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Abstract CD73 is a cell-surface ectoenzyme that hydrolyzes the conversion of extracellular adenosine monophosphate to adenosine, which in turn can promote resistance to immune checkpoint blockade therapy. Immune response may therefore be improved by targeting tumor CD73, and this possibility underlines the need to non-invasively assess tumor CD73 level. In this study, we developed a cysteine site-specific 89Zr-labeled anti-CD73 (89Zr-CD73) IgG immuno-PET technique that can image tumor CD73 expression in living bodies. Anti-CD73 IgG was reduced with tris(2-carboxyethyl)phosphine, underwent sulfohydryl moiety–specific conjugation with deferoxamine–maleimide, and was radiolabeled with 89Zr. CT26 mouse colon cancer cells, CT26/CD73 cells engineered to constitutively overexpress CD73, and 4T1.2 mouse breast cancer cells underwent cell binding assays and western blotting. Balb/c nude mice bearing tumors underwent 89Zr-CD73 IgG PET imaging and biodistribution studies. 89Zr-CD73 IgG showed 20-fold higher binding to overexpressing CT26/CD73 cells compared to low-expressing CT26 cells, and moderate expressing 4T1.2 cells showed uptake that was 38.9 ± 1.51% of CT26/CD73 cells. Uptake was dramatically suppressed by excess unlabeled antibody. CD73 content proportionately increased in CT26 and CT26/CD73 cell mixtures was associated with linear increases in 89Zr-CD73 IgG uptake. 89Zr-CD73 IgG PET/CT displayed clear accumulation in CT26/CD73 tumors with greater uptake compared to CT26 tumors (3.13 ± 1.70%ID/g vs. 1.27 ± 0.31%ID/g at 8 days; P = 0.04). Specificity was further supported by low CT26/CD73 tumor-to-blood ratio of 89Zr-isotype-IgG compared to 89Zr-CD73 IgG (0.48 ± 0.08 vs. 2.68 ± 0.52 at 4 days and 0.53 ± 0.07 vs. 4.81 ± 1.02 at 8 days; both P

The South Korean aquaculture industry has incurred considerable production losses due to various infectious diseases. Artificially synthesized polyinosinic–polycytidylic acid (poly I:C), structurally similar to double-stranded RNA (dsRNA) and cytidine–phosphate–guanosine oligodeoxynucleotides, can enhance immune responses and protect against diseases. Here, we investigated dsRNA molecules with different cytidine–phosphate–guanosine (CpG) motifs (dsRNA-CpGMix) as fused agents to treat Miamiensis avidus and viral hemorrhagic septicemia virus (VHSV) infection in olive flounders. We further investigated the efficacy of specific sequence motifs in dsRNA in modulating immunostimulatory effects. Fish treated with poly I:C or dsRNA-CpGMix exhibited higher survival rates than the control group. Olive flounder leukocytes stimulated with poly I:C or dsRNA-CpGMix showed increased scuticocidal activity in the presence of inactivated immune sera. dsRNA with CpG motif sequences induced higher resistance against M. avidus and VHSV infections than dsRNA without CpG motif sequences, and the dsRNA-CpGMix group showed upregulated ISG15 or Mx compared to the dsRNA-GFP group. Thus, dsRNA containing CpG motifs can be used as effective immunostimulants to enhance resistance against viral and parasitic diseases in olive flounder. The specific sequences of the CpG motifs in dsRNA may be important for enhancing immune responses and resistance against M. avidus and VHSV infections in olive flounders.

Liver cancer stem cells (LCSCs) contribute to the initiation, metastasis, treatment resistance, and recurrence of hepatocellular carcinoma (HCC). Therefore, exploring potential anticancer agents targeting LCSCs may offer new therapeutic options to overcome HCC treatment failure. Hovenia dulcis Thunberg (HDT), a tree from the buckthorn family found in Asia, exhibits various biological activities, including antifatigue, antidiabetic, neuroprotective, hepatoprotective, and antitumor activities. However, the therapeutic effect of HDT in eliminating LCSCs remains to be confirmed. In this study, we evaluated the inhibitory activity of ethanol, chloroform, and ethyl acetate extracts from HDT branches on the growth of Huh7-derived LCSCs. The ethyl acetate extract of HDT (EAHDT) exhibited the most potent inhibitory activity against the growth of Huh7 LCSCs among the three HDT extracts. EAHDT suppressed the in vitro self-renewal ability of Huh7 LCSCs and reduced tumor growth in vivo using the Huh7 LCSC-transplanted chick embryo chorioallantoic membrane model. Furthermore, EAHDT not only arrested the cell cycle in the G0/G1 phase but also induced receptor-interacting protein kinase 3/mixed-lineage kinase domain-like protein-mediated necroptosis and caspase-dependent apoptosis in Huh7 LCSCs in a concentration-dependent manner. Furthermore, the growth inhibitory effect of EAHDT on Huh7 LCSCs was associated with the downregulation of c-MET-mediated downstream signaling pathways and key cancer stemness markers. Based on these findings, we propose that EAHDT can be used as a new natural drug candidate to prevent and treat HCC by eradicating LCSCs.

Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer and has a poor prognosis. As standardized TNBC treatment regimens cause drug resistance and tumor recurrence, the development of new TNBC treatment strategies is urgently required. Bufotalin is a bufadienolide isolated from the skin and parotid venom glands of the toad Bufo gargarizan, and has several pharmacological properties, including antiviral, anti-inflammatory, and anticancer activities. However, the anticancer effect and underlying molecular mechanisms of action of bufotalin in TNBC have not been fully studied. In the current study, we investigated the effects of bufotalin on the growth and metastasis of MDA-MB-231 and HCC1937 TNBC cells. Bufotalin potently inhibited the proliferation of both TNBC cell lines by promoting cell cycle arrest and caspase-mediated apoptosis. Furthermore, bufotalin effectively suppressed the migration and invasion of both TNBC cell lines by regulating the expression of key epithelial-mesenchymal transition (EMT) biomarkers, matrix metalloproteinases (MMPs), and integrin α6. Notably, the anticancer effect of bufotalin in TNBC cells was associated with the downregulation of the signal transducer and activator of the transcription 3 (STAT3) signaling pathway. Collectively, our results suggest that the natural compound bufotalin may exert antiproliferative and antimetastatic activities in TNBC cells by modulating the apoptotic pathway and the STAT3/EMT axis.

Abstract Background This study was focused on the measurement of anticancer properties of six medicinal plants from western Nepal in three cell lines; HeLa, Hep3B, and HCT116, and anti-inflammatory properties in RAW 264.7 cell line through NO, PGE2, and TNF-α production. In addition, the phytochemical screening, total phenolic, flavonoid content, and antioxidant properties were evaluated. Results The qualitative phytochemical analysis revealed the presence of different secondary metabolite and range of total phenolic and total flavonoid content. The highest antioxidant activities were observed in Bergenia pacumbis against both DPPH (IC50 = 25.97 ± 0.19 μg/mL) and ABTS (IC50 = 14.49 ± 0.40 μg/mL). Furthermore, the highest antiproliferative effect against cervical, liver, and colon cancer cell lines were observed in Melia azedarach as IC50 values of 10.50, 5.30, and 1.57 μg/mL respectively, while the strongest anti-metastatic potential on liver cancer cell line was found in Pleurospermum benthamii. In addition, P. benthamii showed the most potent anti-inflammatory effect in RAW264.7 murine macrophage cells. Conclusion This study provided the evidence for M. azedarach and P. benthamii to have great anticancer potential and finding builds the enough scientific backgrounds in future to isolate and purify the bioactive compounds for further applications.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Relapse is frequent and rapid due to glioblastoma stem-like cells (GSCs) that induce tumor initiation, drug resistance, high cancer invasion, immune evasion, and recurrence. Therefore, suppression of GSCs is a powerful therapeutic approach for GBM treatment. Natural compounds berbamine and arcyriaflavin A (ArcA) are known to possess anticancer activity by targeting calcium/calmodulin-dependent protein kinase II gamma (CaMKIIγ) and cyclin-dependent kinase 4 (CDK4), respectively. In this study, we evaluated the effects of concurrent treatment with both compounds on GSCs. Combined treatment with berbamine and ArcA synergistically inhibited cell viability and tumorsphere formation in U87MG- and C6-drived GSCs. Furthermore, simultaneous administration of both compounds potently inhibited tumor growth in a U87MG GSC-grafted chick embryo chorioallantoic membrane (CAM) model. Notably, the synergistic anticancer effect of berbamine and ArcA on GSC growth is associated with the promotion of reactive oxygen species (ROS)- and calcium-dependent apoptosis via strong activation of the p53-mediated caspase cascade. Moreover, co-treatment with both compounds significantly reduced the expression levels of key GSC markers, including CD133, integrin α6, aldehyde dehydrogenase 1A1 (ALDH1A1), Nanog, Sox2, and Oct4. The combined effect of berbamine and ArcA on GSC growth also resulted in downregulation of cell cycle regulatory proteins, such as cyclins and CDKs, by potent inactivation of the CaMKIIγ-mediated STAT3/AKT/ERK1/2 signaling pathway. In addition, a genetic knockdown study using small interfering RNAs (siRNAs) targeting either CaMKIIγ or CDK4 demonstrated that the synergistic anticancer effect of the two compounds on GSCs resulted from dual inhibition of CaMKIIγ and CDK4. Collectively, our findings suggest that a novel combination therapy involving berbamine and ArcA could effectively eradicate GSCs.

Abstract Background Live fish import may lead to the unintended introduction of pathogens. We examined the monthly distribution of microbial pathogens in ornamental finfish imported into South Korea over a 6-month period. Results Vibrio alginolyticus was detected in one lemon damsel in June and July; V. vulnificus was detected in one lemon damsel, one caerulean damsel, and one pearl-spot chromis and one ocellaris clownfish in July, April, and May, respectively; Photobacterium damselae was detected in one ocellaris clownfish and one caerulean damsel in June and July, respectively; V. anguillarum was detected in one pearl-spot chromis in February; V. harveyi was detected in one ocellaris clownfish and two mandarin fish in February and April, respectively; Yersinia ruckeri was detected in a pearlscale goldfish group in June and July and in two colored carp groups in July; and Lactococcus garvieae was detected in a lemon damsel group and a sutchi catfish group in July and May, respectively. European catfish virus, the only viral pathogen detected, was found in two sutchi catfish groups in May. Conclusion This study is the first to identify pathogenic species and the presence or absence of pathogens (non-quarantine diseases) in imported ornamental finfish. These results demonstrate that various pathogens with the potential to harm indigenous fish populations can accompany ornamental finfish imported into South Korea.

Abstract Background Umbelliferone, also known as 7-hydroxycoumarin, is a phenolic metabolite found in many familiar plants. Its derivatives have been shown to have various pharmacological and chemo-preventive effects on human health. A uridine diphosphate glycosyltransferase YjiC from Bacillus licheniformis DSM 13, a cytochrome P450BM3 (CYP450 BM3) variant namely mutant 13 (M13) from Bacillus megaterium, and an O-methyltransferase from Streptomyces avermitilis (SaOMT2) were used for modifications of umbelliferone. Results Three umbelliferone derivatives (esculetin, skimmin, and herniarin) were generated through enzymatic and whole cell catalysis. To improve the efficiencies of biotransformation, different media, incubation time and concentration of substrate were optimized and the production was scaled up using a 3-L fermentor. The maximum yields of esculetin, skimmin, and herniarin were 337.10 μM (67.62%), 995.43 μM (99.54%), and 37.13 μM (37.13%), respectively. The water solubility of esculetin and skimmin were 1.28-folds and 3.98-folds as high as umbelliferone, respectively, whereas herniarin was 1.89-folds less soluble than umbelliferone. Moreover, the antibacterial and anticancer activities of herniarin showed higher than umbelliferone, esculetin and skimmin. Conclusions This study proves that both native and engineered enzymes could be employed for the production of precious compounds via whole cell biocatalysis. We successfully produced three molecules herniarin, esculetin and skimmin in practical amounts and their antibacterial and anticancer properties were accessed. One of the newly synthesized molecules the present research suggests that the combinatorial biosynthesis of different biosynthetic enzymes could rapidly promote to a novel secondary metabolite.

In this study, we determined the effects of clove oil concentration and water temperature on the anesthesia and recovery times, hematological factors, and physiological responses in the olive flounder (Paralichthys olivaceus). To determine these times at three temperatures (17, 21, and 25 °C), fish (mean length [± standard deviation]: 30.0 ± 1.0 cm, mean weight [± standard deviation]: 245.0 ± 27.1 g) were exposed to clove oil at concentrations of 40, 80, 120, 160, 200, 240, and 280 mg L−1. At 0.5, 1, 3, 6, and 24 h, blood samples were collected from 10 fish in the 160 mg L−1 clove oil-treated group (AG) and the non-anesthetic group (NAG) maintained at 21 °C. Subsequently, the following parameters were analyzed: hematocrit and hemoglobin levels; red blood cell count; osmolality; cortisol, glucose, lactic acid, Na+, K+, and Cl- concentrations; and aspartate aminotransferase and alanine aminotransferase activities. Clove oil concentration and temperature exhibited interdependent effects on the anesthesia and recovery times. As the water temperature increased, the anesthesia and recovery times decreased, and as the clove oil concentration increased, the anesthesia time decreased and recovery time was prolonged. AG showed significantly higher plasma cortisol and glucose concentrations than those of NAG at 0.5 and 1 h after anesthesia, but significantly lower concentrations than those of NAG at 24 h after anesthesia. In addition, the lactic acid concentration in AG was lower than that in NAG at 0.5, 1, and 3 h. Intergroup differences for all other blood factors and osmolality were not significant before or after anesthesia. Based on stage III behavior, the anesthesia and recovery times should be less than 180 and 300 s, respectively, to prevent hypoxia in fish under anesthesia. Therefore, the stable anesthesia concentrations for olive flounder (245.0 ± 27.1 g) were 120–160 mg L−1 at 21 °C and 80–240 mg L−1 at 25 °C. Moreover, as 18–21 °C was determined to be the optimal water temperature for olive flounder rearing (˜245.0 g), we suggest that as an optimal anesthetic, clove oil can be optimally used at 160 mg L−1 concentration at 21 °C and that it is a reasonable alternative to other anesthetics. Keywords: Optimal concentration, Recovery time, Hypoxia, Hematological factor

Intestinal tuberculosis (ITB) remains prevalent in Asia. An interferon-γ assay (QuantiFERON-TB gold test [QFT]) is considered to be an effective supplementary tool for diagnosing ITB. We retrospectively analyzed the clinical features of ITB patients based on the initial results of QFT. A total of 109 patients with ITB were enrolled, and 82 patients (75.2%) showed positive QFT results. In the QFT-positive group, the mean age (44.1±12.0 years) was significantly higher than that in the QFT-negative group (37.0±14.8, p=0.0096). Abdominal pain (p=0.006) and diarrhea (p=0.030) were more frequent in the QFT-negative group. Further, C-reactive protein (CRP) levels were significantly higher in the QFT-negative group (6.4±9.9 mg/dL) than in the QFT-positive group (1.3±2.3, p

Targeting angiogenesis is an attractive strategy for the treatment of angiogenesis-related diseases, including cancer. We previously identified 23-demethyl 8,13-deoxynargenicin (compound 9) as a novel nargenicin A1 analog with potential anticancer activity. In this study, we investigated the antiangiogenic activity and mode of action of compound 9. This compound was found to effectively inhibit in vitro angiogenic characteristics, including the proliferation, invasion, capillary tube formation, and adhesion of human umbilical vein endothelial cells (HUVECs) stimulated by vascular endothelial growth factor (VEGF). Furthermore, compound 9 suppressed the neovascularization of the chorioallantoic membrane of growing chick embryos in vivo. Notably, the antiangiogenic properties of compound 9 were related to the downregulation of VEGF/VEGFR2-mediated downstream signaling pathways, as well as matrix metalloproteinase (MMP)-2 and MMP-9 expression in HUVECs. In addition, compound 9 was found to decrease the in vitro AGS gastric cancer cell-induced angiogenesis of HUVECs by blocking hypoxia-inducible factor-1α (HIF-1α) and VEGF expression in AGS cells. Collectively, our findings demonstrate for the first time that compound 9 is a promising antiangiogenic agent targeting both VEGF/VEGFR2 signaling in ECs and HIF-1α/VEGF pathway in tumor cells.

Melanoma is the most serious type of skin cancer and remains highly drug-resistant. Therefore, the discovery of novel effective agents against melanoma is in high demand. Herein, we investigated the cytotoxic activities in melanoma cells and underlying molecular mechanisms of beauvericin (BEA) and its analogue beauvericin G1 (BEA G1), which are cyclohexadepsipeptides isolated from fungi. BEA and BEA G1 significantly suppressed the growth, clonogenicity, migration, and invasion of A375SM human melanoma cells and promoted caspase-dependent apoptosis through upregulation of death receptors, as well as modulation of pro- and anti-apoptotic Bcl-2 family members. Furthermore, the effects of BEA and BEA G1 were associated with the suppression of multiple molecular targets that play crucial roles in melanoma oncogenesis, including ERK, JNK, p38, NF-κB, STAT3, and MITF. Notably, the cytotoxic efficacy of BEA G1 against A375SM cells was stronger than that of BEA. These findings suggest that BEA and BEA G1 can be further investigated as potent cytotoxic natural compounds for the suppression of melanoma progression.

Increasing studies have reported that cancer stem cells (CSCs) play critical roles in therapeutic resistance, recurrence, and metastasis of tumors, including cervical cancer. Pterostilbene, a dimethylated derivative of resveratrol, is a plant polyphenol compound with potential chemopreventive activity. However, the therapeutic effect of pterostilbene against cervical CSCs remains unclear. In this study, we compared the anticancer effects of resveratrol and pterostilbene using both HeLa cervical cancer adherent and stem-like cells. Pterostilbene more effectively inhibited the growth and clonogenic survival, as well as metastatic ability of HeLa adherent cells than those of resveratrol. Moreover, the superior inhibitory effects of pterostilbene compared to resveratrol were associated with the enhanced activation of multiple mechanisms, including cell cycle arrest at S and G2/M phases, induction of ROS-mediated caspase-dependent apoptosis, and inhibition of matrix metalloproteinase (MMP)-2/-9 expression. Notably, pterostilbene exhibited a greater inhibitory effect on the tumorsphere-forming and migration abilities of HeLa cancer stem-like cells compared to resveratrol. This greater effect was achieved through more potent inhibition of the expression levels of stemness markers, such as CD133, Oct4, Sox2, and Nanog, as well as signal transducer and activator of transcription 3 signaling. These results suggest that pterostilbene might be a potential anticancer agent targeting both cancer cells and cancer stem-like cells of cervical cancer via the superior bioavailability to resveratrol.

Background/AimsAmong the many complications that can occur following therapeutic endoscopy, bleeding is the most serious, which occurs in 1.0-6.1% of all colonoscopic polypectomies. The aim of this study was to identify risk factors of delayed post-polypectomy bleeding (PPB).MethodsWe retrospectively reviewed the data of patients who underwent colonoscopic polypectomy between January 2003 and December 2012. We compared patients who experienced delayed PPB with those who did not. The control-to-patient ratio was 3:1. The clinical data analyzed included polyp size, number, location, and shape, patient' body mass index (BMI), preventive hemostasis, and endoscopist experience.ResultsOf 1,745 patients undergoing colonoscopic polypectomy, 21 (1.2%) experienced significant delayed PPB. We selected 63 age- and sex-matched controls. Multivariate logistic regression analysis showed that polyps >10 mm (odds ratio [OR], 2.605; 95% confidence interval [CI], 1.035-4.528; P=0.049), a pedunculated polyp (OR, 3.517; 95% CI, 1.428-7.176; P=0.045), a polyp located in the right hemicolon (OR, 3.10; 95% CI, 1.291-5.761; P=0.013), and a high BMI (OR, 3.681; 95% CI, 1.876-8.613; P=0.013) were significantly associated with delayed PPB.ConclusionsAlthough delayed PPB is a rare event, more caution is needed during colonoscopic polypectomies performed in patients with high BMI or large polyps, pedunculated polyps, or polyps located in the right hemicolon.

In the course of searching for angiogenesis inhibitors from microorganisms, two cyclic peptides, PF1171A (1) and PF1171C (2) were isolated from the soil fungus Penicillium sp. FN070315. In the present study, we investigated the antiangiogenic efficacy and associated mechanisms of 1 and 2 in vitro using human umbilical vein endothelial cells (HUVECs). Compounds 1 and 2 inhibited the proliferation of HUVECs at concentrations not exhibiting cytotoxicity. Moreover, 1 and 2 significantly suppressed vascular endothelial growth factor (VEGF)-induced migration, invasion, proliferation and tube formation of HUVECs as well as neovascularization of the chorioallantoic membrane in developing chick embryos. We also identified an association between the antiangiogenic activity of 1 and 2 and the downregulation of both the phosphorylation of VEGF receptor 2 and the expression of hypoxia inducible factor-1α at the protein level. Taken together, these results further suggest that compounds 1 and 2 will be promising angiogenesis inhibitors.

Background/AimsAntibiotic usage and increasingly aging populations have led to increased incidence of Clostridium difficile infection (CDI) in worldwide. Recent studies in Korea have also reported increasing CDI incidence; however, there have been no reports on the long-term outcomes of CDI. We therefore investigated the long-term clinical outcomes of patients with CDI, including delayed recurrence, associated risk factors and mortality.MethodsHospitalized patients diagnosed with CDI at Seoul Paik Hospital between January 2007 and December 2008 were included. Their medical records were retrospectively investigated. 'Delayed recurrence' was defined as a relapse 8 weeks after a successful initial treatment. Multivariate logistic regression analysis was performed to identify risk factors for the delayed recurrence. Kaplan-Meier curves were used to analyze mortality rates.ResultsA total of 120 patients were enrolled; among them, 87 were followed-up for at least 1 year, with a mean follow-up period of 34.1±25.1 months. Delayed recurrence was observed in 17 patients (19.5%), and significant risk factors were age (over 70 years, P=0.049), nasogastric tube insertion (P=0.008), and proton pump inhibitor or H2-blocker treatments (P=0.028). The 12- and 24-month mortality rates were 24.6% and 32.5%, respectively. No deaths were directly attributed to CDI.ConclusionsDelayed recurrence of CDI was not rare, occurring in 19.5% of the study population. Although CDI-related mortality was not reported, 2-year (32.5%) mortality rate of CDI patients implies that a CDI diagnosis may predict severe morbidity and poor prognosis of the underlying disease.

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and is a leading cause of cancer-related death worldwide. Therefore, exploring effective anticancer agents and their modes of action is essential for the prevention and treatment of HCC. Glycosylation can significantly improve the physicochemical and biological properties of small molecules, such as high solubility, stability increase, and lower toxicity. In this study, for the first time, we evaluated the anticancer activities of mangostin-3-O-β-d-2-deoxyglucopyranoside (Man-3DG) and mangostin 6-O-β-d-2-deoxyglucopyranoside (Man-6DG), glycosides of mangostin, against human hepatoma Hep3B cells. Our results demonstrated that Man-3DG and Man-6DG significantly suppressed growth and migration of Hep3B cells. In addition, they induced apoptosis of Hep3B cells by regulating apoptosis-related proteins of mitochondria. Noticeably, Man-3DG and Man-6DG also caused autophage, while cotreatment of the mangostin glycosides with an autophage inhibitor 3MA enhanced the inhibitory effect on Hep3B cell growth, compared to single agent treatment. Moreover, Man-3DG and Man-6DG inhibited the c-Met signaling pathway, which plays a critical role in the pathogenesis of liver cancer. Furthermore, the mangostin glycosides decreased tumor cell-induced angiogenesis in vitro through downregulation of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF). These findings suggest that Man-3DG and Man-6DG might be promising anticancer agents for HCC treatment with superior pharmacological properties than parent molecule mangostin.

Malignant melanoma is the deadliest form of skin cancer and highly chemoresistant. Melittin, an amphiphilic peptide containing 26 amino acid residues, is the major active ingredient from bee venom (BV). Although melittin is known to have several biological activities such as anti-inflammatory, antibacterial and anticancer effects, its antimelanoma effect and underlying molecular mechanism have not been fully elucidated. In the current study, we investigated the inhibitory effect and action mechanism of BV and melittin against various melanoma cells including B16F10, A375SM and SK-MEL-28. BV and melittin potently suppressed the growth, clonogenic survival, migration and invasion of melanoma cells. They also reduced the melanin formation in α-melanocyte-stimulating hormone (MSH)-stimulated melanoma cells. Furthermore, BV and melittin induced the apoptosis of melanoma cells by enhancing the activities of caspase-3 and -9. In addition, we demonstrated that the antimelanoma effect of BV and melittin is associated with the downregulation of PI3K/AKT/mTOR and MAPK signaling pathways. We also found that the combination of melittin with the chemotherapeutic agent temozolomide (TMZ) significantly increases the inhibition of growth as well as invasion in melanoma cells compared to melittin or TMZ alone. Taken together, these results suggest that melittin could be potentially applied for the prevention and treatment of malignant melanoma.

A glycoside derivative of compound K (CK) was synthesized by using a glycosyltransferase, and its biological activity was tested against various cancer-cell lines. A regiospecific, β-1,4-galactosyltransferase (LgtB) converted 100% of 0.5 mmol CK into a galactosylated product in 3 h. The structure of the synthesized derivative was revealed with high performance liquid chromatography, mass spectroscopy, as well as nuclear magnetic resonance analyses, and it was recognized as 20-O-β-D-lactopyranosyl-20(S)-protopanaxadiol (CKGal). Out of the four cancer-cell lines tested (gastric carcinoma (AGS), skin melanoma (B16F10), cervical carcinoma (HeLa), and brain carcinoma (U87MG)), CKGal showed the best cytotoxic ability against B16F10 and AGS when compared to other ginsenosides like compound K (20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol), Rh2 (3-O-β-D-glucopyranosyl-20(S)-protopanaxadiol), and F12 (3-O-β-D-glucopyranosyl-12-O-β-D-glucopyranosyl-20(S)-protopanaxadiol). Thus, the synthesized derivative (CKGal) is a pharmacologically active ginsenoside.

Nargenicin A1 is major secondary metabolite produced by Nocardia sp. CS682, with an effective antibacterial activity against various Gram-positive bacteria. Most Nocardia spp. have metabolic ability to produce compounds of diverse nature, so one-strain-many-compounds (OSMAC) approach can be applied for obtaining versatile compounds from these strains. In this study, we characterized a novel 1, 3, 6, 8-tetrahydroxynaphthalene (THN) derivative by metabolic engineering approach leading to the inactivation of nargenicin A1 biosynthesis. By using genome mining, metabolite profiling, and bioinformatics, the biosynthetic gene cluster and biosynthetic mechanism were elucidated. Further, the antibacterial, anticancer, melanin formation, and UV protective properties for isolated THN compound were performed. The compound did not exhibit significant antibacterial and cytotoxic activities, but it exhibited promising UV protection effects. Thus, metabolic engineering is an effective strategy for discovering novel bioactive molecules.

Anthraquinones, naturally occurring bioactive compounds, have been reported to exhibit various biological activities, including anti-inflammatory, antiviral, antimicrobial, and anticancer effects. In this study, we biotransformed three selected anthraquinones into their novel O-glucoside derivatives, expressing a versatile glycosyltransferase (YjiC) from Bacillus licheniformis DSM 13 in Escherichia coli. Anthraflavic acid, alizarin, and 2-amino-3-hydroxyanthraquinone were exogenously fed to recombinant E. coli as substrate for biotransformation. The products anthraflavic acid-O-glucoside, alizarin 2-O-β-d-glucoside, and 2-amino-3-O-glucosyl anthraquinone produced in the culture broths were characterized by various chromatographic and spectroscopic analyses. The comparative anti-proliferative assay against various cancer cells (gastric cancer-AGS, uterine cervical cancer-HeLa, and liver cancer-HepG2) were remarkable, since the synthesized glucoside compounds showed more than 60% of cell growth inhibition at concentrations ranging from ~50 μM to 100 μM. Importantly, one of the synthesized glucoside derivatives, alizarin 2-O-glucoside inhibited more than 90% of cell growth in all the cancer cell lines tested.

Silver materials have been widely used in diverse fields. However, their toxicity and their mechanism, especially in different forms, have not been studied sufficiently. Thus, cytotoxicity, apoptosis, and interleukin-1beta (IL-1β) production were investigated using macrophage-like THP-1 cells in the presence of Ag microparticles (AgMPs, 2.7 µm), Ag submicroparticles (AgSMPs, 150 nm), and Ag wires (AgWs, 274 nm×5.3 µm). The levels of cytotoxicity, apoptosis, and IL-1β production by AgWs were higher than those by the other two AgSMPs and AgMPs. This trend was also observed with each step of the signaling mechanism for IL-1β production, which is a single pathway affiliated with ROS generation or lysosomal rupture or both, cathepsin B, caspase-1 (NALP3 inflammasome), and finally IL-1β production in THP-1 cells. All these results suggest that, for development of safe and effective silver materials, the shape or form of silver materials should be considered, especially for macrophage cell lines because epithelial cell lines are not overly sensitive to silver materials.

Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient's surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.

Gastric cancer stem cells (GCSCs) are a major cause of radioresistance and chemoresistance in gastric cancer (GC). Therefore, targeting GCSCs is regarded as a powerful strategy for the effective treatment of GC. Atorvastatin is a widely prescribed cholesterol-lowering drug that inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate-limiting enzyme in the mevalonate pathway. The anticancer activity of atorvastatin, a repurposed drug, is being investigated; however, its therapeutic effect and molecular mechanism of action against GCSCs remain unknown. In this study, we evaluated the anticancer effects of atorvastatin on MKN45-derived GCSCs. Atorvastatin significantly inhibited the proliferative and tumorsphere-forming abilities of MKN45 GCSCs in a mevalonate pathway-independent manner. Atorvastatin induced cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade. Furthermore, atorvastatin exerted an antiproliferative effect against MKN45 GCSCs by inhibiting the expression of cancer stemness markers, such as CD133, CD44, integrin α6, aldehyde dehydrogenase 1A1, Oct4, Sox2, and Nanog, through the downregulation of β-catenin, signal transducer and activator of transcription 3, and protein kinase B activities. Additionally, the combined treatment of atorvastatin and sorafenib, a multi-kinase targeted anticancer drug, synergistically suppressed not only the proliferation and tumorsphere formation of MKN45 GCSCs but also the in vivo tumor growth in a chick chorioallantoic membrane model implanted with MKN45 GCSCs. These findings suggest that atorvastatin can therapeutically eliminate GCSCs. [ABSTRACT FROM AUTHOR]

A 6-year-old, male Yorkshire Terrier dog presented with persistent regurgitation and severe weight loss. Based on the clinical signs, physical and other various diagnostic examinations, including fluoroscopy, were performed. Myasthenia gravis was ruled out through a serum acetylcholine receptor antibody titre measurement and a negative response to neostigmine bromide (0.02 mg/kg) within 4 hours of injection. The dog was diagnosed with idiopathic oesophageal achalasia. As a treatment, a modified Heller's oesophagomyotomy was performed, and the dog recovered well with no signs of recurrence for 18 months. [ABSTRACT FROM AUTHOR]

Angiogenesis plays a very important role in tumor progression through the creation of new blood vessels. Therefore, angiogenesis inhibitors could contribute to cancer treatment. Here, we show that a microbial metabolite, elaiophylin, exhibits potent antiangiogenic activity from in vitro and in vivo angiogenesis assays. Elaiophylin dramatically suppressed in vitro angiogenic characteristics such as proliferation, migration, adhesion, invasion and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by vascular endothelial growth factor (VEGF) at non-toxic concentrations. In addition, elaiophylin immensely inhibited in vivo angiogenesis of the chorioallantoic membrane (CAM) from growing chick embryos without cytotoxicity. The activation of VEGF receptor 2 (VEGFR2) in HUVECs by VEGF was inhibited by elaiophylin, resulting in the suppression of VEGF-induced activation of downstream signaling molecules, Akt, extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38, nuclear factor-κB (NFκB), matrix metalloproteinase (MMP)-2 and -9 which are closely associated with VEGF-induced angiogenesis. We also found that elaiophylin blocked tumor cell-induced angiogenesis both in vitro and in vivo. Elaiophylin downregulated the expression of VEGF by inhibiting hypoxia inducible factor-1α (HIF-1α) accumulation in tumor cells. To our knowledge, these results for the first time demonstrate that elaiophylin effectively inhibits angiogenesis and thus may be utilized as a new class of natural antiangiogenic agent for cancer therapy. [ABSTRACT FROM AUTHOR]

TiO2-coated boron particles were prepared by a wet ball milling method, with the particle size distribution and average particle size being easily controlled by varying the milling operation time. Based on the results from X-ray photoelectron spectroscopy, transmission electron microscopy, energy dispersive X-ray analysis, and Fourier transform infrared spectroscopy, it was confirmed that the initial oxide layer on the boron particles surface was removed by the wet milling process, and that a new B-O-Ti bond was formed on the boron surface. The uniform TiO2 layer on the 150 nm boron particles was estimated to be 10 nm thick. Based on linear sweep voltammetry, cyclic voltammetry, current-time amperometry, and electrochemical impedance analyses, the potential for the application of TiO2-coated boron particles as a photoelectrochemical catalyst was demonstrated. A current of 250 μA was obtained at a potential of 0.5 V for hydrogen evolution, with an onset potential near to 0.0 V. Finally, a current of 220 μA was obtained at a potential of 1.0 V for oxygen evolution. [ABSTRACT FROM AUTHOR]

Background: Cytochrome P450 monooxygenase constitutes a signiicant group of oxidative enzymes that can introduce an oxygen atom in a high regio- and stereo-selectivity mode. We used the Bacillus megaterium cytochrome P450 BM3 (CYP450 BM3) and its variants namely mutant 13 (M13) and mutant 15 (M15) for the hydroxylation of diverse class of lavonoids. Results: Among 20 flavonoids, maximum seven lavonoids were hydroxylated by the variants while none of these molecules were accepted by CYP450 BM3 in in vitro reaction. Moreover, M13 exhibited higher conversion of substrates than M15 and CYP450 BM3 enzymes. We found that M13 carried out regiospeciic 3'-hydroxylation reaction of naringenin with the highest conversion among all the tested lavonoids. The apparent Km and kcat values of M13 for naringenin were 446 µM and 1.955 s-1, respectively. In whole-cell biotransformation experiment with 100 µM of naringenin in M9 minimal medium with 2 % glucose in shake lask culture, M13 showed 2.14- and 13.96-folds higher conversion yield in comparison with M15 (16.11 %) and wild type (2.47 %). The yield of eriodictyol was 46.95 µM [~40.7 mg (13.5 mg/L)] in a 3-L volume lab scale fermentor at 48 h in the same medium exhibiting approximately 49.81 % conversion of the substrate. In addition, eriodictyol exhibited higher antibacterial and anticancer potential than naringenin, lavanone and hesperetin. Conclusions: We elucidated that eriodictyol being produced from naringenin using recombinant CYP450 BM3 and its variants from B. megaterium, which shows an approach for the production of important hydroxylated compounds of various polyphenols that may span pharmaceutical industries. [ABSTRACT FROM AUTHOR]

Background: The combination of tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) has. been the first choice nucleoside reverse transcriptase inhibitor (NRTI) according to many reliable antiretroviral treatment (ART) guidelines because of its high efficacy. However, TDF-related renal toxicity reported in Western countries is a challenging issue regarding clinical use. We conducted this study to evaluate the incidence and characteristics of an ac니te increase in serum creatinine (Cr) level > 1.5 mg/dL among TDF/FTCbased highly active antiretroviral treatment (HAART)-treated patients. Materials and Methods: We retrospectively reviewed the medical records of 205 HIV-infected patients treated with TDF/FTC-containing regimens between 1 February 2010 and 30 April 2014. Three groups of TDF/FTC + ritonavir-boosted protease inhibitor (PI/ r), TDF/FTC + non-nucleoside reverse transcriptase inhibitor (NNRTI), and TDF/FTC + integrase strand transfer inhibitor (INSTI), and three Pl/r subgroups of TDF/FTC + lopinavir (l_PV)/r, TDF/FTC + atazanavir (ATV)/r, TDF/FTC + darunavir (DRV)/r were evaluated. Results: A total 136 patients (91 in the TDF/FTC + Pl/r group, 20 in the TDF/FTC + NNRTI group and 25 in the TDF/FTC + INSTI group) were included in the statistical analysis. Four cases (4.9%; all in the TDF/FTC + Pl/r group) among 136 patients showed an acute increase in serum Cr more than 1.5 mg/dL, so the overall incidence was 2.8 cases per 100 patient-years. One case was a patient treated with TDF/FTC + LPV/r, and the others were treated with TDF/FTC + ATV/r. No case of an acute increase in serum Cr was observed in the TDF/FTC + DRV/r group. The incidence of serum Cr increase more than 1.5 mg/dL in TDF/FTC + Pl/r group was 4.0 cases per 100 patient-years. Conclusion: Although only a small number of patients were evaluated retrospectively from a single center, the TDF/FTC + Pl/r regimen may have been related with relatively higher tendency of increment of serum Cr level. These findings reinforce the importance of close follow-ups of HIV-infected patients treated with the TDF/FTC + Pl/r regimens. [ABSTRACT FROM AUTHOR]

Background/Aims: Antibiotic usage and increasingly aging populations have led to increased incidence of Clostridium difficile infection (CDI) in worldwide. Recent studies in Korea have also reported increasing CDI incidence; however, there have been no reports on the long-term outcomes of CDI. We therefore investigated the long-term clinical outcomes of patients with CDI, including delayed recurrence, associated risk factors and mortality. Methods: Hospitalized patients diagnosed with CDI at Seoul Paik Hospital between January 2007 and December 2008 were included. Their medical records were retrospectively investigated. 'Delayed recurrence' was defined as a relapse 8 weeks after a successful initial treatment. Multivariate logistic regression analysis was performed to identify risk factors for the delayed recurrence. Kaplan-Meier curves were used to analyze mortality rates. Results: A total of 120 patients were enrolled; among them, 87 were followed-up for at least 1 year, with a mean follow-up period of 34.1±25.1 months. Delayed recurrence was observed in 17 patients (19.5%), and significant risk factors were age (over 70 years, P=0.049), nasogastric tube insertion (P=0.008), and proton pump inhibitor or H2-blocker treatments (P=0.028). The 12- and 24-month mortality rates were 24.6% and 32.5%, respectively. No deaths were directly attributed to CDI. Conclusions: Delayed recurrence of CDI was not rare, occurring in 19.5% of the study population. Although CDI-related mortality was not reported, 2-year (32.5%) mortality rate of CDI patients implies that a CDI diagnosis may predict severe morbidity and poor prognosis of the underlying disease. [ABSTRACT FROM AUTHOR]

Boron-CeO2 hybrid materials were prepared by dry and wet ball-milling methods and characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), FT-IR and X-ray photoelectron spectroscopy (XPS). We also demonstrated CO oxidation activity of the material using temperature-programmed reaction mass spectrometry, and found an interesting synergic effect of CO oxidation in response to hybridization of boron and CeO2. The oxidation state of Ce was found to have changed from +4 to +3 during the milling process, which was confirmed by the Ce 3d XPS scan result. These findings indicate that boron maintains the convertible oxidation states (Ce3+↔Ce4+) of Ce at the interface between boron and CeO2 during the CO oxidation reaction. The present study highlights the potential application of boron and CeO2 hybrid materials to pollution control, chemical industry and propulsion energy sources. [ABSTRACT FROM AUTHOR]

We established the first complete ice core processing method and analytical procedures for fundamental proxies, using a 40.2 m long ice core drilled on the Mt. Tsambagarav glacier in the Mongolian Altai mountains in July 2008. The whole core was first divided into two sub ice core sections and the measurements of the visual stratigraphy and electrical conductivity were performed on the surface of these sub core sections. A continuous sequence of samples was then prepared for chemical analyses (stable isotope ratios of oxygen (18O/16O) and hydrogen (²H/¹H), soluble ions and trace elements). A total of 29 insoluble dust layers were identified from the measurement of visual stratigraphy. The electrical conductivity measurement (ECM) shows 11 peaks with the current more than 0.8 μA. Comparing the profiles of SO42- and Cl- concentrations to correlate with known volcanic eruptions, the first two ECM peaks appear to be linked to the eruptions (January and June 2007) of Kliuchevskoi volcano on the Kamchatka Peninsula of Russia, which supports the reliability of our ECM data. Finally, the composition of stable isotopes (δ18O and δD) shows a well-defined seasonal variation, suggesting that various chemical proxies may have been well preserved in the successive ice layers of Tsambagarav ice core. Our ice core processing method and analytical procedures for fundamental proxies are expected to be used for paleoclimate and paleoenvironmental studies from polar and alpine ice cores. [ABSTRACT FROM AUTHOR]

Recent reports have shown that Ca2+/calmodulin (Ca2+/CaM) signaling plays a crucial role in angiogenesis. We previously developed a new Ca2+/CaM antagonist, HBC (4-{3,5-bis-[2-(4-hydroxy-3-methoxyphenyl)ethyl]-4,5-dihydropyrazol-1-yl}benzoic acid), from a curcumin-based synthetic chemical library. Here, we investigated its anti-angiogenic activity and mode of action. HBC potently inhibited the proliferation of human umbilical vascular endothelial cells with no cytotoxicity. Furthermore, HBC blocked in vitro characteristics of angiogenesis such as tube formation and chemoinvasion, as well as neovascularization of the chorioallantoic membrane of growing chick embryos in vivo. Notably, HBC markedly inhibited expression of hypoxia-inducible factor-1α (HIF-1α) at the translational level during hypoxia, thereby reducing HIF-1 transcriptional activity and expression of its major target gene, vascular endothelial growth factor. In addition, combination treatment with HBC and various HIF-1 inhibitors, including suberoylanilide hydroxamic acid, rapamycin, and terpestacin, had greater anti-angiogenic activity than treatment with each single agent. Collectively, our findings indicate that HBC is a new anti-angiogenic agent targeting HIF that can be used to explore the biological role of Ca2+/CaM in angiogenesis. [ABSTRACT FROM AUTHOR]

Cellular oxygen sensing is required for hypoxia-inducible factor-1α stabilization, which is important for tumor cell survival, proliferation, and angiogenesis. Here we find that terpestacin, a small molecule previously identified in a screen of microbial extracts, binds to the 13.4-kDa subunit (UQCRB) of mitochondrial Complex III, resulting in inhibition of hypoxia-induced reactive oxygen species generation. Consequently, such inhibition blocks hypoxia-inducible factor activation and tumor angiogenesis in vivo, without inhibiting mitochondrial respiration. Overexpression of UQCRB or its suppression using RNA interference demonstrates that it plays a crucial role in the oxygen sensing mechanism that regulates responses to hypoxia. These findings provide a novel molecular basis of terpestacin targeting UQCRB of Complex III in selective suppression of tumor progression. [ABSTRACT FROM AUTHOR]

Takotsubo cardiomyopathy (TC) is characterized by reversible left ventricular (LV) apical ballooning and no significant coronary artery stenosis. New variants of TC with localized wall motion abnormality or inversed pattern with hyperdynamic apex have been reported. We present the case of a 24-year-old female with atypical presentation of TC occurring in the setting of paranasal sinus surgery under local anaesthesia with post-surgical nasal packing. She did not demonstrated ST-segment elevation on electrocardiogram, but transient moderate LV systolic dysfunction and localized wall motion abnormality affecting basal to mid-ventricular anterior and anteroseptal wall. She rapidly and completely recovered without sequelae. [ABSTRACT FROM PUBLISHER]

Gram-negative bacteria derived extracellular vesicles (EVs), also known as outer membrane vesicles, have attracted significant attention due to their pathogenic roles in various inflammatory diseases. We recently demonstrated that EVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) can cross the blood-brain barrier (BBB) and that their extracellular RNA cargo can promote the secretion of proinflammatory cytokines, such as IL-6 and TNF-α, in the brain. To gain more insight into the relationship between periodontal disease (PD) and neuroinflammatory diseases, we investigated the effect of Aa EVs in a mouse model of ligature-induced PD. When EVs were administered through intragingival injection or EV-soaked gel, proinflammatory cytokines were strongly induced in the brains of PD mice. The use of TLR (Toll-like receptor)-reporter cell lines and MyD88 knockout mice confirmed that the increased release of cytokines was triggered by Aa EVs via TLR4 and TLR8 signaling pathways and their downstream MyD88 pathway. Furthermore, the injection of EVs through the epidermis and gingiva resulted in the direct retrograde transfer of Aa EVs from axon terminals to the cell bodies of trigeminal ganglion (TG) neurons and the subsequent activation of TG neurons. We also found that the Aa EVs changed the action potential of TG neurons. These findings suggest that EVs derived from periodontopathogens such as Aa might be involved in pathogenic pathways for neuroinflammatory diseases, neuropathic pain, and other systemic inflammatory symptoms as a comorbidity of periodontitis.

Methyltransferases transfer a methyl group to a diverse group of natural products, thus providing structural diversity, stability, and altered pharmacological properties to the molecules. A limited number of regiospecific sugar-O-methyltransferases are functionally characterized. Thus, discovery of such an enzyme could solve the difficulties of biological production of methoxy derivatives of glycosylated molecules. In the current study, a regiospecific sugar-O-methyltransferase, ThnM1, belonging to the biosynthetic gene cluster (BGC) of 1-(α-L-(2-O-methyl)-6-deoxymannopyranosyloxy)-3,6,8-trimethoxynaphthalene produced by Nocardia sp. strain CS682, was analyzed and functionally characterized. ThnM1 demonstrated promiscuity to diverse chemical structures such as rhamnose-containing anthraquinones and flavonoids with regiospecific methylation at the 2′-hydroxyl group of the sugar moiety. Compared with other compounds, anthraquinone rhamnosides were found to be the preferred substrates for methylation. Thus, the enzyme was further employed for whole-cell biotransformation using engineered Escherichia coli to produce a methoxy-rhamnosyl derivative of quinizarin, an anthraquinone derivative. The structure of the newly generated derivative from Escherichia coli fermentation was elucidated by liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopic analyses and identified as quinizarin-4-O-α-l-2-O-methylrhamnoside (QRM). Further, the biological impact of methylation was studied by comparing the cytotoxicity of QRM with that of quinizarin against the U87MG, SNU-1, and A375SM cancer cell lines. [ABSTRACT FROM AUTHOR]

An O-methyltransferase SpOMT2884, originating from Streptomyces peucetius ATCC 27952, was cloned, expressed, and applied for the production of target metabolite from Escherichia coli. Biochemical characterization of the 25 kDa recombinant protein by in vitro and in vivo experiments showed that SpOMT2884 was an S-adenosyl-l-methionine-dependent O-methyltransferase. SpOMT2884 catalyzed O-methylation of different classes of flavonoids such as flavones (7,8-dihydroxyflavone (7,8-DHF), luteolin), flavonols (quercetin, rutin), flavanone (naringenin), and isoflavonoids (daidzein, formononetin). Biotransformation of 7,8-DHF, a preferred substrate of SpOMT2884, in a grown-induced culture of E. coli BL21 (DE3) harboring the recombinant pET-28a-SpOMT2884 stoichiometrically converted 7,8-DHF into 7-hydroxy-8-methoxyflavone, which was confirmed by liquid chromatography, mass spectrometry and various nuclear magnetic resonance (NMR) spectroscopy analyses. In order to improve the biotransformation substrate, time and media parameters were optimized and the production was scaled up using a 3-L fermentor. The maximum yield of 7-hydroxy-8-methoxyflavone was 192 μM (52.57 mg/L), representing almost 96% bioconversion within 12 h, when 200 μM of 7,8-DHF was supplemented in the culture. Further, the 7-hydroxy-8-methoxyflavone was purified in large scale and was used as a substrate separately for in vitro glycosylation to produce glucose, galactose and 2-deoxyglucose conjugated at 7th hydroxyl position of 7-hydroxy-8-methoxyflavone. Biological activity showed that 7-hydroxy-8-methoxyflavone had long term cytoprotective and antioxidant effects compared to 7,8-DHF suggesting that methylation enhances the stability of substrate and glycosylation has proved to increase the water solubility. [ABSTRACT FROM AUTHOR]

Adipocyte-derived leucine aminopeptidase (A-LAP) is a novel member of the M1 family of zinc metallopeptidases, which has been reported to play a crucial role in angiogenesis. In the present study, we conducted a target-based screening of natural products and synthetic chemical libraries using the purified enzyme to search novel inhibitors of A-LAP. Amongst several hits isolated, a natural product purpurin was identified as one of the most potent inhibitors of A-LAP from the screening. In vitro enzymatic analyses demonstrated that purpurin inhibited A-LAP activity in a non-competitive manner with a Ki value of 20 M. In addition, purpurin showed a strong selectivity toward A-LAP versus another member of M1 family of zinc metallopeptidase, aminopeptidase N (APN). In angiogenesis assays, purpurin inhibited the vascular endothelial growth factor (VEGF)-induced invasion and tube formation of human umbilical vein endothelial cells (HUVEC). Moreover, purpurin inhibited in vivo angiogenesis in zebrafish embryo without toxicity. These data demonstrate that purpurin is a novel specific inhibitor of A-LAP and could be developed as a new anti-angiogenic agent. [ABSTRACT FROM AUTHOR]