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9-cis Retinoic acid (9-CRA) is a lipophilic molecule that binds to the retinoid X receptor (RXR). Although retinoic acid (RA) has been known to regulate neutrophil differentiation, a specific role for 9-CRA in chemokine-mediated cellular processes remains obscure. We investigated the effects of 9-CRA on expression of CC chemokine receptors (CCRs) in human monocytic THP-1 cells and peripheral blood monocytes. RNase protection assay was performed to examine the mRNA levels of CCRs in 9-CRA-treated THP-1 cells. mRNA expression of CCR1 and CCR2 was induced in both a dose and time dependent manner. CCR1 and CCR2 mRNA expression began to increase from 6h after a 100nM 9-CRA treatment and reached a maximal level at 12h. Surface expression of CCRs was monitored by flow cytometry. CCR1 and CCR2 surface expression increased in 9-CRA-treated THP-1 cells, but not in untreated cells. Calcium mobilization and chemotactic activity were determined to examine the effect of 9-CRA on cell movement. The intracellular Ca(2+) concentration and the chemotactic activity increased in 9-CRA-treated cells in response to the CCR1-dependent chemokines Lkn-1, MIP-1alpha, and RANTES, and the CCR2-specific chemokine MCP-1. Increased surface expression of CCR1 and the Ca(2+) influx due to 9-CRA were confirmed in peripheral blood monocytes. Taken together, 9-CRA increases the expression levels of mRNA and protein of both CCR1 and CCR2, and the cell migration ability in THP-1 cells and peripheral blood monocytes, indicating that 9-CRA may regulate inflammatory processes through an increased response to CCR1- and CCR2-dependent chemokines.

Simple Summary: Prostate cancer is a serious threat to male health worldwide; however, chemotherapy remains an urgent problem because of resistance and insensitivity to androgen-deprivation therapy. Therefore, discovering novel molecules and chemotherapy can provide a new strategy. Pyruvate kinase M2 (PKM2) is not only a vital enzyme in regulating cancer glycolysis, but is also a transcription that regulates gene expression through nuclear translocation, so the targeting PKM2 can be a promising therapy for prostate cancer. In this experiment, we found a novel specific PKM2 inhibitor—compound 3h—and conducted molecular and cellular experiments to evaluate its anticancer effect. Our results indicated that proliferation of LNCaP cells can be inhibited by compound 3h through inducing apoptosis and autophagy, and inhibiting glycolysis and mitochondria respiration. Therefore, our results provide a new way of developing novel chemotherapy for prostate cancer. Pyruvate kinase M2 (PKM2) is a key enzyme involved in the regulation of glycolysis. Although PKM2 is overexpressed in various tumor tissues, its functional role in cancer chemotherapy remains unexplored. In this study, we investigated the anticancer activity of a new PKM2 inhibitor, compound 3h, through the cell metabolism and associated signaling pathways in prostate cancer cells. To evaluate the molecular basis of specific PKM2 inhibitors, the interactions of compounds 3h and 3K with the PKM2 protein were assessed via molecular docking. We found that, compared to compound 3K, compound 3h exhibited a higher binding affinity for PKM2. Moreover, compound 3h significantly inhibited the pyruvate kinase activity and PKM2 expression. Cytotoxicity and colony formation assays revealed the potent anticancer activity of compound 3h against LNCaP cells. Compound 3h significantly increased the apoptotic and autophagic cell death in LNCaP cells. In addition, compound 3h induced AMPK activation along with the inhibition of the mTOR/p70S6K pathway. Furthermore, compound 3h significantly inhibited glycolysis and mitochondrial respiration, as determined by analyzing the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) production. Our results revealed that compound 3h caused apoptotic and autophagic cell death in LNCaP cells by inhibiting cancer cell metabolism. Therefore, blocking glycolytic pathways using specific PKM2 inhibitors can target cancer cell metabolism in PKM2-overexpressed prostate cancer cells. [ABSTRACT FROM AUTHOR]

Objectives: Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) entry into the host cells depends on the expression of angiotensin‐converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2). We investigated the distribution of ACE2‐ and TMPRSS2‐expressing cells in various oral tissues to identify the underlying mechanism of oral manifestations in patients with coronavirus disease 2019. Subjects: We analyzed the expression patterns of ACE2 and TMPRSS2 in the oral mucosa (tongue, palate, and buccal mucosa), trigeminal ganglion, vessels, and salivary glands of 9 Sprague‐Dawley rats using immunohistochemistry and immunofluorescence. Results: ACE2 and TMPRSS2 were strongly expressed in the intermediate layer of the squamous epithelia of tongue papillae and buccal mucosa. ACE2‐ and TMPRSS2‐positive cells were observed in the taste buds of the tongue. Additionally, ACE2 and TMPRSS2 were co‐expressed in the ductal epithelium and acinar cells of salivary glands. Furthermore, both ACE2 and TMPRSS2 were stained in the neuronal cell body of trigeminal ganglia, but not in Schwann cells. Moreover, ACE2 and TMPRSS2 were expressed in capillaries, but not in venules/arterioles. Conclusions: SARS‐CoV‐2 can spread the suprabasal area of squamous epithelia of the oral mucosa, invades taste bud, trigeminal nerve, parotid gland, and microvessel, resulting in oral manifestations. [ABSTRACT FROM AUTHOR]

Simple Summary: Patients with low-glycolytic hepatocellular carcinoma (HCC) show better clinical outcomes than those with hypoxic and high-glycolytic HCC. Low-glycolytic HCCs seem to utilize carbon sources other than glucose for metabolic fuel and tumor growth. However, by increasing tumor size, its outgrowth perfusion generates hypoxic foci inside the tumor and becomes more aggressive and resistant to therapy. In this study, we found that SLC13A5/NaCT is an important solute carrier (SLC) in low-glycolytic HCCs. To adapt to hypoxic conditions, low-glycolytic cancer cells have to switch metabolism from oxidative phosphorylation to hypoxia-induced glycolysis by the upregulation of HIF1α. However, extracellular citrate treatment in HCCs with high SLC13A5/NaCT expression had reduced glucose uptake due to HIF1α degradation, inducing the failure of metabolic adaptation to hypoxia, resulting in anti-cancer effects in in vitro and in vivo animal models. HCC is well known for low glycolysis in the tumors, whereas hypoxia induces glycolytic phenotype and tumor progression. This study was conducted to evaluate the expression of SLCs in human HCCs and investigated whether extracellular nutrient administration related to SLCs in low-glycolytic HCC can prevent hypoxic tumor progression. SLCs expression was screened according to the level of glycolysis in HCCs. Then, whether extracellular nutrient treatment can affect hypoxic tumor progression, as well as the mechanisms, were evaluated in an in vitro cell line and an in vivo animal model. Low-glycolytic HCCs showed high SLC13A5/NaCT and SLC16A1/MCT1 but low SLC2A1/GLUT1 and HIF1α/HIF1α expression. Especially, high SLC13A5 expression was significantly associated with good overall survival in the Cancer Genome Atlas (TCGA) database. In HepG2 cells with the highest NaCT expression, extracellular citrate treatment upon hypoxia induced HIF1α degradation, which led to reduced glycolysis and cellular proliferation. Finally, in HepG2-animal models, the citrate-treated group showed smaller tumor with less hypoxic areas than the vehicle-treated group. In patients with HCC, SLC13A5/NaCT is an important SLC, which is associated with low glycolysis and good prognosis. Extracellular citrate treatment induced the failure of metabolic adaptation to hypoxia and tumor growth inhibition, which can be a potential therapeutic strategy in HCCs. [ABSTRACT FROM AUTHOR]

Simple Summary: Activation of YAP/TAZ (mediators of Hippo signaling) is associated with the development of liver cancer. The expression level of YAP is known to relate to chemoresistance. However, the therapeutic effect of YAP/TAZ inhibition when combined with sorafenib and conventional chemotherapy in HCC is not known. Recent studies have highlighted the importance of the tumor microenvironment (TME) in chemoresistance. The multicellular tumor spheroid (MCTS) model has emerged as a promising tool for studying cancer drugs as it mimics actual TME. Here, we aimed at assessing the YAP/TAZ expression level of HCCs and identifying the therapeutic effects of CA3 (novel YAP inhibitor) when combined with sorafenib using a patient-derived MCTS model. We established patient-derived MCTS and confirmed that patient-derived MCTS with high YAP/TAZ expression responded more sensitively to the combination therapy (sorafenib with CA3) than MCTS with low or medium YAP/TAZ expression. These findings suggest that the YAP/TAZ inhibitor may serve as a potential therapeutic strategy to enhance sensitivity to sorafenib in HCCs with high YAP/TAZ expression. Purpose: To assess the expression levels of YAP and TAZ in patient-derived HCC tissue and identify the effects of YAP/TAZ inhibition depending on the baseline YAP/TAZ expression when combined with sorafenib using a patient-derived multicellular tumor spheroid (MCTS) model. Methods: Primary HCC cell lines were established from patient-derived tissue. Six patient-derived HCC cell lines were selected according to YAP/TAZ expression on Western blot: high, medium, low. Then, MCTS was generated by mixing patient-derived HCC cells and stroma cells (LX2, WI38, and HUVECs) and YAP/TAZ expression was assessed using Western blot. Cell viability of MCTS upon 48 h of drug treatment (sorafenib, sorafenib with CA3 0.1 µM, and CA3 (novel YAP1 inhibitor)) was analyzed. Results: Out of six patient-derived HCC cell lines, cell lines with high YAP/TAZ expression at the MCTS level responded more sensitively to the combination therapy (Sorafenib + CA3 0.1 μM) despite the potent cytotoxic effect of CA3 exhibited in all of the patient-derived HCCs. Conclusion: Targeting YAP/TAZ inhibition using the novel YAP1 inhibitor CA3 could be a promising therapeutic strategy to enhance sensitivity to sorafenib especially in HCCs with high YAP/TAZ expression in MCTS. [ABSTRACT FROM AUTHOR]

This study was to investigate the effects of Biyeom-go (BYG, an herbal formula) on immune biomarkers present in the nasal mucosa of patients with allergic rhinitis under exposure to particulate matter 2.5 (PM2.5), and on changes in goblet cells and immune biomarkers in mice under exposure to Korea diesel particulate matter (KDP20). Thirty patients showing characteristic allergic rhinitis symptoms were enrolled in Jeonju-si, Korea, and treated with BYG thrice a day for four weeks. Changes in the expression of immune biomarkers (interleukin 4 (IL-4), IL-5, IL-8, IL-13, IL-33, and thymic stromal lymphopoietin (TSLP) mRNA), total nasal symptom scores (TNSS), mini-rhinitis-specific quality of life questionnaire (RQLQ) results, and visual analog scale scores were evaluated after 4 weeks of treatment. Additionally, the difference in PM2.5 concentrations in the air in Jeonju-si, Korea (November, 2019 ∼ March, 2020), was analyzed to determine the change in TNSS. KDP20 (100 μg/mL) was exposed to C57BL/6 mice for 10 days; 0.05% Nasonex (a positive control, mometasone furoate), or BYG was administrated for 5 days twice a day. The expression of inflammatory factors was detected via qRT-PCR using nasopharynx tissue samples of mice. BYG treatment was found to be associated with significant improvement in total nasal symptoms, especially itching and sneezing (p < 0.0001), and mini-RQLQ after 4 weeks. IL-8 (p < 0.01), IL-33 (p < 0.01), and TSLP (p < 0.001) expression levels decreased after BYG treatment. In mice, administration of BYG reduced the number of goblet cells increased through KDP20 treatment. KDP20-induced immune biomarkers (IL-33, TSLP, tumor necrosis factor alpha, and IL-8) were also significantly downregulated in the nasopharynx tissue after BYG treatment. Therefore, BYG may show therapeutic effects against allergic rhinitis in humans, and it was confirmed that the expression of PM-induced inflammatory factors in mice was decreased via BYG treatment. [ABSTRACT FROM AUTHOR]

Cigarette smoking is a major cause of lung cancer. Although tobacco smoking-induced genotoxicity has been well established, there is apparent lack of abundance functional epigenetic effects reported On cigarette smoke-induced lung carcinogenesis. The aim of this study was to determine effects of intratracheal administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) utilizing target gene expression DNA methylation patterns in lung tissues of mice following twice weekly for 8 weeks treatment. An unbiased approach where genomic regions was undertaken to assess early methylation changes within mouse pulmonary tissues. A methylated-CpG island recovery assay (MIRA) was performed to map the DNA methylome in lung tissues, with the position of methylated DNA determined using a Genome Analyzer (MIRA-SEQ). Alterations in epigenetic-regulated target genes were confirmed with quantitative reverse transcription-PCR, which revealed 35 differentially hypermethylated genes including Cdkn1C, Hsf4, Hnf1a, Cdx1, and Hoxa5 and 30 differentially hypomethylated genes including Ddx4, Piwi1, Mdm2, and Pce1 in NNK-exposed lung tissue compared with controls. The main pathway of these genes for mediating biological information was analyzed using the Kyoto Encyclopedia of Genes and Genomes database. Among them, Rssf1 and Mdm2 were closely associated with NNK-induced lung carcinogenesis. Taken together, our data provide valuable resources for detecting cigarette smoke-induced lung carcinogenesis. [ABSTRACT FROM AUTHOR]

Background. Herbal medicine is widely recommended to treat viral infectious diseases. Over 123,000,000 individuals have been infected with the coronavirus since a worldwide pandemic was declared in March 2020. We conducted this research to confirm the potential of herbal medicine as a treatment for coronavirus. Methods. We infected the A549 cell line with betacoronavirus OC43 and then treated it with 100 μg/mL Hyunggaeyungyo-tang (HGYGT) or distilled water with a control of HGYGT. We measured the mRNA expression levels of proinflammatory cytokines and interferon stimulated genes (ISGs) to confirm the effectiveness of HGYGT upon coronavirus infection. Results. We found that the effects of HYGYT decrease the expression level of pPKR, peIF2α, IFI6, IFI44, IFI44L, IFI27, IRF7, OASL, and ISG15 when administered to cells with coronavirus infection. The expressions of IL-1, TNF-α, COX-2, NF-κB, iNOS, and IKK mRNA were also significantly decreased in the HGYGT group than in the control group. Conclusion. Through the reduction of the amount of coronavirus RNA, our research indicates that HGYGT has antiviral effects. The reduction of IKK and iNOS mRNA levels indicate that HGYGT reduces coronavirus RNA expression and may inhibit the replication of coronavirus by acting on NF-kB/Rel pathways to protect oxidative injury. In addition, decreases in mRNA expression levels of proinflammatory cytokines indicate that the HGYGT may relieve the symptoms of coronavirus infections. [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]

Artemisia gmelinii Web. ex Stechm. (AG), a popular medicinal herb in Asia, has been used as a common food ingredient in Korea and is traditionally known for its anti-inflammatory properties. Therefore, in this study, we aimed to investigate whether AG relieves IBD, a classic chronic inflammatory disease of the gastrointestinal tract. We identified 35 chemical compounds in AG ethanol extract using ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry. In mice with DSS-induced IBD, AG administration attenuated the disease activity index and the serum and colonic levels of inflammatory cytokines and chemokines. AG treatment decreased nuclear factor- κ B (NF- κ B) signaling, a key mediator of inflammation, in the mouse colons. Additionally, AG extract enhanced immune responses in lymphoid tissues such as spleen and Peyer's patches. Thus, AG consumption potently ameliorated IBD symptoms and improved immune signaling in lymphoid tissues. [ABSTRACT FROM AUTHOR]

Background: Developing personalized strategies for cancer has shown good efficacies. Methods: We assessed the molecular targets programmed death ligand 1 (PD-L1), microsatellite instability (MSI), and PIK3CA. Seventy-four patients with liposarcomas who underwent curative resection were assessed for PD-L1 expression in the tumor and tumor-infiltrating lymphocytes (TILs), mismatch repair proteins (MLH1, PMS2, MSH2, and MSH6) by immunohistochemistry, MSI using polymerase chain reaction, and PIK3CA mutation/amplification using pyrosequencing and fluorescence in situ hybridization. Results: Seventeen (23%) cases were TIL+ (≥1 + expression) and associated with longer 5-year overall survival than those with TIL– tumors (84.4 vs. 60.8%, p = 0.007). Six (35.3%) PD-L1+ tumors were detected only in TIL+ cases, with none detected in tumor cells. Two well-differentiated liposarcomas showed MSI, one low and one high with concurrent loss of MLH1, MSH6, and PMS2. PIK3CA mutation was detected in 7 (9.5%) [exon 9 (n = 4) and exon 20 (n = 3)] and only 1 Q546K mutation was a PD-L1+ tumor. PIK3CA copy number gain was detected in 18 (24.4%) and was associated with TIL+ tumors (p = 0.045). Conclusions: Our comprehensive immuno-molecular panel suggests that liposarcoma should be categorized based on the molecular genomic subtype for precision medicine. [ABSTRACT FROM AUTHOR]

Chronic prostatitis typically occurs in aging men, and its symptoms include frequent and painful urination. In recent study, several studies have shown that Korean red ginseng (KRG) can be used in the prevention and treatment of various diseases. The objective of this study is to investigate whether KRG can play a role in repressing the development of chronic nonbacterial prostatitis (CNP) in male Wistar rats. To induce CNP, rats were castrated and beta-estradiol (0.25 mg/kg) was subcutaneously (s.c.) injected daily. 7-week-old male Wistar rats were divided into 5 groups (the normal group, CNP group, positive group, and KRG group (0.25g/kg) and another KRG (0.50g/kg) group. After 4 weeks, all rats were sacrificed and their prostate and serum were analyzed. Compared to the positive group, the KRG groups (0.25g/kg and 0.50g/kg) showed similar protective properties on CNP based on the histopathologic morphology of the prostate and the inflammation cytokines in the prostate tissue. Also, results of the immunohistochemistry staining showed that expression levels of vascular endothelial growth factor A (VEGFA), interleukin 6 (IL6), interleukin 1 beta (IL-1ß), tumor necrosis factor (TNF-alpha), and cytochrome c oxidase subunit II (COX2) were also decreased in KRG group (0.25g/kg) and KRG group (0.50g/kg). These results suggested that KRG inhibited the development of CNP and might a useful herbal treatment or functional food for CNP. [ABSTRACT FROM AUTHOR]

Recent studies have shown that overexpression of metastasis-associated in colon cancer 1 (MACC1) is significantly associated with adverse prognoses of patients with different kinds of cancer. However, the exact survival effect of MACC1 on epithelial ovarian cancer (EOC) patients has not yet been established. Thus, the objective of this study was to explore the prognostic role of MACC1 mRNA in EOC by using Kaplan-Meier (KM) plotter and ONCOMINE database. Our results indicated that MACC1 mRNA high expression was significantly associated with unfavorable overall survival (hazard ratio (HR) = 1.51 (95% confidence interval (CI): 1.21 – 1.88), P = 0.00025) and progression-free survival (HR = 1.53 (95% CI: 1.24 – 1.89), P = 5.8e-05) in EOC patients. We also found that the expression of MACC1 mRNA in EOC was 2.5 times higher than that in normal surface ovarian epithelium, which was statistically significant (P = 2.86e-7). Our results suggest that MACC1 expression might be a biomarker for poor prognosis in individual EOC patients. [ABSTRACT FROM AUTHOR]

Root skin color is one of the economically important traits in radish (Raphanus sativus), and the pigmentation in red skin varieties is largely attributable to anthocyanin accumulation. Pelargonidin was found as a major anthocyanin pigment accumulated in the sub-epidermal layer of red radish roots. In the 20 F2 population generated from the F1 with red root skins, root skins with red and white colors segregated in a 3:1 ratio. Additionally, a test cross between a red F3 individual and a white skin individual gave rise to 1:1 segregation of red and white, indicating that the root skin color of radish is determined by a single locus and red color is dominant over white. We performed association mapping for root skin color using SNPs obtained from RNA-seq analysis. Segregation analysis on the 152 F3 test-cross population revealed an RsMyb1 transcription factor as a candidate gene to determine root skin color. A PCR marker based on the polymorphism within 2 kb of RsMyb1 was developed and tested on 12 and 152 individuals from F2 and F3 test cross populations, respectively, and red and white root skin colors were completely distinguished corresponding to the genotypes. Expression levels of RsMyb1 in red or purple root cultivars were significantly higher than in white root cultivars. These findings suggest that RsMyb1 is a crucial determinant for anthocyanin biosynthesis in radish roots, and the molecular marker developed in this study will be useful for marker-assisted selection for red skin individuals at early seedling stages. [ABSTRACT FROM AUTHOR]

Objective: This study analyzed the associations between measured levels of aquaporin (AQP) mRNAs and clinical manifestations in patients with various types of otitis media (OM). Methods: AQP1, 2, 3, 4, 5, 6, 8, and 10 mRNA levels were assayed by real-time PCR from 57 patients with chronic otitis media (COM), 24 patients with cholesteatomatous otitis media (choleOM), and 82 patients with otitis media with effusion (OME). The relationships of these mRNA levels with the presence of bacteria, the type of hearing loss, and clinical manifestations of OM types were evaluated. Results: All eight AQP mRNAs were expressed in inflammatory tissue, chole matrix, and effusion fluid obtained from all 163 patients with OM. The levels in OME of AQP2, 4, 6, and 10 mRNA; and the levels in choleOM of AQP1, 3, 4, and 10 mRNA were elevated significantly compared to the corresponding levels in COM (p < .05). The expression level of AQP8 mRNA did not differ among OM types. Among samples positive for bacterial culture, AQP1 mRNA was significantly higher in OME than in COM and choleOM, AQP5 mRNA was significantly lower in OME than in COM and choleOM, and AQP10 mRNA was significantly higher in OME and choleOM than in COM (p < .05). Conclusions: The levels of expression of AQP mRNA are associated with the pathophysiology of OM. [ABSTRACT FROM AUTHOR]

Outcome of host-pathogen encounter is determined by the complex interplay between protective bacterial and host defense strategies. This complexity further amplifies with the existence of cell-to-cell phenotypic heterogeneity in pathogens which remains largely unexplored. In this study, we illustrated that heterogeneous expression of pneumolysin (Ply), a pore-forming toxin of the meningeal pathogen, S. pneumoniae (SPN) gives rise to stochastically different bacterial subpopulations with variable fate during passage across blood-brain barrier (BBB). We demonstrate that Ply mediated damage to pneumococcus containing vacuolar (PCV) membrane leads to recruitment of cytosolic “eat-me” signals, galectin-8 and ubiquitin, targeting SPN for autophagic clearance. However, a majority of high Ply producing subset extensively damages autophagosomes leading to pneumococcal escape into cytosol and efficient clearance by host ubiquitination machinery. Interestingly, a low Ply producing subset halts autophagosomal maturation and evades all intracellular defense mechanisms, promoting its prolonged survival and successful transcytosis across BBB, both in vitro and in vivo. Ply therefore acts as both, sword and shield implying that its smart regulation ensures optimal disease manifestation. Our elucidation of heterogeneity in Ply expression leading to disparate infection outcomes attempts to resolve the dubious role of Ply in pneumococcal pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Anterior cruciate ligament (ACL) reconstruction is the current standard of care for ACL tears. However, the results are not consistently successful; autografts or allografts have certain disadvantages; and synthetic grafts have had poor clinical results. Purpose: To determine if recellularization of decellularized tendons combined with mechanical stimulation in a bioreactor could replicate the mechanical properties of the native ACL and be successfully used for ACL reconstruction in vivo. Study Design: Controlled laboratory study. Methods: Porcine tibialis tendons were decellularized and then recellularized with human adult bone marrow–derived stem cells. Tendons were cultured in a tissue bioreactor that provided biaxial cyclic loading for up to 7 days. To reproduce mechanical stresses similar to those experienced by the ACL within the knee joint, the tendons were subjected to simultaneous tension and torsion in the bioreactor. Expression of tendon-specific genes and newly synthesized collagen and glycosaminoglycan were used to quantify the efficacy of recellularization and dynamic bioreactor culture. The ultimate tensile load to failure and stiffness of recellularized constructs were measured after dynamic stimulation. Finally, the tissue-engineered tendons were used to reconstruct the ACL in 24 pigs, and ultimate tensile load to failure and stiffness were assessed after 3 months. Results: Dynamic bioreactor culture significantly increased the expression of tendon-specific genes, the quantity of newly synthesized collagen and glycosaminoglycan, and the ultimate tensile load and stiffness of recellularized tendons. After in vivo reconstruction, the ultimate tensile load and stiffness of the tissue-engineered tendons increased significantly up to 3 months after surgery and were within 80% of the ultimate tensile load of the natural ACL. Conclusion: This translational study indicates that recellularization and dynamic mechanical stimuli can significantly enhance matrix synthesis and ultimate tensile load of decellularized porcine tibialis tendons. This approach to tissue engineering can be very useful for ACL reconstruction and may overcome some of the disadvantages of autografts and allografts. Clinical Relevance: Dynamic bioreactor cultivation of tissue-engineered tendons may overcome the limitations of autografts and allografts. [ABSTRACT FROM AUTHOR]

Abstract: Obesity poses a substantial threat of a worldwide epidemic and requires better understanding of adipose‐tissue biology as well as necessitates research into the etiology and therapeutic interventions. In this study, Medicarpin (Med), a natural pterocarpan, was selected (by screening) as a small‐molecule inducer of adipocyte differentiation among 854 candidates by using C3H10T1/2 mesenchymal stem cell; a cellular model of adipogenesis. Med induced the expression of brown‐adipocyte commitment marker Bmp7 as well as the early regulators of brown fat fate Pparγ, Prdm16, and Pgc‐1α during differentiation of C3H10T1/2 mesenchymal stem cells. Med also induced the expression of a key thermogenic marker—uncoupling protein 1 (UCP1)—along with expression of other brown‐fat–specific markers and beige‐fat–specific markers. Of note, Med significantly reduced the expression of white fat markers too. Furthermore, Med treatment promoted formation of multilocular lipid droplets (LDs), expression of mitochondrial‐biogenesis–related genes, and increased oxygen consumption. Gene silencing study revealed that Med promotes the development of brown‐ and beige‐adipocyte characteristics in C3H10T1/2 mesenchymal stem cells through activation of the AMPK pathway, and our data allow us to propose Med as a candidate for therapeutics against obesity or related metabolic disorders. © 2017 BioFactors, 44(2):168–179, 2018 [ABSTRACT FROM AUTHOR]

Menopause is strongly associated with an increased risk of metabolic dysfunctions due to the decline in estrogen. Here, we hypothesized that dietary HT051, containing the roots of Pueraria lobata and Rehmannia glutinosa, has beneficial effects on ovariectomized (OVX) rats by regulating lipid metabolism. Forty-eight female Sprague-Dawley rats were randomly divided into 4 groups: sham-operated (Sham), OVX, OVX with low-dose HT051 supplementation, and OVX with high-dose HT051 supplementation. The rats were fed with a modified AIN-93G diet or an HT051-containing modified AIN-93G diet for 8 weeks. Body weight, fat mass, and serum levels of total cholesterol, triglyceride, glucose, alanine transaminase, and aspartate transaminase decreased in HT051-fed OVX rats. Dietary HT051 supplementation significantly decreased the mRNA expression of lipogenesis-related genes, including sterol regulatory element-binding protein 1c and fatty acid synthase, and increased the mRNA expression of β-oxidation-related genes, including peroxisome proliferator-activated receptor and carnitine palmitoyl transferase 1 in the liver of OVX rats. Moreover, the expression of genes involved in adipogenesis and inflammation was significantly lower in the adipose tissue of OVX rats fed with HT051 than in the OVX group. These findings suggest that HT051 may be a potential natural alternative for the management of postmenopausal metabolic dysfunctions. [ABSTRACT FROM AUTHOR]

Korean diesel particulate matter 20 (KDP20) is a pollutant comprising a complex mixture of carbon and chemical irritants. Although particulate matter and nasal inflammation are strongly associated, the underlying molecular mechanism based on systematic transcriptome analysis remains unknown. In this study, genome-wide gene expression profiles of mouse nasal tissues were determined following exposure to KDP20 for 5 and 10 days and compared with those of the control (n = 4/group). We identified 758 significant differentially expressed genes (DEGs) and classified them as 5-day-specific, 10-day-specific, and common among groups based on their expression patterns. The terms "regulation of alpha-beta T cell differentiation," "macrophage differentiation," and "cell adhesion mediated by integrin" were significantly enriched in each group. Receiver operating characteristic analysis revealed six genes as potential predictive biomarkers. The differential expression of these six genes was validated using quantitative RT-PCR (n = 3/group). Furthermore, a possible mechanism for nasal inflammation was suggested through the binding analysis between metal ions and genes. The genes identified in this study may play important roles in regulating the mechanism of nasal inflammation induced by diesel particles, especially immune cell regulation, and may function as markers for diesel particle-induced nasal inflammation. [Display omitted] • KDP20 exposure for 5–10 days causes nasal inflammation in mice. • DEGs between groups are associated with immune cell differentiation & inflammation. • Six genes were identified as biomarkers of KDP20-induced nasal inflammation. [ABSTRACT FROM AUTHOR]

Central nervous system (CNS) infection continues to be an important cause of mortality and morbidity, necessitating new approaches for investigating its pathogenesis, prevention and therapy. Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, which develops following penetration of the blood–brain barrier (BBB). By chemical library screening, we identified epidermal growth factor receptor (EGFR) as a contributor to E. coli invasion of the BBB in vitro. Here, we obtained the direct evidence that CNS-infecting E. coli exploited sphingosine 1-phosphate (S1P) for EGFR activation in penetration of the BBB in vitro and in vivo. We found that S1P was upstream of EGFR and participated in EGFR activation through S1P receptor as well as through S1P-mediated up-regulation of EGFR-related ligand HB-EGF, and blockade of S1P function through targeting sphingosine kinase and S1P receptor inhibited EGFR activation, and also E. coli invasion of the BBB. We further found that both S1P and EGFR activations occurred in response to the same E. coli proteins (OmpA, FimH, NlpI), and that S1P and EGFR promoted E. coli invasion of the BBB by activating the downstream c-Src. These findings indicate that S1P and EGFR represent the novel host targets for meningitic E. coli penetration of the BBB, and counteracting such targets provide a novel approach for controlling E. coli meningitis in the era of increasing resistance to conventional antibiotics. [ABSTRACT FROM AUTHOR]

Cohesin is a highly-conserved protein complex that plays important roles in sister chromatid cohesion, chromatin structure, gene expression, and DNA repair. In humans, cohesin is a ubiquitously expressed, multi-subunit protein complex composed of core subunits SMC1A, SMC3, RAD21, STAG1/2 and regulatory subunits WAPL, PDS5A/B, CDCA5, NIPBL, and MAU2. Recent studies have demonstrated that genes encoding cohesin subunits are somatically mutated in a wide range of human cancers. STAG2 is the most commonly mutated subunit, and in a recent analysis was identified as one of only 12 genes that are significantly mutated in four or more cancer types. In this review we summarize the findings reported to date and comment on potential functional implications of cohesin mutation in the pathogenesis of human cancer. [ABSTRACT FROM AUTHOR]

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells. [ABSTRACT FROM AUTHOR]

The medicinal plants Artemisia iwayomogi (A. iwayomogi) and Curcuma longa (C. longa) radix have been used to treat metabolic abnormalities in traditional Korean medicine and traditional Chinese medicine (TKM and TCM). In this study we evaluated the effect of the water extract of a mixture of A. iwayomogi and C. longa (ACE) on high-fat diet-induced metabolic syndrome in a mouse model. Four groups of C57BL/6N male mice (except for the naive group) were fed a high-fat diet freely for 10 weeks. Among these, three groups (except the control group) were administered a high-fat diet supplemented with ACE (100 or 200 mg/kg) or curcumin (50 mg/kg). Body weight, accumulation of adipose tissues in abdomen and size of adipocytes, serum lipid profiles, hepatic steatosis, and oxidative stress markers were analyzed. ACE significantly reduced the body and peritoneal adipose tissue weights, serum lipid profiles (total cholesterol and triglycerides), glucose levels, hepatic lipid accumulation, and oxidative stress markers. ACE normalized lipid synthesis-associated gene expressions (peroxisome proliferator-activated receptor gamma, PPARγ; fatty acid synthase, FAS; sterol regulatory element-binding transcription factor-1c, SREBP-1c; and peroxisome proliferator-activated receptor alpha, PPARα). The results from this study suggest that ACE has the pharmaceutical potential reducing the metabolic abnormalities in an animal model. [ABSTRACT FROM AUTHOR]

Histone deacetylases (HDACs) play a pivotal role in eukaryotic gene expression by modulating the levels of acetylation of chromatin and related transcription factors. In contrast to class I HDACs (HDAC1, -2, -3 and -8), the class IIa HDACs (HDAC4, -5, -7 and -9) harbor cryptic deacetylases activity and recruit the SMRT-HDAC3 complex to repress target genes in vivo. In this regard, the specific interaction between the HDAC domain of class IIa HDACs and the C-terminal region of SMRT repression domain 3 (SRD3c) is known to be critical, but the molecular basis of this interaction has not yet been addressed. Here, we used an extensive mutant screening system, named the “partitioned one- plus two-hybrid system”, to isolate SRD3c interaction-defective (SRID) mutants over the entire catalytic domains of HDAC4 (HDAC4c) and -5. The surface presentation of the SRID mutations on the HDAC4c structure revealed that most of the mutations were mapped to the rim surface of the catalytic entry site, strongly suggesting this mutational hot-spot region as the major binding surface of SRD3c. Notably, among the HDAC4c surface residues required for SRD3c binding, some residues (C667, C669, C751, D759, T760 and F871) are present only in class IIa HDACs, providing the molecular basis for the specific interactions between SRD3c and class IIa enzymes. To investigate the functional consequence of SRID mutation, the in vitro HDAC activities of HDAC4 mutants immuno-purified from HEK293 cells were measured. The levels of HDAC activity of the HDAC4c mutants were substantially decreased compared to wild-type. Consistent with this, SRID mutations of HDAC4c prevented the association of HDAC4c with the SMRT-HDAC3 complex in vivo. Our findings may provide structural insight into the binding interface of HDAC4 and -5 with SRD3c, as a novel target to design modulators specific to these enzymes. [ABSTRACT FROM AUTHOR]

Previously, AIMP3 (aminoacyl-tRNAsynthetase-interacting multifunctional protein-3) was shown to be involved in the macromolecular tRNA synthetase complex or to act as a tumor suppressor. In this study, we report a novel role of AIMP3/p18 in the cellular aging of human mesenchymal stem cells (hMSCs). We found that AIMP3/p18 expression significantly increased in senescent hMSCs and in aged mouse bone marrow-derived MSCs (mBM-MSCs). AIMP3/p18 overexpression is sufficient to induce the cellular senescence phenotypes with compromised clonogenicity and adipogenic differentiation potential. To identify the upstream regulators of AIMP3/p18 during senescence, we screened for potential epigenetic regulators and for miRNAs. We found that the levels of miR-543 and miR-590-3p significantly decreased under senescence-inducing conditions, whereas the AIMP3/p18 protein levels increased. We demonstrate for the first time that miR-543 and miR-590-3p are able to decrease AIMP3/p18 expression levels through direct binding to the AIMP/p18 transcripts, which further compromised the induction of the senescence phenotype. Taken together, our data demonstrate that AIMP3/p18 regulates cellular aging in hMSCs possibly through miR-543 and miR-590-3p. [ABSTRACT FROM AUTHOR]

Human papillomavirus ( HPV) infection plays a significant role in the development and progression of head and neck squamous cell carcinoma ( HNSCC). Expression of mi R-21 has a prognostic role in a wide variety of cancers. The upregulation of mi R-21 suppresses a number of target genes, including phosphatase tensin homologue ( PTEN) and programmed cell death 4 ( PDCD4). We investigated the association between the expression of mi R-21 and the clinical features of HNSCC using stratified analysis based on HPV infection status. HPV status and mi R-21 expression in HNSCC tissues from 167 patients were evaluated using in situ hybridization. The expression of PDCD4 and PTEN was examined by immunohistochemistry. The up-regulation of stromal mi R-21 expression occurred in 40.6% of HPV-negative samples and 28.3% of the HPV-positive group. In HPV-stratified multivariate analysis, high mi R-21 expression was associated with poor cancer-specific survival in HPV-negative tumors, but not in HPV-positive tumors. There was a significant association between mi R-21 and cytoplasmic PDCD4 overexpression in HPV-negative HNSCCs. We suggest that stromal mi R-21 expression is an independent prognostic factor in HPV-negative tumors and mi R-21 may play different roles depending on HPV infection status. [ABSTRACT FROM AUTHOR]

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing. [ABSTRACT FROM AUTHOR]

Human immunoglobulin heavy chain variable domains (VH) are promising scaffolds for antigen binding. However, VH is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the VH domain, we performed in vivo protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of E. coli. After screening a human germ-line VH library, 95% of the VH proteins obtained were identified as VH3 family members; one VH protein, MG2x1, stood out among separate clones expressing individual VH variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in VH. Comparison of the crystal structures of the VH variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the VH interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered VH acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the VH variant as a scaffold was qualified as most of VH clones selected randomly were expressed as soluble form in E. coli regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected VH conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are multicompetent lymphocytes of the innate immune system that play a central role in host defense and immune regulation. Although increasing evidence suggests that innate immunity plays a key role in the pathogenesis of chronic rhinosinusitis (CRS), the role of NK cells in CRS has been poorly studied. This study aimed to characterize the peripheral blood NK cells from patients with CRS, and to compare the functions of these cells with those from non-CRS controls. The correlation between NK cell functional activity and prognosis was also assessed. Eighteen CRS patients and 19 healthy non-CRS controls were included. The patients with CRS were classified into two subgroups, namely a treatment-responsive group and recalcitrant group. NK cell degranulation was determined by measuring the cell surface expression of CD107a against 721.221 and K562 cells. Intracytoplasmic cytokine production was determined by flow cytometry. Compared to the controls, the NK cells of CRS group had an impaired ability to degranulate and to produce cytokines such as IFN-γ and TNF-α. The recalcitrant subgroup showed the most severe defects in NK cell effector functions. Moreover, the decreased NK cell functions in patients with CRS were associated with poor prognostic factors such as concomitant asthma and peripheral blood eosinophilia. NK cells, which were originally named for their ability to mediate spontaneous cytotoxicity towards diseased cells including infected cells, may play an important role in regulating the inflammatory process in CRS pathogenesis. [ABSTRACT FROM AUTHOR]

Genomic analyses have revealed the enormous heterogeneity in essentially all cancer types. However, the identification of precise subtypes, which are biologically informative and clinically useful, remains a challenge. The application of integrative analysis of multilayered genomic profiles to define the chromosomal regions of genomic copy number alterations with concomitant transcriptional deregulation is posited to provide a promising strategy to identify driver targets. In this study, we performed an integrative analysis of the DNA copy numbers and gene expression profiles of hepatocellular carcinoma (HCC). By comparing DNA copy numbers between HCC subtypes based on gene expression pattern, we revealed the DNA copy number alteration with concordant gene expression changes at 6p21–p24 particularly in the HCC subtype of aggressive phenotype without expressing stemness genes. Among the genes at 6p21–p24, we identified IER3 as a potential driver. The clinical utility of IER3 copy numbers was demonstrated by validating its clinical correlation with independent cohorts. In addition, short hairpin RNA–mediated knock-down experiment revealed the functional relevance of IER3 in liver cancer progression. In conclusion, our results suggest that genomic copy number alterations with transcriptional deregulation at 6p21–p24 identify an aggressive HCC phenotype and a novel functional biomarker. [ABSTRACT FROM AUTHOR]

Activated B-cell-like subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic active B-cell receptor signaling and a constitutive activation of the nuclear factor κB pathway. As a driving force of nuclear factor κB overactivity, myeloid differentiation primary response gene 88 (MYD88) L265P mutation occurs in activated B-cell-like DLBCL. However, the MYD88 L265P mutation has not yet been studied in conjunction with MYD88 protein expression in DLBCL cases. Thus, we investigated MYD88 expression in 124 DLBCL specimens by immunohistochemistry. In addition, the MYD88 L265P mutation was examined by polymerase chain reaction and direct DNA sequencing. MYD88 was overexpressed in 38.7% (48/124) of DLBCL cases. MYD88 was expressed in the cytoplasm of lymphoma cells. MYD88 overexpression was associated with older age (P = .016) and tumor recurrence (P = .007). In univariate analysis, MYD88 overexpression was correlated with shortened disease-free survival (DFS) of DLBCL (P = .013) and non-germinal center B-cell-like DLBCL (P = .034). In multivariate analysis, MYD88 overexpression was an independent factor for reduced DFS (P = .025). MYD88 L265P mutation was found in 6.5% (8/124) of DLBCL cases. All but 1 case were of non-germinal center B-cell-like subtype. The MYD88 L265P mutation was not associated with MYD88 expression (Spearman ρ = .0.074) and clinicopathologic parameters of DLBCL. The data indicate that high MYD88 expression is significantly associated with tumor recurrence and shortened DFS in patients with DLBCL. In addition, the incidence of MYD88 L265P mutation in Korea is much lower than previously reported. Thus, MYD88 may be crucial for lymphoma progression, independent of MYD88 L265P mutation. [ABSTRACT FROM AUTHOR]

The specific properties of silver nanoparticles (AgNPs), such as antimicrobial activity and electrical conductivity, allow them to be used in many fields. However, their expanding application is also raising health, environmental and safety concerns. Previous in vivo AgNP toxicity studies have indicated a gender-different accumulation of silver in the kidneys, with 2-3 times more silver in female kidneys compared to male kidneys. However, no other studies have further addressed this gender difference. Accordingly, the current study investigated the gender-dependent effect of AgNPs on the kidney gene level based on toxicogenomic studies of kidneys obtained from rats exposed to AgNPs via inhalation for 12 weeks. When compared with the fresh air control, the silver nanoparticle-exposed kidneys included 104 genes with a more than 1.3-fold expression increase. For the male rat kidneys exposed to a low or high dose of silver nanoparticles, 96 genes exhibited expression changes, where six genes changed with both the low and high dose; four increased and two decreased. Meanwhile, for the female rat kidneys exposed to a low or high dose of silver nanoparticles, 66 genes exhibited expression changes, where 11 genes changed with both the low and high dose; nine increased and two decreased. Gender-dependent gene expression changes of more than 2-fold were linked to 163 genes, with 79 genes in the male kidneys and 84 genes in the female kidneys, plus gender-dependent gene expression changes of more than 5-fold were linked to 21 genes. However, no genes involved in apoptosis or the cell cycle were activated by the 12-week silver nanoparticle inhalation exposure. Overall, the male rat kidneys showed a higher expression of genes involved in xenobiotic metabolism, while the female rat kidneys showed a higher expression of genes involved in extracellular signaling. [ABSTRACT FROM AUTHOR]

Background: The effectiveness of molecular targeted agents is modest in hepatocellular carcinoma (HCC). Efficacy of molecular targeted therapies has been better in cancer patients with high expression of actionable molecules defined as cognate target molecules. However, patient stratification based on the actionable molecules dictating the effectiveness of targeted drugs has remained understudied in HCC. Experimental Design & Results: Paired tumor and non-tumoral tissues derived from a total of 130 HCC patients were studied. Real-time RT-PCR was used to analyze the mRNA expression of actionable molecules in the tissues. mRNA levels of EGFR, VEGFR2, PDGFRβ, FGFR1, and mTOR were up-regulated in tumors compared to non-tumors in 35.4, 42.3, 61.5, 24.6, and 50.0% of patients, respectively. Up-regulation of EGFR was observed at early stage and tended to gradually decrease toward late stages (BCLC stage A: 41.9%; B: 30.8%; C: 17.6%). Frequency of VEGFR2 expression in tumors at stage C was lower than that in the other stages (BCLC stage A: 45.9%; B: 41.0%; C: 29.4%). PDGFRβ and mTOR were observed to be up-regulated in more than 50% of tumors in all the stages whereas FGFR1 was up-regulated in only about 20% of HCC irrespective of stages. A cluster analysis of actionable gene expression revealed that HCC can be categorized into different subtypes that predict the effectiveness of molecular targeted agents and combination therapies in clinical trials. Analysis of in vitro sensitivity to sorafenib demonstrated that HCC cells with up-regulation of PDGFRβ and c-Raf mRNA are more susceptible to sorafenib treatment in a dose and time-dependent manner than cells with low expression of the genes. Conclusions: mRNA expression analysis of actionable molecules could provide the rationale for new companion diagnostics-based therapeutic strategies in the treatment of HCC. [ABSTRACT FROM AUTHOR]

Abstract: Phenotypic and genotypic changes in Chlamydomonas reinhardtii BafJ5, a starchless mutant, with respect to lipid metabolism was studied in different trophic states under nitrogen (N) sufficient and limited conditions. Interestingly, cellular lipid content increased linearly with input acetate concentration with highest lipid content (∼42%) under nitrogen limitation and mixotrophic state. RT-qPCR studies indicate that key fatty acid biosynthesis genes are down-regulated under N limitation but not under mixotrophic state, whereas, ACS2, encoding Acetyl-CoA synthetase, and DGTT4, encoding Diacylglycerol O-acyltransferase, are up-regulated under all conditions. These results collectively indicate that acetate is the limiting factor and central molecule in lipid droplet synthesis. The study also provides further evidence of the presence of a chloroplast pathway for triacylglycerol synthesis in microalgae. [Copyright &y& Elsevier]

Summary: It has been reported that HER2 expression is different in gastric and breast cancers, and a gastric cancer scoring system (GCSS) has recently been suggested. We investigated HER2 protein expression using GCSS and a breast cancer scoring system (BCSS) and correlated it with HER2 gene amplification. HER2 status was evaluated in 1091 cases by analyzing tissue microarrays constructed using 2 different cores from each case. Polyclonal (HercepTest) and monoclonal (Pathway) antibodies were used for immunohistochemistry (IHC), and results were scored by BCSS and GCSS. Gene amplification was evaluated by automated dual-color silver-enhanced in situ hybridization (SISH) in all cases and correlated with the results from fluorescence in situ hybridization (FISH) in 590 cases. The concordance between the IHC results using polyclonal and monoclonal antibodies was high (κ = 0.785). The results of dual-color SISH and FISH showed very high concordance as well (κ = 0.918). GCSS was significantly more sensitive for detecting SISH positivity than was BCSS in both antibodies (polyclonal, P = .003; monoclonal, P < .001), but specificity was higher in BCSS than GCSS (polyclonal, P = .004; monoclonal, P < .001). It has been recently shown that HER2-overexpressing patients with unresectable gastric cancer benefited from trastuzumab therapy. Because IHC is recommended before gene amplification studies in HER2 testing, GCSS should be used for evaluating HER2 expression in gastric cancers. [ABSTRACT FROM AUTHOR]

Summary This study investigated the effect of local glucagon-like peptide-1 (GLP-1) production within mouse islets on cytoprotection in vitro and in vivo by gene transfer of GLP-1. Transduction of recombinant adenovirus vector expressing GLP-1 (rAd-GLP-1) induced a significant increase in bioactive GLP-1 in the mouse islet culture, whereas transduction with adenovirus vector expressing β-galactosidase (rAd-LacZ), as a control, had no effect on GLP-1 secretion. Islets transduced with rAd-GLP-1 were protected from H2O2-induced cell damage in vitro. In addition, glucose-stimulated insulin secretion was significantly increased in rAd-GLP-1-transduced islets. When transplanted under the kidney capsule of diabetic syngeneic mice, islet grafts retrieved 4 or 7 days after transplantation revealed that the rAd-GLP-1-transduced group had significantly more Ki67-positive cells as compared with the rAd-LacZ-transduced group. Regarding blood glucose control, diabetic mice transplanted with a marginal mass of rAd-GLP-1-transduced islets became normoglycemic more rapidly and 78% of the recipients were normoglycemic at 35 days post-transplant, whereas only 48% of the mice transplanted with rAd-LacZ-transduced islets were normoglycemic ( P < 0.05). In conclusion, delivery of the GLP-1 gene to islets enhanced islet cell survival during the early post-transplant period, and preserved islet mass and functions over time in the transplants. [ABSTRACT FROM AUTHOR]

Abstract: Paraquat (PQ) is the third most extensively used herbicide in the world, causing thousands of deaths due to accidental or intentional self-poisoning in developing countries. Although many therapeutic treatments for PQ-induced poisonings have been proposed and developed, the efficacy of these treatments is still poor and requires further investigation. Methylprednisolone (trade name Solumedrol, hereinafter MP) is a widely used steroid for the treatment of various diseases but the function of MP has not yet been studied in the context of PQ-induced intoxication. The aim of this study was to determine if MP can ameliorate PQ-induced toxicity in an alveolar A549 cell line by inducing ATP-dependent transporter P-glycoprotein (P-gp) expression. P-gp expression and activity in the PQ-treated A549 cell line were enhanced by MP treatment and cytotoxicity by PQ was dramatically decreased. We also found that MP per se or together with PQ induced P-gp expression by both Western blot and qRT-PCR analyses. In addition, induced P-gp transporter was shown to improve the efflux effect on PQ-treated A549 cell lines as was demonstrated using the Calcein-AM fluorescence accumulation assay. In summary, MP induces the transmembrane ATP-dependent transporter P-gp expression, which greatly improves PQ-treated A549 cell viability, reduces accumulation of intracellular PQ and prevents PQ induced cytotoxicity but it should be further evaluated in in vivo studies. [Copyright &y& Elsevier]

Abstract: Cytotoxic necrotizing factor 1 (CNF1), the paradigm of Rho GTPase activating bacterial toxins has been shown to promote E. coli invasion of human brain microvascular endothelial cells (HBMEC), which constitute the blood–brain barrier, but its synthesis and secretion is unclear. In this study, we performed mini Tn5 mutagenesis screen to identify genetic requirements for CNF1 production and secretion. Transposon mutagenesis screen of meningitis-causing E. coli K1 strain RS218 revealed that CNF1 production was markedly decreased in a transposon mutant (NBC-28G9) where transposon insertion occurred in the 5′ end of gidA gene. In contrast, total deletion of gidA gene has less drastic effect on the production of CNF1. The N-terminus truncated GidA exhibited dominant negative effect on the production of CNF1. The inhibition of CNF1 production by N-terminus truncated GidA was shown to occur at the translational level. This was supported by our demonstrations that cnf1 mRNA transcription levels did not differ between strains RS218 and NBC-28G9; and the production of recombinant CNF1 under the control of artificial promoter was also repressed by truncated GidA. Progressive deletion of DNA regions in cnf1 gene identified two putative regions that were responsible for translational inhibition mediated by truncated GidA. [Copyright &y& Elsevier]

Mitochondrial adenine nucleotide translocator (ANT) plays important roles in the regulation of mitochondrial permeability transition and cell bioenergetics. The mouse has three ANT isoforms (1, 2 and 4) showing tissue-specific expression patterns. Although ANT1 is known to have a pro-apoptotic property, the specific functions of ANT2 have not been well determined. In the present study, ANT2 expression was significantly lower in the aged rat liver and in a liver fibrosis model. To explore the protective role of ANT2 in the liver, we established a hepa1c1c7 cell line overexpressing ANT2. Overexpression of ANT2 caused hepa1c1c7 cells to be more resistant to oxidative stress, and mitochondrial membrane potential (MMP, ∆Ψm) was relatively intact in ANT2-overexpressing cells under oxidative stress. In addition, ANT2 was found to increase ATP production by influencing mitochondrial bioenergetics. These results imply that the hepatoprotective effect of ANT2 is due to the stabilization of MMP and enhanced ATP production, and thus, maintaining ANT2 levels in the liver might be important to enhance resistance to aging and oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play pivotal role in antiviral innate immunity of a host. The present in-vivo experiment was conducted to investigate the role of these innate immune factors in early phase as well as during recovery of viral haemorrhagic septicaemia virus (VHSV) infection by quantitative real-time reverse transcriptase polymerase chain reaction. A less lethal VHSV infection was generated in olive flounder (Paralichthys olivaceus) and was sampled at 3, 6, and 12h post infection (hpi), and 1, 2, 4, and 7 days post infection (dpi). At 3 hpi, the VHSV N gene was detected in three out of five fish and all five fish showed a relative fold increase of TLR 2, TLR 7, interleukin 8 (IL 8), IFN regulatory factor 3 (IRF 3), IRF 7, and ISG 15. Viral copies rapidly increased at 12 hpi then remained high until 2 dpi. When viral copy numbers were high, a higher expression of immune genes IL 1β, IRF 3, IRF 7, Type I IFN, ISG 15 and Mx was observed. Viral copies were drastically reduced in 4 and 7 dpi fish, and also the immune response was considerably reduced but remained elevated, except for ISG 15 which found equal to control in 7 dpi fish. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. A fish with ascites sampled at 7 dpi displayed high viral copy but under-expressed immune genes except for Mx. When viral copies were high at 1 and 2 dpi, both TLR 2 and TLR 7 were down-regulated, perhaps indicating immune suppression by the virus. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against VHSV. [Copyright &y& Elsevier]

Paecilomyces tenuipes is a popular medicinal mushroom, and has received extensive attention for medical application because of its various physiological activities. However, there is limited information about the anticancer and immunomodulatory activities of Paecilomyces tenuipes . This study attempted to evaluate the effect of an extract from the cultured fruiting bodies of Paecilomyces tenuipes (PTE) on the expression of the interleukin-18 (IL-18) gene in rat pheochromocytoma (PC) 12 cells and rat brain. Related mRNA levels were determined by reverse transcription-polymerase chain reaction (RT-PCR). Protein levels were measured by Western blot and immunohistochemistry. Our results demonstrated that PTE induced IL-18 gene expression both in vitro and in vivo. Treatment of PC12 cells and rat brain cells with 10 μg/ml and 20 mg/kg PTE, respectively, yielded significant increases of IL-18 levels. Significantly, IL-18-immunoreactive neurons were detected in the Purkinje cells of the cerebellum. IL18-immunohistochemical staining was markedly enhanced in animals treated with PTE compared to findings in the untreated controls. These results suggest that PTE could be a potential candidate as an immune activator or anticancer drug. [ABSTRACT FROM AUTHOR]

Myelocytomatosis oncogene (c-MYC) is a well-known nuclear oncoprotein having multiple functions in cell proliferation, apoptosis and cellular transformation. Chromosomal modification is also important to the differentiation and growth of stem cells. Histone deacethylase (HDAC) and polycomb group (PcG) family genes are well-known chromosomal modification genes. The aim of this study was to elucidate the role of c-MYC in the expression of chromosomal modification via the HDAC family genes in human mesenchymal stem cells (hMSCs). To achieve this goal, c-MYC expression was modified by gene knockdown and overexpression via lentivirus vector. Using the modified c-MYC expression, our study was focused on cell proliferation, differentiation and cell cycle. Furthermore, the relationship of c-MYC with HDAC2 and PcG genes was also examined. The cell proliferation and differentiation were checked and shown to be dramatically decreased in c-MYC knocked-down human umbilical cord blood-derived MSCs, whereas they were increased in c-MYC overexpressing cells. Similarly, RT-PCR and Western blotting results revealed that HDAC2 expression was decreased in c-MYC knocked-down and increased in c-MYC overexpressing hMSCs. Database indicates presence of c-MYC binding motif in HDAC2 promoter region, which was confirmed by chromatin immunoprecipitation assay. The influence of c-MYC and HDAC2 on PcG expression was confirmed. This might indicate the regulatory role of c-MYC over HDAC2 and PcG genes. c-MYCs' regulatory role over HDAC2 was also confirmed in human adipose tissue-derived MSCs and bone-marrow derived MSCs. From this finding, it can be concluded that c-MYC plays a vital role in cell proliferation and differentiation via chromosomal modification. [ABSTRACT FROM AUTHOR]

Abstract: Cytotoxic necrotizing factor 1 (CNF1) plays an important role in meningitis-causing Escherichia coli. Mini-Tn5 mutagenesis of meningitis-causing E. coli revealed that transposon mutants of selA and selB genes failed to express CNF1. We subsequently showed that SelB and selenocysteine, however, are not essential for the expression of CNF1, but the deletion of 47 amino acids of SelB at its C terminus has a dominant negative effect on CNF1 expression at the translational level. Bioinformatic analysis of the mRNA of cnf1 predicted two putative selenocysteine incorporation sequence (SECIS) elements, but we failed to detect any selenocysteine in CNF1 protein. These findings suggest that SelB is involved in translational regulation of CNF1 expression but without incorporation of selenocysteine in CNF1 protein. [Copyright &y& Elsevier]

Abstract: Early growth response-1 (Egr-1), a zinc finger transcription factor, plays a key regulatory role in pathological cardiovascular processes including atherosclerosis. Here, we investigate whether Egr-1 expression and foam cell formation require toll-like receptor 9 (TLR9) and myeloid differentiation factor 88 (MyD88). CpG DNA and its related synthetic CpG oligodeoxynucleotides (CpG ODN) play an important role in immunity and inflammation. CpG ODN increased expression of Egr-1 and formation of foam cells in Raw264.7 cells or bone marrow-derived macrophages. Egr-1 siRNA decreased foam cell formation by CpG ODN compared to that of control siRNA. In addition, when TLR9 or MyD88 was knocked down, CpG ODN-induced Egr-1 expression was also attenuated. CpG ODN increased ERK1/2 phosphorylation. U0126, a MEK pathway inhibitor, suppressed activation of Egr-1 expression by CpG ODN. CpG ODN-induced expression of tissue factor (TF) and NGFI-A binding protein 2 (Nab2), and the expression of both genes is blocked by knockdown of TLR9 or MyD88 siRNA or MEK inhibition. These results suggest that CpG ODN activates the TLR9–MyD88–ERK1/2 pathway causing expression of Egr-1 and its target genes such as TF and Nab2, thus inducing foam cell formation. [Copyright &y& Elsevier]

Abstract: To explore cellular changes in sensory neurons after nerve injury and to identify potential target genes contributing to different stages of neuropathic pain development, we used Affymetrix oligo arrays to profile gene expression patterns in L5/6 dorsal root ganglia (DRG) from the neuropathic pain model of left L5/6 spinal nerve ligation at different stages of neuropathic pain development. Our data indicated that nerve injury induced changes in expression of genes with similar biological functions in a temporal specific manner that correlates with particular stages of neuropathic pain development, indicating dynamic neuroplasticity in the DRG in response to peripheral nerve injury and during neuropathic pain development. Data from post-array validation indicated that there was a temporal correlation between injury-induced expression of the glial fibrillary acidic protein (GFAP), a marker for activated astrocytes, and neuropathic pain development. Spinal nerve ligation injury in GFAP knockout mice resulted in neuropathic pain states with similar onset, but a shortened duration compared with that in age, and gender-matched wild-type littermates. Intrathecal GFAP antisense oligonucleotide treatment in injured rats with neuropathic pain states reversed injury-induced behavioral hypersensitivity and GFAP upregulation in DRG and spinal cord. Together, these findings indicate that injury-induced GFAP upregulation not only serves as a marker for astrocyte activation, but it may also play a critical, but yet identified, role in the maintenance of neuropathic pain states. [Copyright &y& Elsevier]

The skin sensitizer 2,4-dinitrofluorobenzene (DNFB) provokes delayed hypersensitivity responses as a result of topical application to the skin. Here, we demonstrate that DNFB modifies proteins in RAW 264.7 cells and skin tissues in NC/Nga mice; we also show the functional involvement of DNFB-induced modification of cellular proteins in the DNFB-induced macrophage inflammatory protein (MIP)-2 gene expression in RAW 264.7 cells. In addition, we demonstrate that DNFB strongly induces reactive oxygen species (ROS) production. Our RT-PCR analysis and reporter gene assays reveal that the DNFB-induced intracellular ROS production is necessary for MIP-2 gene expression by DNFB. We observed that the vitamin C and chemical oxidant scavenger N-acetyl-cysteine have an inhibitory effect on the generation of ROS, the activation of MAP kinase pathways, and the MIP-2 gene expression in DNFB-treated RAW 264.7 cells. These results provide insight into the mechanisms involved in DNFB-induced contact hypersensitivity. [ABSTRACT FROM AUTHOR]

Abstract: This study presents a novel approach to detect biological associations for gene pairs with cell cycle-specific expression profiles. Previous studies have shown that periodic transcription is commonly regulated by transcription factors that are also periodically transcribed, and there is a growing number of examples where cell cycle regulated genes are conserved in yeast and mammalian cells. Some genes have periodicity for their oscillatory activity throughout cell division. These cell cycle-specific oscillatory activities could be explained by a biological phenomenon in terms of efficiency and logical order. In the yeast data used in this study, about 13% of genes behave in this manner based on a previous yeast study. Microarrays have been applied to determine genome-wide expression patterns during the cell cycle of a number of different cells. Moreover, several previous studies have shown that many pairs of genes, which have linearly correlated expression profiles, have similar cellular roles or physical interactions. Based on this point of view, the traditional clustering methods have focused on similar expression profiles based on the premise that genes with similar expression profiles have similar biological functions or relevant biological interactions. However, there are a number of previous studies indicating that the expression of some genes may be delayed compared to others due to a time lag in their transcriptional control. Therefore, we propose a novel clustering method, named as phase-synchronization clustering, based on the theory of phase synchronization for detecting biological associations using cell cycle-specific expression profiles. We evaluate phase-synchronization clustering here using Saccharomyces cerevisiae microarray data. Phase-synchronization clustering is able to detect biologically associated gene pairs that have linearly correlated (simultaneous and inverted) as well as time-delayed expression profiles. The performance of phase-synchronization clustering is compared with other conventional clustering methods. The likelihood of finding relevant biological associations by phase-synchronization clustering is significantly higher than other clustering methods. Therefore, phase-synchronization clustering is more efficient for detecting known biological interactions for gene pairs than other conventional clustering methods for analyzing cell cycle-specific expression data. The evaluation analysis of the results by phase-synchronization clustering also suggests that the cellular activities during the cell division process could be understood as a phenomenon of collective synchronization. [Copyright &y& Elsevier]

Abstract: To gain insight into the mechanism by which Gli-similar 2 (Glis2) regulates transcription, we performed yeast-two hybrid cDNA library screening using Glis2 as bait. This screening identified β-catenin as a potential Glis2-interacting protein. Mammalian two-hybrid, co-immunoprecipitation, and GST-pulldown analyses supported the interaction between Glis2 and β-catenin. Pulldown analyses with several Glis2 deletion mutants indicated that the 1st zinc finger motif of Glis2 is critical for its interaction with β-catenin, while the armadillo repeats of β-catenin are important in its interaction with Glis2. Reporter analyses showed that Glis2 represses T-cell factor (TCF)-mediated transcriptional activation. In addition, Glis2 represses the expression of the TCF target gene cyclin D1. Our results indicate that Glis2 interacts with β-catenin and suggest that Glis2 functions as a negative modulator of β-catenin/TCF-mediated transcription. [Copyright &y& Elsevier]