHOR]

The CRISPR–Cas9 system is a widely used gene-editing tool, offering unprecedented opportunities for treating various diseases. Controlling Cas9/dCas9 activity at specific location and time to avoid undesirable effects is very important. Here, we report a conditionally active CRISPR–Cas9 system that regulates target gene expression upon sensing cellular environmental change. We conjugated the oxygen-sensing transcription activation domain (TAD) of hypoxia-inducing factor (HIF-1α) with the Cas9/dCas9 protein. The Cas9-TAD conjugate significantly increased endogenous target gene cleavage under hypoxic conditions compared with that under normoxic conditions, whereas the dCas9-TAD conjugate upregulated endogenous gene transcription. Furthermore, the conjugate system effectively downregulated the expression of SNAIL, an essential gene in cancer metastasis, and upregulated the expression of the tumour-related genes HNF4 and NEUROD1 under hypoxic conditions. Since hypoxia is closely associated with cancer, the hypoxia-dependent Cas9/dCas9 system is a novel addition to the molecular tool kit that functions in response to cellular signals and has potential application for gene therapeutics. [ABSTRACT FROM AUTHOR]

Coloration, a major factor contributing to apple quality, attracts consumer attraction, thereby increasing the sale of fruits in the market. Among the fruit pigments, anthocyanins and carotenoids are the most important pigments that impart coloration to apples. Pigment accumulation, which is regulated by transcriptional factors in the anthocyanin and carotenoid biosynthesis pathways, varies according to genetics, transcription, and developmental stage. To understand the regulation of color development in apples, we evaluated the expression of the genes in the anthocyanin and carotenoid biosynthetic pathways and analyzed the accumulation of pigment, including anthocyanin and carotenoid in the skin and flesh of the red-fleshed apple 'Enbu' cultivar at different fruit development stages (30, 60, 90, 120, and 150 days after full bloom, and ripe). Color development increased according to the developmental stage, with considerable variation in both the transcript levels and pigment concentrations observed in tissues, skin, and flesh. Moreover, we identified key transcription factors among the anthocyanin and carotenoid biosynthesis genes that regulated pigment accumulation. Pearson's correlation analysis showed a strong correlation between the coloration patterns and the expression levels of anthocyanin biosynthesis-related genes (MdPAL, MdCHI, and MdF3H) and carotenoid biosynthesis-related genes (MdGGPPS, MdPSY, MdZDS, MdCRTISO, MdCRHβ, and MdZEP). This study provides insight into the molecular mechanisms underlying pigment biosynthesis for breeding high-quality red-fleshed apple varieties to cater to consumer attention and preference. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CXEs) catalyze the hydrolysis of carboxylic acid esters into the corresponding acids and alcohols. Previously, a carboxylesterase gene was isolated from pepper (Capsicum annuum L.) and designated as PepEST. This gene is highly expressed during the incompatible interaction between ripe fruits of pepper and an anthracnose fungus (Colletotrichum gloeosporioides), and the PepEST protein exhibits an antifungal activity by suppressing fungal growth with the inhibition of appressorium formation. In this study, a PepEST-homologous gene was newly isolated from the pepper genome, which is designated as PepEST2 (the original PepEST gene is re-named here as PepEST1). Compared with PepEST1 whose expression is only induced by inoculation of the fungus, PepEST2 is not induced by fungus infection, rather expressed mainly in roots. Subcellular localization analysis using tobacco protoplasts showed that both PepEST1 and PepEST2 proteins are localized in the cytoplasm, and enzymatic activity assays showed similar Vmax and Km values of PepEST2 to those of PepEST1. Antifungal activity assays against C. gloeosporioides demonstrated that not only PepEST1 but also PepEST2 was able to suppress the fungal growth by inhibiting the appressorium formation. In addition, the antifungal activity of PepEST2 was higher than that of PepEST1. Collectively, this study suggests that two closely related members of pepper CXEs are functionally diversified due to the differential expression patterns, and also provides PepEST2 as an alternative gene to confer disease resistance to plants. [ABSTRACT FROM AUTHOR]

Simple Summary: Endometrial cancer has become increasingly common owing to the recent westernization of diet and lifestyle, with 1.7% of cancer patients dying annually. Furthermore, the 5-year endometrial cancer survival rate is barely 15%. Nectin-4 has emerged as a possible biomarker and therapeutic target. Nectin-4 is highly expressed in various cancers. However, no studies have been conducted to determine the clinical importance of Nectin-4 expression in endometrial cancer. Here, we examined 320 tissue samples from patients with endometrial cancer to determine the relevance of Nectin-4 expression in the diagnosis and prognosis of endometrial cancer. Our findings emphasize the importance of Nectin-4 as a novel diagnostic tool and screening marker for assessing endometrial cancer and improving the accuracy of approaches used to predict high-risk endometrial cancer. The adhesion molecule Nectin-4 is a new potential therapeutic target for different types of cancer; however, little is known about its diagnosis significance in endometrial cancer (EC). We found that Nectin-4 expression was significantly higher in EC tissues than in nonadjacent normal tissue. The area under the receiver operating characteristic curve value of 0.922 indicated good diagnostic accuracy for Nectin-4 expression in EC. Furthermore, Nectin-4 expression was associated with DNA mismatch repair (MMR) protein deficiency. Notably, the high Nectin-4 expression group of patients with MSH2/6-deficient EC had shorter progression-free survival than that of the low Nectin-4 expression group. The number of lymphovascular space invasion-positive patients in groups with MMR deficiency and high Nectin-4 expression was also increased compared with that in the low Nectin-4 expression group. Bioinformatics analysis revealed that alteration in Nectin-4 and MMR genes is associated with Nectin-4 expression in EC. To the best of our knowledge, this is the first study to show that Nectin-4 expression may be a potential biomarker for EC diagnosis and that high Nectin-4 expression in MMR-deficient patients with EC can predict short progression-free survival, thus providing clues to identify patients for adjuvant therapy. [ABSTRACT FROM AUTHOR]

Background: Early‐onset colorectal cancer (EOCRC) has been increasing in incidence worldwide but its genomic pathogenesis is mostly undetermined. This study aimed to identify robust EOCRC‐specific gene expression patterns in non‐familial adenomatous polyposis (FAP) and non‐hereditary nonpolyposis colorectal cancer syndrome (HNPCC) EOCRC. Method: We first performed gene expression profiling analysis using RNA sequencing of discovery cohort comprised of 49 EOCRC (age <50) and 50 late‐onset colorectal cancer (LOCRC) (age >70) specimens. To obtain robust gene expression data from this analysis, we validated differentially expressed genes (DEGs) through TCGA cohort (EOCRC:59 samples, LOCRC:229 samples) and our validation cohort (EOCRC:72 samples, LOCRC:43 samples) using real‐time RT‐PCR. After the validation of DEGs, we validated the selected gene at protein levels using Western blotting. To identify whether genomic methylation regulates the expression of a particular gene, we selected methylation sites using The Cancer Genome Atlas (TCGA) datasets and validated them by pyrosequencing in our validation cohort. Results: The EOCRC patients included in this study had significantly more prominent family history of cancer than the LOCRC patients (23 [46.9%] vs. 13 [26%], p = 0.050). Alanyl aminopeptidase (ANPEP) was significantly downregulated in the EOCRC tissues (FC = 1.78, p = 0.0007) and was also commonly downregulated in the TCGA cohort (FC = −1.08, p = 0.0021). Moreover, the ANPEP mRNA and protein expression levels were significantly downregulated in the EOCRC tissues of our validation cohort (p = 0.037 and 0.027). In comparisons of the normal and tumor tissues in public datasets, the ANPEP level was significantly lower in the tumor tissue in the TCGA dataset (p < 2.2 × 10−16) and GSE196006 dataset (p = 0.0005). Furthermore, the ANPEP expression level did not show a decreasing tendency at a young age in the normal colon tissue of the GTEx dataset. Lastly, the hypermethylation of cg26222247 in ANPEP was identified to be weakly associated with reduced ANPEP expression in our EOCRC cohort. Conclusion: The reduced expression of ANPEP was identified as a novel biomarker of non‐FAP and non‐HNPCC EOCRC. [ABSTRACT FROM AUTHOR]

Simple Summary: Garlic juice is one of the most popular health drinks in Asia. Garlic juice processing waste (GJPW) is a potential feed ingredient for aquaculture, because it is rich in bioactive chemicals, which makes it an attractive alternative to synthetic antibiotics and/or antioxidants. This study investigates the effects of various dietary levels of garlic juice processing waste on the growth performance and health status of juvenile black (Sebastes schlegelii). On the basis of the results, rockfish fed diets supplemented with GJPW show improvement in growth performance, digestive enzyme activity, and growth and antioxidant-related gene expression. Therefore, considering the effects on the overall performance of juvenile rockfish, GJPW is a promising useful additive, and 2.5 g kg−1 dietary GJPW was found to be a suitable dietary level for juvenile rockfish black rockfish. An 8-week feeding trial was conducted to evaluate the effects of various dietary levels of garlic juice processing waste (GJPW) on the growth, feed utilization, digestive and antioxidant enzyme activity, growth- and antioxidant-related gene expression, and resistance to Streptococcus iniae infection of juvenile black rockfish (Sebastes schlegelii). A total of 450 juvenile rockfish were randomly distributed into 30 L rectangular tanks (30 fish per tank). Five experimental diets were prepared in triplicate. The fish were fed experimental diets supplemented with GJPW at concentrations of 0 (GJPW0, control), 2.5 (GJPW2.5), 5 (GJPW5), 7.5 (GJPW7.5), and 10 g kg−1 (GJPW10) diet. All of the GJPW-supplemented treatments (2.5, 5, 7.5, and 10 g kg−1) significantly enhanced weight gain (WG), specific growth rate (SGR), feed efficiency (FE), protein efficiency ratio (PER), and digestive enzyme activity (amylase, trypsin, and lipase). A decreasing trend was seen in plasma aspartate aminotransferase (ALT), alanine aminotransferase (AST), and glucose (GLU) content with increasing dietary levels of GJPW. In contrast, plasma lysozyme and antioxidant enzyme activities were significantly increased with increasing dietary GJPW levels. Furthermore, GJPW administration significantly upregulated the expression of insulin-like growth factor-1 (IGF-1), superoxide dismutase (SOD), catalase (CAT), and glutathione S-transferase (GST) in the liver of rockfish. A challenge test with S. iniae showed significantly higher resistance in the GJPW-supplemented treatments than in the control. In short, dietary supplementation GJPW enhanced growth performance and antioxidant response in juvenile black rockfish, with suitable effects in fish fed with 2.5 g kg−1 GJPW for 8 weeks. [ABSTRACT FROM AUTHOR]

Matriptases are cell surface proteolytic enzymes belonging to the type II transmembrane serine protease family that mediate inflammatory skin disorders and cancer progression. Matriptases may affect the development of periodontitis via protease-activated receptor-2 activity. However, the cellular mechanism by which matriptases are involved in periodontitis is unknown. In this study, we examined the antiperiodontitis effects of matriptase on Porphyromonas gingivalis-derived lipopolysaccharide (PG-LPS)-stimulated human gingival fibroblasts (HGFs). Matriptase small interfering RNA-transfected HGFs were treated with PG-LPS. The mRNA and protein levels of proinflammatory cytokines and matrix metalloproteinase 1 (MMP-1) were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR) and an enzyme-linked immunosorbent assay (ELISA), respectively. Western blot analyses were performed to measure the levels of Toll-like receptor 4 (TLR4)/interleukin-1 (IL-1) receptor-associated kinase (IRAK)/transforming growth factor β-activated kinase 1 (TAK1), p65, and p50 in PG-LPS-stimulated HGFs. Matriptase downregulation inhibited LPS-induced proinflammatory cytokine expression, including the expression of IL-6, IL-8, tumor necrosis factor-α (TNF-α), and IL-Iβ. Moreover, matriptase downregulation inhibited PG-LPS-stimulated MMP-1 expression. Additionally, we confirmed that the mechanism underlying the effects of matriptase downregulation involves the suppression of PG-LPS-induced IRAK1/TAK1 and NF-κB. These results suggest that downregulation of matriptase PG-LPS-induced MMP-1 and proinflammatory cytokine expression via TLR4-mediated IRAK1/TAK1 and NF-κB signaling pathways in HGFs. [ABSTRACT FROM AUTHOR]

Hepatic diseases, such as hepatonecrosis, hepatitis, and hepatocirrhosis, are associated with mitochondrial dysfunction and increased reactive oxygen species generation and inflammation, ultimately leading to liver failure. In this study, we examined if acupuncture at LR3 can affect mitochondria-related gene expression in a liver damage model of experimentally induced acute liver failure (ALF). ALF was induced by the intraperitoneal injection of D-galactosamine (D-GalN) in experimental rats, who then received either sham (ALF), manual acupuncture (MA), electroacupuncture (EA), or silymarin (PC, positive control) treatment. Liver tissues were extracted from experimental and untreated control rats for histopathological analysis and expression profiling of genes involved in mitochondrial function. Of the 168 mitochondria-related genes profiled, two genes belonging to the solute-carrier transporter family (Slc25a15 and Slc25a25) and Ndufb7 were upregulated. Gamma-glutamylcysteine synthetase was more downregulated in MA than ALF. Furthermore, MA reversed D-GalN-induced inflammatory cell infiltration, destruction of hepatic cell plates, and increase in the levels of the proinflammatory cytokine TNF-α. MA at LR3 can reduce the risk of D-GalN-induced ALF by inducing the expression of metabolic and inflammation-related genes and regulating proinflammatory factor production in hepatic mitochondria. [ABSTRACT FROM AUTHOR]

Simple Summary: Considering that cancer-associated fibroblasts (CAFs) facilitate cancer cell motility, invasiveness, and drug resistance, heterogeneity within the CAF population may be of major significance during cancer progression and metastasis. Through pseudotime trajectory analysis of single-cell RNA sequencing data, we revealed several subpopulations of lung CAFs with distinct gene expression patterns, namely, immunosuppressive, neoantigen-presenting, myofibroblastic, and proliferative CAFs. The knockdown of KPNA2, one of the neoantigen-presenting CAF-specific markers, attenuated CAF invasiveness, suggesting that CAF subtype markers may represent therapeutic targets within the tumor microenvironment of lung cancer patients. Cancer-associated fibroblasts (CAFs) reside within the tumor microenvironment, facilitating cancer progression and metastasis via direct and indirect interactions with cancer cells and other stromal cell types. CAFs are composed of heterogeneous subpopulations of activated fibroblasts, including myofibroblastic, inflammatory, and immunosuppressive CAFs. In this study, we sought to identify subpopulations of CAFs isolated from human lung adenocarcinomas and describe their transcriptomic and functional characteristics through single-cell RNA sequencing (scRNA-seq) and subsequent bioinformatics analyses. Cell trajectory analysis of combined total and THY1 + CAFs revealed two branching points with five distinct branches. Based on Gene Ontology analysis, we denoted Branch 1 as "immunosuppressive", Branch 2 as "neoantigen presenting", Branch 4 as "myofibroblastic", and Branch 5 as "proliferative" CAFs. We selected representative branch-specific markers and measured their expression levels in total and THY1 + CAFs. We also investigated the effects of these markers on CAF activity under coculture with lung cancer cells. This study describes novel subpopulations of CAFs in lung adenocarcinoma, highlighting their potential value as therapeutic targets. [ABSTRACT FROM AUTHOR]

Periodontitis is a Gram-negative bacterial infectious disease. Numerous inflammatory cytokines, including interleukin-1β (IL-1β), regulate periodontitis pathophysiology and cause periodontal tissue destruction. In human gingival fibroblasts (HGFs), IL-1β stimulates the production of matrix metalloproteinases (MMPs) and proinflammatory cytokines via various mechanisms. Several transcription factors, such as signal transducer and activator of transcription 3 (STAT-3), activator protein 1 (AP-1), and nuclear factor-κB (NF-κB), regulate gene expression. Mitogen-activated protein kinases (MAPKs) regulate these transcription factors. However, the MAPK/STAT-3 activation signal in HGFs is unknown. We investigated the potential inhibitory effects of the extract of Evodiae fructus (EFE), the dried, ripe fruit of Evodia rutaecarpa, on MMP and proinflammatory cytokine expression in IL-1β-stimulated HGFs. EFE inhibited the expression of MMP-1, MMP-3, and proinflammatory cytokines (TNF-α, IL-6, and IL-8) in IL-1β-stimulated HGFs through the inhibition of IL-1β-induced MAPK/STAT-3 activation. Also, these results suggest that the EFE may be a useful for the bioactive material for oral care. [ABSTRACT FROM AUTHOR]

Xanthomonas oryzae pv. oryzae (Xoo) is a plant pathogen responsible for causing bacterial blight in rice. The immediate alterations in Xoo upon initial contact with rice are essential for pathogenesis. We studied time-resolved genome-wide gene expression in pathogenicity-activated Xoo cells at the transcriptome and proteome levels. The early response genes of Xoo include genes related to cell motility, inorganic ion transport, and effectors. The alteration of gene expression is initiated as early as few minutes after the initial interaction and changes with time. The time-resolved comparison of the transcriptome and proteome shows the differences between transcriptional and translational expression peaks in many genes, although the overall expression pattern of mRNAs and proteins is conserved. The discrepancy suggests an important role of translational regulation in Xoo at the early stages of pathogenesis. The gene expression analysis using time-resolved transcriptome and proteome provides unprecedented valuable information regarding Xoo pathogenesis. [ABSTRACT FROM AUTHOR]

Fetal growth restriction (FGR) is the failure of the fetus toachieve its genetically determined growth potential, which increasesrisks for a variety of genetic diseases, such as type 2 diabetes mellitus, coronary artery disease, and stroke, during the lifetime. The dysregulation of DNA methylationis known to interact with environmental fluctuations, affect gene expressions comprehensively, and be fatal to fetus development in specific cases. Therefore, we set out to find out epigenetic and transcriptomic alterations associated with FGR development. We found a set of differentially expressed genes associated with differentially methylated regions in placentae and cord blood samples. Using dimensional reduction analysis, the expression and methylation variables of the epigenetically altered genes classified the FGR samples from the controls. These genes were also enriched in the biological pathways such as metabolism and developmental processes related to FGR. Furthermore, three genes of INS, MEG3, and ZFP36L2 are implicated in epigenetic imprinting, which has been associated with FGR. These results strongly suggest that DNA methylation is highly dysregulated during FGR development, and abnormal DNA methylation patterns are likely to alter gene expression. [ABSTRACT FROM AUTHOR]

Pathological changes in the epigenetic landscape of chromatin are hallmarks of cancer. The caudal-type homeobox gene CDX2 is not expressed in normal gastric epithelia but rather in adult intestinal epithelia, and it is overexpressed in intestinal metaplasia (IM). However, it remains unclear how CDX2 transcription is suppressed in normal gastric epithelial cells and overexpressed in IM. Here, we demonstrate that methylation of the CDX2 promoter increases with age in Helicobacter pylori -positive, noncancerous gastric tissue, whereas the promoter is demethylated in paired gastric tumors in which CDX2 is upregulated. Moreover, we also found that the CDX2 promoter is demethylated in IM as well as gastric tumor. Immunohistochemistry revealed that CDX2 is present in foci of parts of the gastric mucosae but highly expressed in IM as well as in gastric tumors, suggesting that the elevated level of CDX2 in IM and gastric tumors may be attributable to promoter demethylation. Our data suggest that CDX2 repression may be associated with promoter methylation in noncancerous H. pylori -positive mucosa but its upregulation might be attributable to increased promoter activity mediated by chromatin remodeling during gastric carcinogenesis. [ABSTRACT FROM AUTHOR]

Temporal gene expression profiles have been widely considered to uncover the mechanism of cancer development and progression. Gene expression patterns, however, have been analyzed for limited stages with small samples, without proper data pre-processing, in many cases. With those approaches, it is difficult to unveil the mechanism of cancer development over time. In this study, we analyzed gene expression profiles of two independent colorectal cancer sample datasets, each of which contained 556 and 566 samples, respectively. To find specific gene expression changes according to cancer stage, we applied the linear mixed-effect regression model (LMER) that controls other clinical variables. Based on this methodology, we found two types of gene expression patterns: continuously increasing and decreasing genes as cancer develops. We found that continuously increasing genes are related to the nervous and developmental system, whereas the others are related to the cell cycle and metabolic processes. We further analyzed connected sub-networks related to the two types of genes. From these results, we suggest that the gene expression profile analysis can be used to understand underlying the mechanisms of cancer development such as cancer growth and metastasis. Furthermore, our approach can provide a good guideline for advancing our understanding of cancer developmental processes. [ABSTRACT FROM AUTHOR]

A Bacillus strain, BS15, showing strong fibrinolytic activity, antibacterial activity, and salt tolerance was isolated from gul (oyster) jeotgal, a Korean fermented sea food. BS15 was identified as B. pumilus. B. pumilus BS15 was able to grow in LB broth with 18% (w/v) NaCl. When culture supernatant was analyzed by SDS-PAGE, 22, 27, 35, and 60 kDa proteins were observed. The 27 kDa protein was determined to be major fibrinolytic enzyme by fibrin zymography. The gene (aprEBS15) was cloned in pHY300PLK, a Bacillus-E. coli shuttle vector. A B. subtilis transformant (TF) harboring pHYBS15 showed higher fibrinolytic activity than B. pumilus BS15, and produced the same 27 kDa protein. aprEBS15 was overexpressed in E. coli BL21 (DE3), and recombinant enzyme (AprEBS15) was purified. The optimum pH and temperature of AprEBS15 were pH 8.0 and 40°C, respectively. Km and Vmax values were 0.26 mM and 21.88 µmol/L/min, respectively. B. pumilus BS15 can be used as a starter for jeotgals and other fermented foods with high salinities. [ABSTRACT FROM AUTHOR]

Ghrelin modulates mesolimbic dopaminergic pathways in the brain in addition to its role in feeding. We investigated what roles ghrelin in the nucleus accumbens (NAcc) core may play in mediating locomotor activating effects of amphetamine (AMPH). First, when rats were administered with AMPH (1 mg/kg, i.p.) following a bilateral microinjection of ghrelin (0.1 or 0.5 μg/side) into the NAcc core, their locomotor activity was significantly enhanced, while these effects were blocked by co-microinjection of ghrelin receptor antagonist (0.5 μg/side) into this site. Second, we pre-exposed rats to saline or amphetamine (1 mg/kg, i.p.) every 2 to 3 days for a total of four times. After 2 weeks of drug-free withdrawal period, we examined the effect of saline, ghrelin (0.5 μg/side), D1 dopamine receptor agonist, SKF81297 (0.5 μg/side) or ghrelin (0.5 μg/side) + SKF81297 (0.5 μg/side) directly microinjected into the NAcc core on locomotor activity. When we measured rats' locomotor activity for 1 hour immediately following microinjections, only ghrelin + SKF81297 produces sensitized locomotor activity, while all others have no effects. These results suggest that ghrelin may have a distinct role in the NAcc core to provoke the sensitized locomotor activity induced by psychomotor stimulants, and further, it may produce these effects by interaction with D1 dopamine receptors. [ABSTRACT FROM AUTHOR]

Direct conversion from fibroblasts to generate hepatocyte like-cells (iHeps) bypassing the pluripotent state has been described in previous reports as an attractive method acquiring hepatocytes for cell-based therapy. The limited proliferation of iHeps, however, has hampered it uses in cell-based therapy. Since hepatic stem cells (HepSCs) possess self-renewal and bipotency with the capacity to differentiate into both hepatocytes and cholangiocytes, they have therapeutic potential for treating liver disease. Here, we investigated the therapeutic effects of induced HepSCs (iHepSCs) on a carbon tetrachloride (CCl4)-induced liver fibrosis model. We demonstrate that Oct4 and Hnf4a are sufficient to convert fibroblasts into expandable iHepSCs. Hepatocyte-like cells derived from iHepSCs (iHepSC-HEPs) exhibit the typical morphology of hepatocytes and hepatic functions, including glycogen storage, low-density lipoprotein (LDL) uptake, Indocyanine green (ICG) detoxification, drug metabolism, urea production, and albumin secretion. iHepSCs-derived cholangiocyte-like cells (iHepSC-CLCs) expressed cholangiocyte-specific markers and formed cysts and tubule-like structures with apical-basal polarity and secretory function in three-dimensional culture condition. Furthermore, iHepSCs showed anti-inflammatory and anti-fibrotic effects in CCl4-induced liver fibrosis. This study demonstrates that Oct4 and Hnf4α-induced HepSCs show typical hepatic and biliary functionality in vitro. It also presents the therapeutic effect of iHepSCs in liver fibrosis. Therefore, directly converting iHepSCs from somatic cells may facilitate the development of patient-specific cell-based therapy for chronic liver damage. [ABSTRACT FROM AUTHOR]

Sageretia thea (S. thea) commonly known as Chinese sweet plum or Chinese bird plum has been used for treating hepatitis and fevers in Korea and China. S. thea has been reported to exert anti-oxidant, anticancer and anti-human immunodeficiency virus activity. However, there is little study on the anti-inflammatory activity of S. thea. Thus, we evaluated the anti-inflammatory effect of extracts of leaves (ST-L) and branches (ST-B) from Sageretia thea in LPS-stimulated RAW264.7 cells. ST-L and ST-B significantly inhibited the production of the pro-inflammatory mediators such as NO, iNOS, COX-2, IL-1 β and IL-6 in LPS-stimulated RAW264.7 cells. ST-L and ST-B blocked LPS-induced degradation of I κ B- α and nuclear accumulation of p65, which resulted in the inhibition of NF- κ B activation in RAW264.7 cells. ST-L and ST-B also attenuated the phosphorylation of ERK1/2, p38 and JNK in LPS-stimulated RAW264.7 cells. In addition, ST-L and ST-B increased HO-1 expression in RAW264.7 cells, and the inhibition of HO-1 by ZnPP reduced the inhibitory effect of ST-L and ST-B against LPS-induced NO production in RAW264.7 cells. Inhibition of p38 activation and ROS elimination attenuated HO-1 expression by ST-L and ST-B, and ROS elimination inhibited p38 activation induced by ST-L and ST-B. ST-L and ST-B dramatically induced nuclear accumulation of Nrf2, but this was significantly reversed by the inhibition of p38 activation and ROS elimination. Collectively, our results suggest that ST-L and ST-B exerts potential anti-inflammatory activity by suppressing NF- κ B and MAPK signaling activation, and activating HO-1 expression through the nuclear accumulation of Nrf2 via ROS-dependent p38 activation. These findings suggest that ST-L and ST-B may have great potential for the development of anti-inflammatory drug to treat acute and chronic inflammatory disorders. [ABSTRACT FROM AUTHOR]

In all organisms, DNA damage must be repaired quickly and properly, as it can be lethal for cells. Because eukaryotic DNA is packaged into nucleosomes, the structural units of chromatin, chromatin modification is necessary during DNA damage repair and is achieved by histone modification and chromatin remodeling. Chromatin remodeling proteins therefore play important roles in the DNA damage response (DDR) by modifying the accessibility of DNA damage sites. Here, we show that mutation in a SWI2/SNF2 chromatin remodeling protein (DDM1) causes hypersensitivity in the DNA damage response via defects in single-strand annealing (SSA) repair of double-strand breaks (DSBs) as well as in the initial steps of homologous recombination (HR) repair. ddm1 mutants such as ddm1-1 and ddm1-2 exhibited increased root cell death and higher DSB frequency compared to the wild type after gamma irradiation. Although the DDM1 mutation did not affect the expression of most DDR genes, it did cause substantial decrease in the frequency of SSA as well as partial inhibition in the γ-H2AX and Rad51 induction, the initial steps of HR. Furthermore, global chromatin structure seemed to be affected by DDM1 mutations. These results suggest that DDM1 is involved in the homology directed repair such as SSA and HR, probably by modifying chromatin structure. [ABSTRACT FROM AUTHOR]

Background: Sageretia thea (S. thea) has been used as the medicinal plant for treating hepatitis and fevers in Korea and China. Recently, anticancer activity of S. thea has been reported, but the potential mechanism for the anti-cancer property of S. thea is still insufficient. Thus, we evaluated whether extracts from the leaves (STL) and branches (STB) of S. thea exert anticancer activity and elucidated its potential mechanism in SW480 cells. Methods: MTT assay was performed for measuring cell viability. Western blot and RT-PCR were used for analyzing the level of protein and mRNA, respectively. Results: Treatment of STL or STB decreased the cell viability and induced apoptosis in SW480 cells. Decreased level of cyclin D1 protein was observed in SW480 cells treated with STL or STB, but no change in cyclin D1 mRNA level was observed with the treatment of STL or STB. MG132 blocked downregulation of cyclin D1 protein by STL or STB. Thr286 phosphorylation of cyclin D1 by STL or STB occurred faster than downregulation of cyclin D1 protein in SW480 cells. When SW480 cells were transfected with T286A-cyclin D1, cyclin D1 degradation by STL or STB did not occur. Inhibition of GSK3β and cyclin D1 nuclear export attenuated STL or STB-mediated cyclin D1 degradation. In addition, STL or STB increased HO-1 expression, and the inhibition of HO-1 attenuated the induction of apoptosis by STL or STB. HO-1 expression by STL or STB resulted from Nrf2 activation through ROS-dependent p38 activation. Conclusions: These results indicate that STL or STB may induce GSK3β-dependent cyclin D1 degradation, and increase HO-1 expression through activating Nrf2 via ROS-dependent p38 activation, which resulted in the decrease of the viability in SW480 cells. These findings suggest that STL or STB may have great potential for the development of anti-cancer drug. [ABSTRACT FROM AUTHOR]

In fasting or diabetes, gluconeogenic genes are transcriptionally activated by glucagon stimulation of the cAMP-protein kinase A (PKA)-CREB signaling pathway. Previous work showed pyruvate dehydrogenase kinase (PDK) inhibition in skeletal muscle increases pyruvate oxidation, which limits the availability of gluconeogenic substrates in the liver. However, this study found upregulation of hepatic PDK4 promoted glucagon-mediated expression of gluconeogenic genes, whereas knockdown or inhibition of hepatic PDK4 caused the opposite effect on gluconeogenic gene expression and decreased hepatic glucose production. Mechanistically, PDK4 deficiency decreased ATP levels, thus increasing phosphorylated AMPK (p-AMPK), which increased p-AMPK-sensitive phosphorylation of cyclic nucleotide phosphodiesterase 4B (p-PDE4B). This reduced cAMP levels and consequently p-CREB. Metabolic flux analysis showed that the reduction in ATP was a consequence of a diminished rate of fatty acid oxidation (FAO). However, overexpression of PDK4 increased FAO and increased ATP levels, which decreased p-AMPK and p-PDE4B and allowed greater accumulation of cAMP and p-CREB. The latter were abrogated by the FAO inhibitor etomoxir, suggesting a critical role for PDK4 in FAO stimulation and the regulation of cAMP levels. This finding strengthens the possibility of PDK4 as a target against diabetes. [ABSTRACT FROM AUTHOR]

Glioma is the most malignant type of primary central nervous system tumors, and has an extremely poor prognosis. One potential therapeutic approach is to induce the terminal differentiation of glioma through the forced expression of pro-neural factors. Our goal is to show the proof of concept of the neuronal conversion of C6 glioma through the combined action of small molecules. We investigated the various changes in gene expression, cell-specific marker expression, signaling pathways, physiological characteristics, and morphology in glioma after combination treatment with two small molecules (CHIR99021, a glycogen synthase kinase 3 [GSK3] inhibitor and forskolin, a cyclic adenosine monophosphate [cAMP] activator). Here, we show that the combined action of CHIR99021 and forskolin converted malignant glioma into fully differentiated neurons with no malignant characteristics; inhibited the proliferation of malignant glioma; and significantly down-regulated gene ontology and gene expression profiles related to cell division, gliogenesis, and angiogenesis in small molecule–induced neurons. In vivo, the combined action of CHIR99021 and forskolin markedly delayed neurological deficits and significantly reduced the tumor volume. We suggest that reprogramming technology may be a potential treatment strategy replacing the therapeutic paradigm of traditional treatment of malignant glioma, and a combination molecule comprising a GSK3 inhibitor and a cAMP inducer could be the next generation of anticancer drugs. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinases (MAPKs) are involved in a variety of intracellular events such as gene expression, cell proliferation, and programmed cell death. MAPKs are activated by dual phosphorylation on threonine and tyrosine residues through sequential activation of protein kinases. Recent studies have shown that the protein kinases involved in MAPK signal transductions might be organized into signaling complexes by scaffold proteins. These scaffold proteins are essential regulators that function by assembling the relevant molecular components in mammalian cells. In this study, we report that dual-specificity phosphatase 22 (DUSP22), a member of the protein tyrosine phosphatase family, acts as a distinct scaffold protein in c-Jun N-terminal kinase (JNK) signaling. DUSP22 increased the phosphorylation in the activation loop of JNK regardless of its phosphatase activity but had no effect on phosphorylation levels of ERK and p38 in mammalian cells. Furthermore, DUSP22 selectively associated with apoptosis signal-regulating kinase 1 (ASK1), MAPK kinase 7 (MKK7), and JNK1/2. Both JNK phosphorylation and JNK-mediated apoptosis increased in a concentration-dependent manner regardless of DUSP22 phosphatase activity at low DUSP22 concentrations, but then decreased at higher DUSP22 concentrations, which is the prominent feature of a scaffold protein. Thus, our data suggest that DUSP22 regulates cell death by acting as a scaffold protein for the ASK1-MKK7-JNK signal transduction pathway independently of its phosphatase activity. [ABSTRACT FROM AUTHOR]