Specific inhibition of an anticancer target, polo-like kinase 1, by allosterically dismantling its mechanism of substrate recognition.

Polo-like kinase 1 (Plk1) is considered an attractive target for anticancer therapy. Over the years, studies on the noncatalytic polo-box domain (PBD) of Plk1 have raised the expectation of generating highly specific protein-protein interaction inhibitors. However, the molecular nature of the canonical PBD-dependent interaction, which requires extensive water network-mediated interactions with its phospholigands, has hampered efforts to identify small molecules suitable for Plk1 PBD drug discovery. Here, we report the identification of the first allosteric inhibitor of Plk1 PBD, called Allopole, a prodrug that can disrupt intracellular interactions between PBD and its cognate phospholigands, delocalize Plk1 from centrosomes and kinetochores, and induce mitotic block and cancer cell killing. At the structural level, its unmasked active form, Allopole-A, bound to a deep Trp-Phe-lined pocket occluded by a latch-like loop, whose adjoining region was required for securely retaining a ligand anchored to the phospho-binding cleft. Allopole-A binding completely dislodged the L2 loop, an event that appeared sufficient to trigger the dissociation of a phospholigand and inhibit PBD-dependent Plk1 function during mitosis. Given Allopole's high specificity and antiproliferative potency, this study is expected to open an unexplored avenue for developing Plk1 PBD-specific anticancer therapeutic agents.

Fluorescent A2A and A3 adenosine receptor antagonists as flow cytometry probes.

Adenosine receptor (AR) ligands are being developed for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A2A and A3ARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled to eight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at A2AAR were Ki 144-316 nM for 10, 12, and 19, and at A3AR affinity of Ki 21.6 nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface A2AAR was imaged using confocal microscopy. Compound 19 MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for A3AR characterization in whole cells by flow cytometry (Kd 11.8 nM), and its bitopic interaction mode with an A3AR homology model was predicted. Given its affinity and selectivity (11-fold vs. A2AAR, ~ 50-fold vs. A1AR and A2BAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the A3AR.

Artificially designed recombinant protein composed of multiple epitopes of foot-and-mouth disease virus as a vaccine candidate.

BACKGROUND: Concerns regarding the safety of inactivated foot-and-mouth disease (FMD) vaccine have been raised since it is produced from cultured live FMD virus (FMDV). To overcome this issue, recombinant protein has been studied as an alternative vaccine. RESULTS AND CONCLUSION: We designed a chimerical multi-epitope recombinant protein (5BT), which is comprised of tandem repeats of five B cell epitopes (residue of VP1 136-162) derived from different FMDV variants and one T-cell epitope (residue of 3A 21-35). To increase solubility and stability of 5BT, it was conjugated with BmpB, the membrane protein B of Brachyspira hyodysenteriae (B5BT). Our results indicated that 5BT was susceptible to degradation by host protease and produced with substantial fraction of inclusion body. The stability and solubility of 5BT was greatly increased by conjugating to BmpB. FMDV specific antibodies were observed in the serum of mice immunized with 5BT and B5BT comparable to inactivated FMD vaccine. Sera from 5BT and B5BT groups also exhibited high epitope-specific antibody titers in peptide specific ELISA, indicating that all five epitopes are exposed to the B cell receptor for the antibody reaction. Thus the multi-epitope recombinant protein designed in this study may be a potential candidate as an alternative vaccine against FMDV epidemic variants.

t-Butyl pyridine and phenyl C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as potent TRPV1 antagonists.

A series of 2-substituted 6-t-butylpyridine and 4-t-butylphenyl C-region analogues of 2-(3-fluoro-4-methylsulfonamidophenyl)propanamides were investigated for hTRPV1 antagonism. The analysis of structure activity relationships indicated that the pyridine derivatives generally exhibited a little better antagonism than did the corresponding phenyl surrogates for most of the series. Among the compounds, compound 7 showed excellent antagonism toward capsaicin activation with Ki=0.1nM and compound 60S demonstrated a strong antiallodynic effect with 83% MPE at 10mg/kg in the neuropathic pain model. The docking study of 7S in our hTRPV1 homology model indicated that the interactions between the A/B-regions of 7S with Tyr511 and the interactions between the t-butyl and ethyl groups in the C-region of 7S with the two hydrophobic binding pockets of hTRPV1 contributed to the high potency.

Influence of Flaxseed Oil on Fecal Microbiota, Egg Quality and Fatty Acid Composition of Egg Yolks in Laying Hens.

Although there have been many attempts to produce ω-3 fatty acid-rich eggs using alpha-linolenic acid (ALA) that is a popular fatty acid in the poultry feed industry, only limited knowledge about the effects of ALA-enriched diets on chicken fecal microbiota is currently available. Herein we examined the changes in the fecal microbiota composition, egg quality traits and fatty acid composition of the egg yolks of laying hens fed ALA-rich flaxseed oil for 8 weeks. The animals fed the experimental diets that contained 0 % (group C), 0.5 % (group T1), and 1.0 % (group T2) of flaxseed oil, respectively, and eggs and feces were obtained for the analyses. ω-3 fatty acids, including ALA, were increased in T1 and T2 compared with C. Furthermore, the freshness of eggs was improved with no side effects on the eggs. The diet also changed the fecal microbiota; Firmicutes was increased in T1 and T2 (48.6 to 83 and 79.6 %) and Bacteroidetes was decreased (40.2 to 8.8 and 4.2 %). Principal coordinate analysis revealed that Lactobacillus, among the 56 examined genera, was the most influenced bacterial group in terms of the fecal microbial community shifts. These results indicate that ALA-rich diets influenced both the egg and fecal microbiota in beneficial manners in laying hens although the association between the fatty acid composition of the egg yolk and the fecal microbiota was not clear. This study is a first step to understand the effect of flaxseed oil as well as intestinal microbiota of laying hens.

Soluble RANKL expression in Lactococcus lactis and investigation of its potential as an oral vaccine adjuvant.

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

Determination of C-Terminal δ-Catenin Responsible for Inducing Dendritic Morphogenesis.

δ-Catenin induces dendritic morphogenesis in several cells and it was reported that deletion of C-terminal 207 amino acid of δ-catenin completely abolished the dendritic morphogenesis. However, exact domain responsible for inducing dendritic morphogenesis in C-terminus of δ-catenin was not mapped. Here, we report that expression of ΔC47 (lacking 47 amino acid of C-terminus: 1-1200), ΔC77 (lacking 77 amino acid of C-terminus: 1-1170) deletion mutants of δ-catenin induced the dendritic morphogenesis of HEK293T and NIH3T3 cells as full-length δ-catenin did. In agreement with previous report, ΔC207 deletion mutant did not show the dendritic morphogenesis of the cells. Interestingly, introducing 107 amino acid deletion of C-terminus (ΔC107 mutant: 1-1140) and 177 amino acid deletion of C-terminus (ΔC177 mutant: 1-1070) showed limited primary and secondary dendritic process and notable spine-like process formation. These results suggest that 1140-1170 amino acid of C-terminal δ-catenin is required for primary and secondary dendrite-like process formation.

A phase III, randomized, double-blind, matched-pairs, active-controlled clinical trial and preclinical animal study to compare the durability, efficacy and safety between polynucleotide filler and hyaluronic acid filler in the correction of crow's feet: a new concept of regenerative filler.

The Rejuran® is a new filler product made from purified polynucleotides. Here we present data from an animal study and a clinical trial to examine the durability, efficacy and safety of the Rejuran® on crow's feet. For the animal study, 25 mice were divided into three groups: Group 1 received phosphate buffered saline (PBS); Group 2 were treated with Yvoire®; and Group 3 were treated with Rejuran®. The durability and efficacy of each treatment were assessed by microscopy and staining. In the clinical trial, 72 patients were randomized to receive Rejuran® treatment for crow's feet on one side and Yvoire-Hydro® on the contralateral side, at a ratio of 1:1. Repeated treatments were performed every two weeks for a total of three times, over a total of 12 weeks' observation. All injections and observations of efficacy and safety were performed by the same two investigators. In the animal study, the Rejuran® group showed similar durability and inflammatory response to the Yvoire® group. Upon efficacy assessment, the Rejuran® group showed the greatest elasticity and collagen composition, and a significant difference in skin surface roughness and wrinkle depth. In the clinical trial, the primary and secondary objective efficacy outcome measure showed no statistical significance between the two groups, and in safety outcomes there were no unexpected adverse effects. Our data suggest that the Rejuran®, as a new regenerative filler, can be useful to reduce wrinkles, by showing evidence for its efficacy and safety.

Formic acid as an alternative reducing agent for the catalytic nitrate reduction in aqueous media.

Formic acid was used for the nitrate reduction as a reductant in the presence of Pd:Cu/gamma-alumina catalysts. The surface characteristics of the bimetallic catalyst synthesized by wet impregnation were investigated by SEM, TEM-EDS. The metals were not distributed homogeneously on the surface of catalyst, although the total contents of both metals in particles agreed well with the theoretical values. Formic acid decomposition on the catalyst surface, its influence on solution pH and nitrate removal efficacy was investigated. The best removal of nitrate (50 ppm) was obtained under the condition of 0.75 g/L catalyst with Pd:Cu ratio (4:1) and two fold excess of formic acid. Formic acid decay patterns resembled those of nitrate removal, showing a linear relationship between k(f) (formic acid decay) and k (nitrate removal). Negligible amount of ammonia was detected, and no nitrite was detected, possibly due to buffering effect of bicarbonate that is in situ produced by the decomposition of formic acid, and due to the sustained release of H2 gas.

Lysyl oxidase-responsive anchoring nanoparticles for modulation of the tumor immune microenvironment.

In the tumor microenvironment, lysyl oxidase (LOX) is known to play a key role in stabilizing the tumor extracellular matrix. Here, we designed LOX-responsive nanoparticles to interact with the collagen matrix of the tumor microenvironment. Collagen-coated and imiquimod-loaded polydopamine nanoparticles (CPN/IQ) could form crosslinked structures with the collagen matrix via LOX. In vitro, anchoring of CPN/IQ nanoparticles was observed with LOX-secreting CT26 cells, but this was blocked by a LOX inhibitor. In CT26 tumor-bearing mice, co-administration of nanoparticles plus the LOX inhibitor did not significantly alter the antitumor efficacy among nanoparticles. In the absence of the LOX inhibitor, however, a single administration of CPN/IQ could provide sustained responsiveness to near-infrared irradiation and ablation of primary tumors. In the primary tumor microenvironment, CPN/IQ lowered the Treg cell population but increased the cytotoxic CD3+CD8+ T cell population. In splenic dendritic cells, CPN/IQ treatment significantly increased the CD11c+CD86+ and CD11c+CD80+ cell populations. In a CT26 distant tumor-rechallenge model, CPN/IQ treatment increased the cytotoxic CD3+CD8+ T cell population and provided 100% survival of mice until 64 days. This study indicates the feasibility of tumor immune microenvironment modulation using LOX-responsive size-transforming nanoparticles. Although we tested the concept in a CT26 cell-derived tumor model, the concept of LOX-responsive collagen matrix- anchoring nanoparticles may be broadly applied to other tumor tissues with LOX-rich tumor microenvironments.

Discovery of Chemical Scaffolds as Lysophosphatidic Acid Receptor 1 Antagonists: Virtual Screening, In Vitro Validation, and Molecular Dynamics Analysis.

Lysophosphatidic acid receptor 1 (LPAR1) is an emerging therapeutic target for numerous human diseases including fibrosis. However, the limited number of available core structures of LPAR1 antagonists has prompted the need for novel chemical templates. In this study, we conducted a high-throughput virtual screening to discover potential new scaffolds. We tested three existing crystal structures alongside an AlphaFold model to evaluate their suitability in structure-based virtual screening, finding that the crystal structures show superior performance compared with the predictive model. Furthermore, we also found that enhancing the precision in the screening process did not necessarily improve the enrichment of hits. From the screening campaign, we identified five structures that were validated using an LPAR1-dependent calcium flux assay. To gain a deeper insight into the protein-ligand interaction, we extensively analyzed the binding modes of these compounds using in silico techniques, laying the groundwork for the discovery of novel LPAR1 antagonists.

Structural Optimization and Anticancer Activity of Polo-like Kinase 1 (Plk1) Polo-Box Domain (PBD) Inhibitors and Their Prodrugs.

Polo-like kinase 1 (Plk1), a mitotic kinase whose activity is widely upregulated in various human cancers, is considered an attractive target for anticancer drug discovery. Aside from the kinase domain, the C-terminal noncatalytic polo-box domain (PBD), which mediates the interaction with the enzyme's binding targets or substrates, has emerged as an alternative target for developing a new class of inhibitors. Various reported small molecule PBD inhibitors exhibit poor cellular efficacy and/or selectivity. Here, we report structure-activity relationship (SAR) studies on triazoloquinazolinone-derived inhibitors, such as 43 (a 1-thioxo-2,4-dihydrothieno[2,3-e][1,2,4]triazolo[4,3-a]pyrimidin-5(1H)-one) that effectively block Plk1, but not Plk2 and Plk3 PBDs, with improved affinity and drug-like properties. The range of prodrug moieties needed for thiol group masking of the active drugs has been expanded to increase cell permeability and mechanism-based cancer cell (L363 and HeLa) death. For example, a 5-thio-1-methyl-4-nitroimidazolyl prodrug 80, derived from 43, showed an improved cellular potency (GI50 4.1 µM). As expected, 80 effectively blocked Plk1 from localizing to centrosomes and kinetochores and consequently induced potent mitotic block and apoptotic cell death. Another prodrug 78 containing 9-fluorophenyl in place of the thiophene-containing heterocycle in 80 also induced a comparable degree of anti-Plk1 PBD effect. However, orally administered 78 was rapidly converted in the bloodstream to parent drug 15, which was shown be relatively stable toward in vivo oxidation due to its 9-fluorophenyl group in comparison to unsubstituted phenyl. Further derivatization of these inhibitors, particularly to improve the systemic prodrug stability, could lead to a new class of therapeutics against Plk1-addicted cancers.

Cell membrane-derived vesicles for delivery of therapeutic agents.

Cell membranes have recently emerged as a new source of materials for molecular delivery systems. Cell membranes have been extruded or sonicated to make nanoscale vesicles. Unlike synthetic lipid or polymeric nanoparticles, cell membrane-derived vesicles have a unique multicomponent feature, comprising lipids, proteins, and carbohydrates. Because cell membrane-derived vesicles contain the intrinsic functionalities and signaling networks of their parent cells, they can overcome various obstacles encountered in vivo. Moreover, the different natural combinations of membranes from various cell sources expand the range of cell membrane-derived vesicles, creating an entirely new category of drug-delivery systems. Cell membrane-derived vesicles can carry therapeutic agents within their interior or can coat the surfaces of drug-loaded core nanoparticles. Cell membranes typically come from single cell sources, including red blood cells, platelets, immune cells, stem cells, and cancer cells. However, recent studies have reported hybrid sources from two different types of cells. This review will summarize approaches for manufacturing cell membrane-derived vesicles and treatment applications of various types of cell membrane-derived drug-delivery systems, and discuss challenges and future directions.

Enhanced Efficacy of Immunization with a Foot-and-Mouth Disease Multi-Epitope Subunit Vaccine Using Mannan-Decorated Inulin Microparticles.

BACKGROUND: Despite the many advantages of recombinant subunit vaccines, they have critical weaknesses that include a low efficacy for promoting cellular and humoral immune responses against antigens because of their poor immunogenicity, and a rapidly cleared properties as a result of proteolytic enzymes in the body. To circumvent these problems, we developed mannan-decorated inulin acetate microparticles (M-IA MPs) that functioned as carriers and adjuvants for immunization with the recombinant foot-and-mouth disease multi-epitope subunit vaccine (M5BT). METHODS: The M5BT-loaded M-IA MPs were obtained by a double-emulsion solvent-evaporation method. Their properties including morphology, size and release ability were determined by field emission scanning electron microscope, dynamic light-scattering spectrophotometer and spectrophotometer. To assess the immunization efficacy of the MPs, mice were immunized with MPs and their sera were analyzed by ELISA. RESULTS: The M-IA MPs obtained by a double-emulsion solvent-evaporation method were spherical and approximately 2-3 µm, and M5BT was encapsulated in the M-IA MPs. The M5BT-loaded M-IA MPs showed higher antigen-specific IgG, IgG1, IgG2a and anti-FMDV antibodies than the M5BT-loaded IA MPs and the Freund's adjuvant as a control. CONCLUSION: The M-IA MPs showed a powerful and multifunctional polymeric system that combined two toll-like receptor agonists compared to the conventional adjuvant.

Discovery of dual-acting opioid ligand and TRPV1 antagonists as novel therapeutic agents for pain.

In order to discover a novel type of analgesic, we investigated dual activity ligands with TRPV1 antagonism and mu-opioid receptor affinity with the goal of eliciting synergistic analgesia while avoiding the side effects associated with single targeting. Based on a combination approach, a series of 4-benzyl-4-(dimethylamino)piperidinyl analogues were designed, synthesized and evaluated for their receptor activities. Among them, compound 49 exhibited the most promising dual-acting activity toward TRPV1 and the mu-opioid receptor in vitro. In vivo,49 displayed potent, dose-dependent antinociceptive activity in both the 1st and 2nd phases in the formalin assay. Consistent with its postulated mechanism, we confirmed that in vivo, as in vitro, compound 49 both antagonized TRPV1 and functioned as a mu-opioid agonist. This result indicates that dual-acting TRPV1 antagonist/mu-opioid ligands can be made and represent a new and promising class of analgesic.

Oral Immunization of FMDV Vaccine Using pH-Sensitive and Mucoadhesive Thiolated Cellulose Acetate Phthalate Microparticles.

Several barriers such as gastric pH, enzymatic degradation and rapid transit should be overcome to orally deliver antigens for taking up by epithelial microfold cells in Peyer's patches of small intestine. To solve the above mentioned problems, we designed pH-sensitive and mucoadhesive polymeric microparticles (MPs) prepared by double emulsion technique using cellulose acetate phthalate (CAP) to enhance immune response of foot-and-mouth disease (FMD) virus (FMDV) subunit vaccine. Thiolation of CAP improved mucoadhesive property of CAP to prolong the MPs transit time through the gastrointestinal tract. Thiolated CAP (T-CAP) also slowed down antigen release in acidic pH of stomach but released more antigens in neutral pH of small intestine due to the pH-sensitivity of the T-CAP. Oral immunization of a chimerical multi-epitope recombinant protein as the FMD subunit vaccine via T-CAP MPs effectively delivered the vaccine to Peyer's patches eliciting mucosal IgA response. It will make a step forward into a promising oral subunit vaccine development in livestock industry.

Prohibition of antibiotic growth promoters has affected the genomic profiles of Lactobacillus salivarius inhabiting the swine intestine.

After the introduction of a ban on the use of antibiotic growth promoters (AGPs) for livestock, the feeding environment, including the composition of animal intestinal microbiota, has changed rapidly. We hypothesized that the microbial genomes have also been affected by this legal prohibition, and investigated an important member of the swine gut microbiota, Lactobacillus salivarius, with a pan-genomic approach. Here, we isolated 21 L. salivarius strains composed of 6 strains isolated before the AGP prohibition (SBPs) and 15 strains isolated after the AGP prohibition (SAPs) at an interval of a decade, and the draft genomes were generated de novo. Several genomic differences between SBPs and SAPs were identified, although the number and function of antibiotic resistance genes were not different. SBPs showed larger genome size and a higher number of orthologs, as well as lower genetic diversity, than SAPs. SBPs had genes associated with the utilization of L-rhamnose and D-tagatose for energy production. Because these sugars are also used in exopolysaccharide (EPS) synthesis, we tried to identify differences in biofilm formation-associated genes. The genes for the production of EPSs and extracellular proteins were different in terms of amino acid sequences. Indeed, SAPs formed dense biofilm and survived better than SBPs in the swine intestinal environment. These results suggest that SAPs have evolved and adapted to protect themselves from new selection pressure of the swine intestinal microenvironment by forming dense biofilms, adopting a distinct antibiotic resistance strategy. This finding is particularly important to understand the evolutionary changes in host-microbe interaction and provide detailed insight for the development of effective probiotics for livestock.

Radiographic evaluation of vessel count and density with quantitative magnetic resonance imaging during external breast expansion in Asian women: A prospective clinical trial.

BACKGROUND: Breast augmentation with fat transfer does not bear the risks associated with silicone implantation. The method can potentially be especially useful in Asian women, who often reject augmentation mammoplasty with implants. This prospective clinical trial evaluated the effects of external breast expansion on breast density and vessel count using magnetic resonance imaging. METHODS: Thirty-four enrolled patients were instructed to apply one of two devices, the conventional BRAVA device (used in the AESTES trial) or a novel external expansion device (EVERA) designed for Asian women, continuously for 8 h per day for 12 weeks. For external expansion, the pressure was set to 25 mmHg. Follow-up examinations were performed for 4 weeks after completion of the expansion. The ratio between the fibroglandular and adipose tissues of the breast was measured using T1-weighted MRI, and the number of vessels in the breast tissue was determined before and after the treatment by contrast MRI. Additionally, the volume of the breast was measured by laser scanning before, during, and after the device application. The obtained measurements were compared within and between the groups at different time points. RESULTS: Six patients dropped out, while 28 completed the trial without major side effects or adverse events. External expansion significantly increased breast vessel count in both the EVERA and AESTES groups (p = 0.019, p = 0.022). However, it did not significantly change breast density in either group (p = 0.186, p = 0.638). No significant intergroup differences were noted in vessel count (p = 0.874) or density (p = 0.482). Breast volume increases after 12 weeks of application were statistically significant in both groups, with mean changes of 81 ± 22 cc (AESTES) and 98 ± 30 cc (EVERA) (p < 0.001 in both cases). CONCLUSIONS: External expansion resulted in a marked increase in breast vessel count but did not affect breast density. The observed increase in breast volume can be considered substantial for Asian women. LEVEL OF EVIDENCE: Level II, therapeutic study.

A Multicenter Noncomparative Clinical Study on Midface Rejuvenation Using a Nonabsorbable Polypropylene Mesh: Evaluation of Efficacy and Safety.

BACKGROUND: Facial rejuvenation can be achieved using a variety of techniques. Since minimally invasive procedures for face lifting have become popular because of their convenience and short operating time, numerous minimally invasive surgical procedures have been developed. In this study, a nonabsorbable polypropylene mesh is introduced as a new face lifting instrument, with the nasolabial fold as the main target area. In this paper, we report the efficacy and safety of a polypropylene mesh in midface rejuvenation. METHODS: Thirty-three subjects with moderate-to-severe nasolabial folds were enrolled from two medical institutions for a noncomparative single-sample study. A mesh was inserted above the superficial muscular aponeurotic system layer, reaching the nasolabial folds through a temporal scalp incision. After 3 weeks, the temporal end of the mesh was pulled to provide a lifting effect. Then, the mesh was fixed to the deep temporal fascia using nonabsorbable sutures. To evaluate efficacy, we compared the scores on the Wrinkle Severity Rating Scale and a visual analog scale for patient satisfaction between the baseline and 7 weeks postoperatively. In addition, we evaluated safety based on the incidence of adverse events. RESULTS: The treatment was deemed effective at improving wrinkles in 23 of 28 cases, and patient satisfaction improved significantly during the study period. There were seven cases of skin or subcutaneous tissue complications, including edema and erythema, but there were no suspected serious adverse events. CONCLUSIONS: Face lifting using a nonabsorbable mesh can improve nasolabial folds without serious adverse effects. Thus, this technique is safe and effective for midface rejuvenation.

Lichen secondary metabolites in Flavocetraria cucullata exhibit anti-cancer effects on human cancer cells through the induction of apoptosis and suppression of tumorigenic potentials.

Lichens are symbiotic organisms which produce distinct secondary metabolic products. In the present study, we tested the cytotoxic activity of 17 lichen species against several human cancer cells and further investigated the molecular mechanisms underlying their anti-cancer activity. We found that among 17 lichens species, F. cucullata exhibited the most potent cytotoxicity in several human cancer cells. High performance liquid chromatography analysis revealed that the acetone extract of F. cucullata contains usnic acid, salazinic acid, Squamatic acid, Baeomycesic acid, d-protolichesterinic acid, and lichesterinic acid as subcomponents. MTT assay showed that cancer cell lines were more vulnerable to the cytotoxic effects of the extract than non-cancer cell lines. Furthermore, among the identified subcomponents, usnic acid treatment had a similar cytotoxic effect on cancer cell lines but with lower potency than the extract. At a lethal dose, treatment with the extract or with usnic acid greatly increased the apoptotic cell population and specifically activated the apoptotic signaling pathway; however, using sub-lethal doses, extract and usnic acid treatment decreased cancer cell motility and inhibited in vitro and in vivo tumorigenic potentials. In these cells, we observed significantly reduced levels of epithelial-mesenchymal transition (EMT) markers and phosphor-Akt, while phosphor-c-Jun and phosphor-ERK1/2 levels were only marginally affected. Overall, the anti-cancer activity of the extract is more potent than that of usnic acid alone. Taken together, F. cucullata and its subcomponent, usnic acid together with additional component, exert anti-cancer effects on human cancer cells through the induction of apoptosis and the inhibition of EMT.