Iron oxide nanozymes stabilize stannous fluoride for targeted biofilm killing and synergistic oral disease prevention.

Dental caries (tooth decay) is the most prevalent human disease caused by oral biofilms, affecting nearly half of the global population despite increased use of fluoride, the mainstay anticaries (tooth-enamel protective) agent. Recently, an FDA-approved iron oxide nanozyme formulation (ferumoxytol, Fer) has been shown to disrupt caries-causing biofilms with high specificity via catalytic activation of hydrogen peroxide, but it is incapable of interfering with enamel acid demineralization. Here, we find notable synergy when Fer is combined with stannous fluoride (SnF 2 ), markedly inhibiting both biofilm accumulation and enamel damage more effectively than either alone. Unexpectedly, our data show that SnF 2 enhances the catalytic activity of Fer, significantly increasing reactive oxygen species (ROS) generation and antibiofilm activity. We discover that the stability of SnF 2 (unstable in water) is markedly enhanced when mixed with Fer in aqueous solutions without any additives. Further analyses reveal that Sn 2+ is bound by carboxylate groups in the carboxymethyl-dextran coating of Fer, thus stabilizing SnF 2 and boosting the catalytic activity. Notably, Fer in combination with SnF 2 is exceptionally effective in controlling dental caries in vivo , preventing enamel demineralization and cavitation altogether without adverse effects on the host tissues or causing changes in the oral microbiome diversity. The efficacy of SnF 2 is also enhanced when combined with Fer, showing comparable therapeutic effects at four times lower fluoride concentration. Enamel ultrastructure examination shows that fluoride, iron, and tin are detected in the outer layers of the enamel forming a polyion-rich film, indicating co-delivery onto the tooth surface. Overall, our results reveal a unique therapeutic synergism using approved agents that target complementary biological and physicochemical traits, while providing facile SnF 2 stabilization, to prevent a widespread oral disease more effectively with reduced fluoride exposure.

Comparative transcriptome and metabolome analyses of four Panax species explore the dynamics of metabolite biosynthesis.

Background: The genus Panax in the Araliaceae family has been used as traditional medicinal plants worldwide and is known to biosynthesize ginsenosides and phytosterols. However, genetic variation between Panax species has influenced their biosynthetic pathways is not fully understood. Methods: Simultaneous analysis of transcriptomes and metabolomes obtained from adventitious roots of two tetraploid species (Panax ginseng and P. quinquefolius) and two diploid species (P. notoginseng and P. vietnamensis) revealed the diversity of their metabolites and related gene expression profiles. Results: The transcriptome analysis showed that 2,3-OXIDOSQUALENE CYCLASEs (OSCs) involved in phytosterol biosynthesis are upregulated in the diploid species, while the expression of OSCs contributing to ginsenoside biosynthesis is higher in the tetraploid species. In agreement with these results, the contents of dammarenediol-type ginsenosides were higher in the tetraploid species relative to the diploid species. Conclusion: These results suggest that a whole-genome duplication event has influenced the triterpene biosynthesis pathway in tetraploid Panax species during their evolution or ecological adaptation. This study provides a basis for further efforts to explore the genetic variation of the Panax genus.

Secondary Fermented Extract of Chaga-Cheonggukjang Attenuates the Effects of Obesity and Suppresses Inflammatory Response in the Liver and Spleen of High-Fat Diet-Induced Obese Mice.

Cheonggukjang and chaga mushrooms have numerous health benefits, and have been used in alternative medicine. Therefore, a powder mixture of 98: Cheonggukjang and 2: Chaga extracts was fermented with Lactobacillus acidophilus KCTC3925 (FCC) and its anti-obesity effects in high-fat diet (HFD)-induced obese mice were determined. Five-week-old male ICR mice were fed a normal diet or HFD in the presence or absence of 3% and 5% FCC by weight (n = 10 per group). After 12 weeks, the mice were sacrificed, and the serum and tissue samples were collected for analysis. Body weight and epididymal fat pad weight were significantly lowered in the 3% and 5% FCC groups compared with those in the HFD control group (p < 0.01). FCC supplementation suppressed serum triglyceride and increased serum HDL-C levels (p < 0.01). Serum GOT, GPT, and leptin levels, hepatic COX-2 mRNA expression, and splenic COX-2 and IL-4 mRNA expression were significantly higher in the HFD groups than in the control group (p > 0.05); however, except for splenic IL-4 levels, the increases were significantly attenuated by FCC supplementation. Expression of ICAM-1, an aortic inflammatory marker, was significantly increased in the HFD group; this effect was suppressed in the 3% FCC group (p < 0.01) but not in the 5% FCC group. FCC suppressed the body weight and epididymal fat pad weight gain, as well as inflammatory responses in the liver and spleen of HFD-fed mice. Thus, FCC supplementation will be beneficial for the treatment of obesity-related effects.

Chest radiography surveillance for lung cancer: Results from a National Health Insurance database in South Korea.

BACKGROUND: Lung cancer screening with low-dose computed tomography reduced mortality in selected high risk patients. However, the use of chest radiography for lung cancer screening in Asian populations is still controversial. We investigated the effectiveness of chest radiographic surveillance using a nationwide health service data in South Korea. METHODS: Data from the Korean National Health Insurance Service examinee cohort of 2004 to 2013 were examined, and 63,228 patients with lung cancer were identified, 38,494 (57%) of whom underwent chest radiography screening. The others did not undergo lung cancer screening and were considered as a control group. Clinical data including age, smoking, screening intervals, lung cancer stages, treatments, and survival were collected. Survival gain from surveillance after adjustment for lead-time bias based on the sojourn time was calculated. Cox-proportional hazard analyses were performed to evaluate the effectiveness of screening and to determine the appropriate screening interval for chest radiography surveillance. RESULTS: Early lung cancer was found in 38% of patients receiving chest radiography versus 26% of those without surveillance. A patient age of more than 65 years (hazard ratio [HR], 1.53; 95% confidence limits [CL], 1.50-1.56), male (HR, 1.66; 95% CL, 1.62-1.70), and high lung cancer stages at the time of diagnosis were independent factors associated with mortality (each, P < 0.001). Chest radiography surveillance was a factor for decreasing mortality in female (HR, 0.81; 95% CL, 0.77-0.84, P < 0.001), with mortality reduction of 10% at the 3- and 5-year survival time-points. In female patients, chest radiography surveillance at intervals of less than 3 years was an independent predictor of improved survival. CONCLUSIONS: Surveillance chest radiography increased survival in a female screened population in South Korea. Chest radiography intervals of less than 3 years may help to detect lung cancer in female patients.

Converting organosulfur compounds to inorganic polysulfides against resistant bacterial infections.

The use of natural substance to ward off microbial infections has a long history. However, the large-scale production of natural extracts often reduces antibacterial potency, thus limiting practical applications. Here we present a strategy for converting natural organosulfur compounds into nano-iron sulfides that exhibit enhanced antibacterial activity. We show that compared to garlic-derived organosulfur compounds nano-iron sulfides exhibit an over 500-fold increase in antibacterial efficacy to kill several pathogenic and drug-resistant bacteria. Furthermore, our analysis reveals that hydrogen polysulfanes released from nano-iron sulfides possess potent bactericidal activity and the release of polysulfanes can be accelerated by the enzyme-like activity of nano-iron sulfides. Finally, we demonstrate that topical applications of nano-iron sulfides can effectively disrupt pathogenic biofilms on human teeth and accelerate infected-wound healing. Together, our approach to convert organosulfur compounds into inorganic polysulfides potentially provides an antibacterial alternative to combat bacterial infections.

Identification of candidate UDP-glycosyltransferases involved in protopanaxadiol-type ginsenoside biosynthesis in Panax ginseng.

Ginsenosides are dammarane-type or triterpenoidal saponins that contribute to the various pharmacological activities of the medicinal herb Panax ginseng. The putative biosynthetic pathway for ginsenoside biosynthesis is known in P. ginseng, as are some of the transcripts and enzyme-encoding genes. However, few genes related to the UDP-glycosyltransferases (UGTs), enzymes that mediate glycosylation processes in final saponin biosynthesis, have been identified. Here, we generated three replicated Illumina RNA-Seq datasets from the adventitious roots of P. ginseng cultivar Cheongsun (CS) after 0, 12, 24, and 48 h of treatment with methyl jasmonate (MeJA). Using the same CS cultivar, metabolomic data were also generated at 0 h and every 12-24 h thereafter until 120 h of MeJA treatment. Differential gene expression, phylogenetic analysis, and metabolic profiling were used to identify candidate UGTs. Eleven candidate UGTs likely to be involved in ginsenoside glycosylation were identified. Eight of these were considered novel UGTs, newly identified in this study, and three were matched to previously characterized UGTs in P. ginseng. Phylogenetic analysis further asserted their association with ginsenoside biosynthesis. Additionally, metabolomic analysis revealed that the newly identified UGTs might be involved in the elongation of glycosyl chains of ginsenosides, especially of protopanaxadiol (PPD)-type ginsenosides.

Suppressive Effect of Fermented Angelica tenuissima Root Extract against Photoaging: Possible Involvement of Hemeoxygenase-1.

Angelica tenuissima root has historically been used as a traditional medicine in Korea. Previous studies have identified the anti-melanogenic effects of the extract of A. tenuissima root fermented by Aspergillus oryzae (FAT). This study investigated the protective effects of FAT against ultraviolet light B exposure (UVB; 30 mJ/cm2) in HaCaT (human keratinocyte) or Hs68 (human foreskin fibroblast) skin cells. FAT treatment was able to stimulate wound healing rate at the basal condition. FAT also favored the maintenance and/or improvement of extracellular matrix impairment caused by UVB irradiation through: 1) upregulation of procollagen Type-1 synthesis and secretion; 2) suppression of MMP-1 and elastase expression. FAT was able to play a role in the attenuation of inflammatory responses caused by UVB irradiation via upregulation of photo-protective hemeoxygease-1 and suppression of proinflammatory cyclooxygenase-2 expression. After further verification of the anti-photoaging potential of FAT, it could be utilized as an effective ingredient in anti-aging and anti-wrinkle cosmetics.

Decursin and Z-Ligustilide in Angelica tenuissima Root Extract Fermented by Aspergillus oryzae Display Anti-Pigment Activity in Melanoma Cells.

The anti-melanogenic effects of the extract of Angelica tenuissima (AT) root and the extract of AT root fermented by Aspergillus oryzae (FAT) were investigated. These effects were determined by measuring the inhibitory activity of AT and FAT on melanin production in B16F10 melanocytes and with in vitro tyrosinase activity assays. The AT extract inhibited melanin production at concentrations above 250 µg/ml, and this inhibitory effect was significantly enhanced by the fermentation process with A. oryzae. HPLC analysis resulted in the isolation of two active compounds from both the AT and FAT extracts. Their chemical structures were identified as decursin and Z-ligustilide through comparison with previously reported NMR data. The decursin and Z-ligustilide contents were increased in the FAT extract and could be responsible for its enhanced inhibitory effects on melanin production and tyrosinase activity compared with that of the AT extract.

The suppressive effect of the three-herb extract mixture on vascular and liver inflammation in atherogenic diet with high fructose-fed mice.

CONTEXT: Cynanchum wilfordii (Maximowicz) Hemsley (Apocynaceae), Arctium lappa L. var. rubescens Frivald (Asteraceae) and Dioscorea opposite Thunb (Dioscoreaceae) root extracts have been widely used as an alternative for intervening obesity. OBJECTIVES: The synergistic effect of three-herb mixture of C. wilfordii, A. lappa and D. opposita was determined on aortic and liver inflammatory responses. MATERIALS AND METHODS: CWE, ALE and DOE were prepared from the root of C. wilfordii, A. lappa and D. opposite by 70% ethanol extraction, respectively. CADE was prepared using a powder mixture of 2 CWE:1 ALE:1 DOE. C57BL/6 mice were fed an atherogenic diet combined with 10% fructose (ATHFR) in the presence of 200 mg/kg/day CWE, ALE, DOE or CADE for 8 weeks (each group, n = 6). Biochemical profiles, protein expression of vascular cell adhesion molecule-1 (VCAM-1) on the aorta and liver were determined. RESULTS: CADE could significantly suppress the protein expression of VCAM-1 in both the aorta and liver (80% reduction) compared to ATHFR-fed mice. Impairment of liver function was significantly ameliorated by CADE supplement, as determined by GOT (60% reduction) and GPT (51% reduction) levels. CONCLUSIONS: CADE should be considered when developing medications to suppress the vascular and liver inflammatory responses for individuals who are either non-responsive or resistant to lipid-lowering drugs.

Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.

The complete chloroplast genome sequence of Korean Lonicera japonica and intra-species diversity.

Lonicera japonica is a traditional medicinal plant well known for its anti-inflammatory effect. The complete chloroplast genome sequence of L. japonica collected from Korea was obtained by de novo assembly using whole genome sequence data. The chloroplast genome is 155,060 bp in length, containing 88,853 bp in a large single copy (LSC), 18,653 bp in a small single copy (SSC) and 23,777 bp in a pair of inverted repeats (IRs). A total of 112 genes including 78 protein-coding genes and 34 structural RNA genes were identified. The sequence comparison of two L. japonica collected from Korea and China revealed 48 single nucleotide polymorphisms (SNPs) and 45 insertions/deletions (InDels). In addition, phylogenetic analysis represented intraspecific diversity within L. japonica species collected in Korea and China.

Biotransformation of Pueraria lobata Extract with Lactobacillus rhamnosus vitaP1 Enhances Anti-Melanogenic Activity.

Isoflavone itself is less available in the body without the aid of intestinal bacteria. In this study, we searched for isoflavone-transforming bacteria from human fecal specimens (n = 14) using differential selection media. Isoflavone-transforming activity as the production of dihydrogenistein and dihydrodaidzein was assessed by high-performance liquid chromatography and we found Lactobacillus rhamnosus, named L. rhamnosus vitaP1, through 16S rDNA sequence analysis. Extract from Pueraria lobata (EPL) and soy hypocotyl extract were fermented with L. rhamnosus vitaP1 for 24 and 48 h at 37°C. Fermented EPL (FEPL) showed enhanced anti-tyrosinase activity and antioxidant capacities, important suppressors of the pigmentation process, compared with that of EPL (p < 0.05). At up to 500 µg/ml of FEPL, there were no significant cell cytotoxicity and proliferation on B16-F10 melanoma cells. FEPL (100 µg/ml) could highly suppress the content of melanin and melanosome formation in B16-F10 cells. In summary, Lactobacillus rhamnosus vitaP1 was found to be able to biotransform isoflavones in EPL. FEPL showed augmented anti-melanogenic potential.

Integrated Transcriptomic and Metabolomic Analysis of Five Panax ginseng Cultivars Reveals the Dynamics of Ginsenoside Biosynthesis.

Panax ginseng C.A. Meyer is a traditional medicinal herb that produces bioactive compounds such as ginsenosides. Here, we investigated the diversity of ginsenosides and related genes among five genetically fixed inbred ginseng cultivars (Chunpoong [CP], Cheongsun [CS], Gopoong [GO], Sunhyang [SH], and Sunun [SU]). To focus on the genetic diversity related to ginsenoside biosynthesis, we utilized in vitro cultured adventitious roots from the five cultivars grown under controlled environmental conditions. PCA loading plots based on secondary metabolite composition classified the five cultivars into three groups. We selected three cultivars (CS, SH, and SU) to represent the three groups and conducted further transcriptome and gas chromatography-mass spectrometry analyses to identify genes and intermediates corresponding to the variation in ginsenosides among cultivars. We quantified ginsenoside contents from the three cultivars. SH had more than 12 times the total ginsenoside content of CS, with especially large differences in the levels of panaxadiol-type ginsenosides. The expression levels of genes encoding squalene epoxidase (SQE) and dammarenediol synthase (DDS) were also significantly lower in CS than SH and SU, which is consistent with the low levels of ginsenoside produced in this cultivar. Methyl jasmonate (MeJA) treatment increased the levels of panaxadiol-type ginsenosides up to 4-, 13-, and 31-fold in SH, SU, and CS, respectively. MeJA treatment also greatly increased the quantity of major intermediates and the expression of the underlying genes in the ginsenoside biosynthesis pathway; these intermediates included squalene, 2,3-oxidosqualene, and dammarenediol II, especially in CS, which had the lowest ginsenoside content under normal culture conditions. We conclude that SQE and DDS are the most important genetic factors for ginsenoside biosynthesis with diversity among ginseng cultivars.

Hepatic anti-inflammatory effect of hexane extracts of Dioscorea batatas Decne: Possible suppression of toll-like receptor 4-mediated signaling.

The hepatic anti-inflammatory potential of hexane extracts of Dioscorea batatas Decne edible part (EDH-1e) and bark (EDH-2b) were investigated in Western-type diet-fed apolipoprotein E null [ApoE (-/-)] mice and HepG2 cells. EDH-1e and EDH-2b suppressed the increased levels of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, transforming growth factor beta 1 (TGF-ß1), vascular cell adhesion protein 1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1), and reduced infiltration of monocytes into liver tissue. The protein levels of Toll-like receptor 4 (TLR4) were also downregulated by EDH-1e and EDH-2b treatment as were the levels of activator protein 1 (AP-1), c-fos, and c-jun in the livers from Western-type diet-fed ApoE (-/-) mice and in lipopolysaccharide-stimulated HepG2 cells. Taken together, EDH-1e and EDH-2b attenuated hepatic inflammation and fibrosis via suppression of the TLR4-AP1-mediated signaling pathway.

Cynanchum wilfordii Radix attenuates liver fat accumulation and damage by suppressing hepatic cyclooxygenase-2 and mitogen-activated protein kinase in mice fed with a high-fat and high-fructose diet.

Excessive consumption of fat and fructose augments the pathological progression of nonalcoholic fatty liver disease through hepatic fibrosis, inflammation, and hepatic de novo lipogenesis. We hypothesized that supplementation with Cynanchum wilfordii extract (CWE) decreases fat accumulation in the liver by suppressing cyclooxygenase-2 (COX-2), the nuclear translocation of nuclear factor κB (NF-κB), and p38 mitogen-activated protein kinase (MAPK). The beneficial effect of CWE was evaluated in a murine model of nonalcoholic fatty liver disease. Mice were fed either a normal diet or an atherogenic diet with fructose (ATHFR) in the presence or absence of CWE (50, 100, or 200 mg/kg; n=6/group). Treatment with ATHFR induced a hepatosplenomegaly-like condition (increased liver and spleen weight); this pathological change was attenuated in the presence of CWE. The ATHFR group exhibited impaired liver function, as evidenced by increased blood levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, fat accumulation in the liver, and lipid profiles. Supplementation of CWE (100 and 200 mg/kg, P<.05) ameliorated these impaired liver functions. Atherogenic diet with fructose increased the protein levels of COX-2 and p38 MAPK, as well as the nuclear translocation of NF-κB. These signaling pathways, which are associated with the inflammatory response, were markedly suppressed after CWE treatment (100 and 200 mg/kg). In summary, CWE supplementation reduced high-fat and high-fructose diet-induced fat accumulation and damage in the liver by suppressing COX-2, NF-κB, and p38 MAPK.

Topical delivery of low-cost protein drug candidates made in chloroplasts for biofilm disruption and uptake by oral epithelial cells.

Protein drugs (PD) are minimally utilized in dental medicine due to high cost and invasive surgical delivery. There is limited clinical advancement in disrupting virulent oral biofilms, despite their high prevalence in causing dental caries. Poor efficacy of antimicrobials following topical treatments or to penetrate and disrupt formed biofilms is a major challenge. We report an exciting low-cost approach using plant-made antimicrobial peptides (PMAMPs) retrocyclin or protegrin with complex secondary structures (cyclic/hairpin) for topical use to control biofilms. The PMAMPs rapidly killed the pathogen Streptococcus mutans and impaired biofilm formation following a single topical application of tooth-mimetic surface. Furthermore, we developed a synergistic approach using PMAMPs combined with matrix-degrading enzymes to facilitate their access into biofilms and kill the embedded bacteria. In addition, we identified a novel role for PMAMPs in delivering drugs to periodontal and gingival cells, 13-48 folds more efficiently than any other tested cell penetrating peptides. Therefore, PDs fused with protegrin expressed in plant cells could potentially play a dual role in delivering therapeutic proteins to gum tissues while killing pathogenic bacteria when delivered as topical oral formulations or in chewing gums. Recent FDA approval of plant-produced PDs augurs well for clinical advancement of this novel concept.

Biosynthetic potential of sesquiterpene synthases: product profiles of Egyptian Henbane premnaspirodiene synthase and related mutants.

The plant terpene synthase (TPS) family is responsible for the biosynthesis of a variety of terpenoid natural products possessing diverse biological functions. TPSs catalyze the ionization and, most commonly, rearrangement and cyclization of prenyl diphosphate substrates, forming linear and cyclic hydrocarbons. Moreover, a single TPS often produces several minor products in addition to a dominant product. We characterized the catalytic profiles of Hyoscyamus muticus premnaspirodiene synthase (HPS) and compared it with the profile of a closely related TPS, Nicotiana tabacum 5-epi-aristolochene synthase (TEAS). The profiles of two previously studied HPS and TEAS mutants, each containing nine interconverting mutations, dubbed HPS-M9 and TEAS-M9, were also characterized. All four TPSs were compared under varying temperature and pH conditions. In addition, we solved the X-ray crystal structures of TEAS and a TEAS quadruple mutant complexed with substrate and products to gain insight into the enzymatic features modulating product formation. These informative structures, along with product profiles, provide new insight into plant TPS catalytic promiscuity.

Influence of Degree-of-Polymerization and Linkage on the Quantification of Proanthocyanidins using 4-Dimethylaminocinnamaldehyde (DMAC) Assay.

Proanthocyanidins (PACs) are naturally occurring flavonoids possessing health beneficial bioactivities. Their quantification often utilizes the 4-dimethylaminocinnamaldehyde (DMAC) spectrophotometric assay with the assumption that molar absorption coefficients (MACs) are similar across the various PAC species. To assess the validity of this assumption, individual PAC monomers and oligomers were examined for their absorbance response with DMAC. Our results have shown that PAC dimers and trimers with interflavan linkage variations exhibited differential absorbance response. Absence of A-type linkage between the terminal and second units in PAC molecule not only impacts absorbance intensity at 640 nm but also elicits a prominent secondary 440 nm absorbance peak. Cranberry (A-type) and cocoa (B-type) oligomeric PACs exhibited differential absorbance (MACs) relationship with degree-of-polymerization. Thus, PAC structural variations have considerable impact on the resulting MAC. The use of DMAC assay in PAC quantification, especially in comparing across specific oligomers and compositions, should not assume MACs are similar.

Hybrid PET/optical imaging of integrin αVß3 receptor expression using a (64)Cu-labeled streptavidin/biotin-based dimeric RGD peptide.

BACKGROUND: Hybrid PET/optical imaging provides quantitative and complementary information for diagnosis of tumors. Herein, we developed a (64)Cu-labeled AlexaFluor 680-streptavidin ((AF)SAv)/biotin-based dimeric cyclic RGD peptide (RGD2) for hybrid PET/optical imaging of integrin αVß3 expression. METHODS: (64)Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-(AF)SAv/biotin-PEG-RGD2 was prepared by formation of a complex comprising DOTA-(AF)SAv and biotin-PEG-RGD2, followed by radiolabeling with (64)Cu. Receptor binding studies of DOTA-(AF)SAv/biotin-PEG-RGD2 were performed using U87MG cells and (125)I-RGDyK as the radioligand, and cellular uptake studies of (64)Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 were also performed. MicroPET imaging followed by optical imaging of U87MG tumor-bearing mice was acquired after injection of the hybrid probe, and region of interest (ROI) analysis of tumors was performed. Ex vivo PET/optical imaging and biodistribution studies of the major tissues were performed after the in vivo imaging, and immunofluorescence staining of the tumor tissue sections was carried out. RESULTS: (64)Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 was prepared in 52.1 ± 5.4 % radiochemical yield and with specific activity of 1.0 ± 0.1 GBq/mg. Receptor binding studies showed that DOTA-(AF)SAv/biotin-PEG-RGD2 had higher binding affinity for integrin αVß3 than RGD2, reflecting a possible polyvalency effect. Moreover, the hybrid probe revealed time-dependent uptake by U87MG cells. In a microPET/optical imaging study, the hybrid probe demonstrated high accumulation in tumors; ROI analysis revealed 2.7 ± 0.2 % ID/g at 1 h and 4.7 ± 0.2 % ID/g at 21 h after injection, and subsequently acquired optical images showed tumors with strong fluorescence intensity. Ex vivo PET/optical images of the major tissues confirmed the in vivo imaging data, and biodistribution studies demonstrated high and specific uptake in tumors (4.8 ± 0.1 % ID/g). Immunofluorescence staining showed the formation of new blood vessels in tumor tissues, suggesting that the tumor uptake was due to specific binding of the hybrid probe to integrin αVß3 expressed on tumor cells. CONCLUSIONS: These results indicate that a (64)Cu-DOTA-(AF)SAv/biotin-PEG-RGD2 is able to provide quantitative information on hybrid PET/optical imaging of integrin αVß3 expression.

Theobromine inhibits differentiation of 3T3-L1 cells during the early stage of adipogenesis via AMPK and MAPK signaling pathways.

Obesity is characterized by hypertrophy and/or by the differentiation or adipogenesis of pre-existing adipocytes. In this study, we investigated the inhibitory effects of theobromine, a type of alkaloid in cocoa, on adipocyte differentiation of 3T3-L1 preadipocytes and its mechanisms of action. Theobromine inhibited the accumulation of lipid droplets, the expression of PPARγ and C/EBPα, and the mRNA expression of aP2 and leptin. The inhibition of adipogenic differentiation by theobromine occurred primarily in the early stages of differentiation. In addition, theobromine arrested the cell cycle at the G0/G1 phase and regulated the expressions of CDK2, p27, and p21. Theobromine treatment increased AMPK phosphorylation and knockdown of AMPKα1/α2 prevented the ability of theobromine to inhibit PPARγ expression in the differentiating 3T3-L1 cells. Theobromine reduced the phosphorylation of ERK and JNK. Moreover, the secretion and the mRNA level of TNF-α and IL-6 were inhibited by theobromine treatment. These data suggest that theobromine inhibits adipocyte differentiation during the early stages of adipogenesis by regulating the expression of PPARγ and C/EBPα through the AMPK and ERK/JNK signaling pathways in 3T3-L1 preadipocytes.