The Development of an Extraction Method for Simultaneously Analyzing Fatty Acids in Macroalgae Using SPE with Derivatization for LC-MS/MS.

Fatty acid analysis is an essential step in evaluating the potential of macroalgae for biodiesel production. An extraction method was developed to simultaneously analyze up to five types of biodiesel-fuel-related fatty acids (myristic acid, palmitic acid, cis-palmitvaccenic acid, stearic acid, and oleic acid) in macroalgae using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Lypophilization and solid-phase extraction (SPE) techniques were applied to improve the extraction efficiency and effectively purify samples. The optimal conditions for SPE were set by comparing the recoveries according to the various solvent conditions for each step (loading, washing, and elution). In addition, the introduction of trimethylaminoethyl (TMAE) derivatives to the hydroxyl group of the target analyte increased the ionization efficiency and sensitivity. The derivatized samples were analyzed using the LC-MS/MS method with electrospray ionization in the positive and multiple-reaction monitoring modes. The target analytes were separated and detected within 13.5 min using a CAPCELL PAK C18 MGII S3 column. Gradient elution was performed using distilled water and acetonitrile containing 5 mM ammonium acetate. This method offers a reliable and sensitive tool for the analysis of macroalgae samples for their potential use in biodiesel production. To the best of our knowledge, this is the first report on the simultaneous determination of fatty acids in macroalgae using LC-MS/MS with TMAE derivatization.

Senescent melanocytes driven by glycolytic changes are characterized by melanosome transport dysfunction.

Rationale: Senescent melanocytes accumulate in photoaged skin and are closely related to skin aging. A better understanding of the molecular characteristics of senescent melanocytes may be the key to controlling skin aging. Methods: We have developed an in vitro model of senescence in melanocytes using UV irradiation and investigated the functional characteristics and molecular mechanisms underlying senescence in UV-irradiated melanocytes. Results: We have highlighted that in vitro senescent melanocytes are characterized by melanosome transport dysfunction resulting in melanin accumulation. The defective melanosome transport was confirmed with the ultrastructural characterization of both in vitro UV-induced senescent melanocytes and in vivo melanocytes of hypopigmented aging skin. A single-cell transcriptomic analysis revealed that the glycolytic metabolism pathway appeared to be significantly upregulated in most senescent phenotypes. Furthermore, the inhibition of glycolysis by pharmacological compounds mitigates the pro-aging effects of melanocytes senescence, suggesting that alterations in cellular glucose metabolism act as a driving force for senescence in melanocytes. Conclusion: These results demonstrate that senescent melanocytes are characterized by glycolytic metabolism changes and a defective melanosome transport process, which may be related to impaired mitochondrial function, highlighting the importance of metabolic reprogramming in regulating melanocyte senescence.

Stimulatory effects of wavelength-dependent photobiomodulation on proliferation and angiogenesis of colorectal cancer.

In recent decades, the laser treatment of cancer has been introduced as a promising treatment option. Because of the maldistribution of optical energy and an ambiguous boundary between the normal and tumor tissues, laser irradiation can stimulate residual cancer cells, leading to a cancer regrowth. As photobiomodulation (PBM) is involved in an extensive range of cellular responses, profound comprehension of photo-stimulated mechanisms against the cancer cells is required to establish a safety margin for PBM. Therefore, we aimed to identify the stimulant effects of PBM at various wavelengths against the tumor cells to establish a safety margin for the laser treatment. CT26 murine colon cancer cells were exposed to either 405 (BL), 635 (VIS), or 808 (NIR) nm laser lights at the fluences of 0, 10, 30, and 50 J/cm2. In addition, CT26 tumor-bearing mice were irradiated with BL, VIS, or NIR at a fluence of 30 J/cm2. Both the proliferation and angiogenesis potential of the CT26 cells and tumors were evaluated using the MTT assay, western blot, and immunohistochemistry (IHC) staining analyses. Although cell viability was not statistically significant, BL significantly induced p-ERK upregulation in the CT26 cells, indicating that PBM with BL can stimulate proliferation. In vivo tests showed that the NIR group exhibited the maximum relative tumor volume, and BL yielded a slight increase compared to the control. In the IHC staining and western blot analyses, both BL and NIR increased the expression of EGFR, VEGF, MMP-9, and HIF-1α, which are related to the proliferation and angiogenesis-related factors. Further investigations will be pursued to clarify the molecular pathways that depend on the cancer cell types and laser wavelengths for the establishment of safety guidelines in clinical environments.

Enhancement of gold nanorods-assisted photothermal treatment on cancer with laser power in stepwise modulation.

OBJECTIVES: Photothermal therapy (PTT) is a minimally invasive or noninvasive method by destructing cancer cells through selective thermal decomposition. However, a long period of laser irradiation to achieve coagulative necrosis often causes unfavorable thermal damage to the surrounding healthy tissue. The current study aims to evaluate the feasibility of temporal power modulation to improve the treatment efficacy of gold nanorods-assisted PTT against tumor tissue. MATERIALS AND METHODS: A total of 25 µg/ml of PEGylated gold nanorods (PEG-GNR) was used as an absorbing agent during 1064 nm laser irradiation for PTT. Temperature monitoring was conducted on the aqueous solution of PEG-GNR for dosimetry comparison. For in vivo tests, CT-26 tumor-bearing murine models with PEG-GNR injected were treated with three irradiation conditions: 3 W/cm2 for 90 s, 1.5 W/cm2 for 180 s, and 3 W/cm2 for 60 s followed by 1.5 W/cm2 for 60 s (modulated). Ten days after the treatments, histology analysis was performed to assess the extent of coagulation necrosis in the treated tissues. RESULTS: The temporal power modulation maintained the tissue temperature of around 50°C for a longer period during the irradiation. Histology analysis confirmed that the modulated group entailed a larger coagulative necrosis area with less thermal damage to the peripheral tissue, compared to the other irradiation conditions. CONCLUSION: Therefore, the power-modulated PTT could improve treatment efficacy with reduced injury by maintaining the constant tissue temperature. Further studies will examine the feasibility of the proposed technique in large animal models in terms of acute and chronic tissue responses and treatment margin for clinical translations.

Ponatinib-induced eruptive nevi and melanocytic proliferation.

Ponatinib, an oral third-generation tyrosine kinase inhibitor, is indicated for the treatment of imatinib-resistant leukemia. We experienced a case of ponatinib-induced eruptive nevi, and the biologic effects of ponatinib on melanocytes were investigated. Treatment with ponatinib significantly increased the proliferation of normal human melanocyte or melanoma cells through the upregulation of the extracellular signal-regulated kinase and protein kinase B signaling pathways. The downstream molecules of cyclin B1 and D1 were significantly increased in ponatinib-treated melanocytes. These results demonstrate the capacity of ponatinib to induce the proliferation and tumorigenesis of melanocytes.

Senescent Fibroblast-Derived GDF15 Induces Skin Pigmentation.

Senescent fibroblasts play a role in aging pigmentation. In this study, we found that GDF15 expression levels are increased in UV-irradiated senescent fibroblasts and photoaged hyperpigmented skin. To investigate the effects of GDF15 on melanogenesis, normal human melanocytes were cocultured with fibroblasts infected with the GDF15 lentivirus or GDF15 short hairpin RNA. It was found that GDF15 stimulates melanogenesis in melanocytes through MITF/tyrosinase upregulation via ß-catenin signaling. The stimulatory action of GDF15 during pigmentation was further confirmed in ex vivo cultured skin and in a reconstituted human skin sample. These results suggest that senescent fibroblast-derived GDF15 stimulates skin pigmentation and may play a role in aging-associated pigmentation.

Dasatinib, a second-generation tyrosine kinase inhibitor, induces melanogenesis via ERK-CREB-MITF-tyrosinase signaling in normal human melanocytes.

Dasatinib, a second-generation tyrosine kinase inhibitor, is indicated for the therapy of imatinib-resistant leukemia and also for the treatment of solid cancers. Here, we report a novel effect of dasatinib of inducing differentiation in normal human melanocytes. Treatment with dasatinib significantly increased the melanin content and tyrosinase activity through the up-regulation of MITF and tyrosinase expressions. Consistently, dasatinib had clear stimulatory action in the pigmentation of ex vivo cultured skin. The molecular mechanism underlying the melanogenic effect of dasatinib was associated with the ERK-dependent phosphorylation of CREB. The ERK inhibitor PD98059 not only inhibited the phosphorylation of CREB but also abrogated dasatinib-induced melanocyte differentiation. These results demonstrate for the first time the capacity of dasatinib to induce differentiation in normal human melanocytes depending on the activation of ERK-CREB-MITF-tyrosinase signaling cascades.

The Knockdown of TREK-1 in Hippocampal Neurons Attenuate Lipopolysaccharide-Induced Depressive-Like Behavior in Mice.

TWIK-related potassium channel-1 (TREK-1) is broadly expressed in the brain and involved in diverse brain diseases, such as seizures, ischemia, and depression. However, the cell type-specific roles of TREK-1 in the brain are largely unknown. Here, we generated a Cre-dependent TREK-1 knockdown (Cd-TREK-1 KD) transgenic mouse containing a gene cassette for Cre-dependent TREK-1 short hairpin ribonucleic acid to regulate the cell type-specific TREK-1 expression. We confirmed the knockdown of TREK-1 by injecting adeno-associated virus (AAV) expressing Cre into the hippocampus of the mice. To study the role of hippocampal neuronal TREK-1 in a lipopolysaccharide (LPS)-induced depression model, we injected AAV-hSyn-BFP (nCTL group) or AAV-hSyn-BFP-Cre (nCre group) virus into the hippocampus of Cd-TREK-1 KD mice. Interestingly, the immobility in the tail suspension test after LPS treatment did not change in the nCre group. Additionally, some neurotrophic factors (BDNF, VEGF, and IGF-1) significantly increased more in the nCre group compared to the nCTL group after LPS treatment, but there was no difference in the expression of their receptors. Therefore, our data suggest that TREK-1 in the hippocampal neurons has antidepressant effects, and that Cd-TREK-1 KD mice are a valuable tool to reveal the cell type-specific roles of TREK-1 in the brain.

A Silyl-Nickel Moiety as a Metal-Ligand Cooperative Site.

Nickel(II) complexes supported by a diphosphinosilyl ligand, [PhSi(2-PiPr2C6H4)2]- (PhSiP2), reveal unusual metal-ligand cooperativity (MLC). While (PhSiP2)Ni(NHMes) (2a) was cleanly isolated at room temperature, a nickel triisopropylphenylamido species, (PhSiP2)Ni(NHTrip) (2b), slowly transformed into a nickel(II) phenyl species, [(ArNH)SiP2]Ni(Ph) (3b), where (ArNH)SiP2- = [(NHTrip)Si(2-PiPr2C6H4)2]-. The X-ray crystallographic data of 3b exhibit a Si-N bond generated from Si-N coupling between the silyl moiety and amino group, along with cleavage of a Si-C bond. Because substoichiometric amounts of π-acidic ligands, such as isocyanide, enhance the conversion rate of 2 to 3 (kobs = 0.28 vs 0.44 h-1), this reaction may involve reductive elimination (RE) and oxidative addition (OA) operating at the silyl-nickel moiety. This is further supported by the fact that the presence of excess π-acidic ligands results in the generation of demetalated ligands (ArNH)PhSiP2 (4) having both Si-N and Si-Ph bonds and the nickel(0) species. Theoretical evaluations also agree on such a pathway. Interestingly, the reaction of a nickel phenyl species (3) with gaseous carbon dioxide (CO2(g)) produces a nickel(II) carbamato complex, (PhSiP2)Ni(OC(O)NHAr) (6), which may involve a RE-OA process occurring at a nickel center. Although 2 and 3 might be in equilibrium, the reaction of 3 with CO2 does not follow this pathway. Instead, a CO2 interaction induces RE at the silyl-nickel moiety, followed by amide group transfer, to give a carbamato product, 6, based on our experimental and theoretical evaluations. These results highly support that group transfer involving MLC can be managed via a RE-OA pathway at the silyl-nickel(II) moiety.

The E3 ubiquitin ligase, NEDD4L (NEDD4-2) regulates bestrophin-1 (BEST1) by ubiquitin-dependent proteolysis.

The bestrophin family comprises well-known Ca2+-activated chloride channels (CaCC) that are expressed in a variety tissues including the brain, eye, gastrointestinal tract, and muscle tissues. Among the family members, bestrophin-1 (BEST1) is known to exist mainly in retinal pigment epithelium cells, but we recently reported that BEST1 mediates Ca2+-activated Cl- currents in hippocampal astrocytes. Despite its critical roles in physiological processes, including tonic γ-aminobutyric acid release and glutamate transport, the mechanisms that regulate BEST1 are poorly understood. In this study, we identified NEDD4L (NEDD4-2), an E3 ubiquitin ligase, as a binding partner of BEST1. A series of deletion constructs revealed that the WW3-4 domains of NEDD4L were important for interaction with BEST1. We observed that BEST1 underwent ubiquitin-dependent proteolysis and found that the conserved lysine370 residue in the C-terminus of BEST1 was important for its ubiquitination. Finally, we demonstrated that NEDD4L inhibited whole cell currents mediated by BEST1 but not by the BEST1(K370R) mutant. Collectively, our data demonstrated that NEDD4L played a critical role in regulating the surface expression of BEST1 by promoting its internalization and degradation.

Senescent fibroblasts drive ageing pigmentation: ​A potential therapeutic target for senile lentigo.

Cutaneous ageing is an important extrinsic process that modifies the pigmentary system. Because cellular senescence is a fundamental ageing mechanism, we examined the role of senescent cells in ageing pigmentation. Methods: Biopsies obtained from senile lentigo and perilesional normal skin were assayed for a marker of cellular senescence, p16INK4A. To determine the secretory phenotypes of senescent fibroblasts, we performed microarray, RNA sequencing and methylation array analyses in senile lentigo and senescent fibroblasts. To further investigate the impact of senescent cells on ageing-related pigmentation, an intervention that targeted senescent cells using radiofrequency was performed. Results:In vivo, senescent fibroblasts accumulated at the sites of age-related pigmentation. Phenotype switching of the cells resulted in the repression of stromal-derived factor 1 (SDF1) by promoter methylation. SDF1 induced melanocyte differentiation via stromal-epithelial interactions, ultimately driving skin pigmentation. Furthermore, the elimination of senescent fibroblasts from pigmented skin using radiofrequency was accompanied by skin lightening, rendering it a potential target for treatment. Conclusion: Aged pigmented skin contains an increasing proportion of senescent fibroblasts. Cells with phenotype switching exhibited a loss of SDF1, which stimulates the melanogenic process and thereby contributes to aging pigmentation. These data may promote the development of new therapeutic paradigms, such as a stroma-targeting therapy for pigmentary disorders.

A P-P Bond as a Redox Reservoir and an Active Reaction Site.

The carbonylation of a nickel(II) anilido species 2 led to the formation of a dinickel(0)-CO complex (P2 P-PP2 ){Ni(CO)}2 3 with a P-P bond along with isocyanate generation. In this reaction, the central phosphide moiety of an anionic PPP ligand (PPP- =- P[2-Pi Pr2 C6 H4 ]2 ) acts as a single-electron donor to form a P radical. Alternatively, 3 can be synthesized from the reduction of (PPP)NiCl (1) in the presence of CO; thus, the reaction proceeds by radical coupling of a . P-Ni0 -CO species. The reverse reaction occurred to generate 1 when 3 was treated with AgCl. Since the P-P bond is light-sensitive, its homolysis is possible and was explored by EPR spectroscopy and DFT analysis. Finally, various bond-activation reactions of 3 occurred under visible-light conditions, thus indicating that a P-P bond can act as an active reaction site.

Construction of Uniform Monolayer- and Orientation-Tunable Enzyme Electrode by a Synthetic Glucose Dehydrogenase without Electron-Transfer Subunit via Optimized Site-Specific Gold-Binding Peptide Capable of Direct Electron Transfer.

Direct electron transfer (DET) between enzymes and electrodes is a key issue for practical use of bioelectrocatalytic devices as a bioenergy process, such as enzymatic electrosynthesis, biosensors, and enzyme biofuel cells. To date, based on the DET of bioelectrocatalysis, less than 1% of the calculated theoretical current was transferred to final electron acceptor due to energy loss at enzyme-electrode interface. This study describes the design and construction of a synthetic glucose dehydrogenase (GDH; α and γ subunits) combined with a gold-binding peptide at its amino or carboxy terminus for direct contact between enzyme and electrode. The fused gold-binding peptide facilitated stable immobilization of GDH and constructed uniform monolayer of GDH onto a Au electrode. Depending on the fused site of binding peptide to the enzyme complex, nine combinations of recombinant GDH proteins on the electrode show significantly different direct electron-transfer efficiency across the enzyme-electrode interface. The fusion of site-specific binding peptide to the catalytic subunit (α subunit, carboxy terminus) of the enzyme complex enabled apparent direct electron transfer (DET) across the enzyme-electrode interface even in the absence of the electron-transfer subunit (i.e., ß subunit having cytochrome domain). The catalytic glucose oxidation current at an onset potential of ca. (-)0.46 V vs Ag/AgCl was associated with the appearance of an flavin adenine dinucleotide (FAD)/FADH2 redox wave and a stabilized bioelectrocatalytic current of more than 100 µA, determined from chronoamperometric analysis. Electron recovery was 7.64%, and the catalytic current generation was 249 µA per GDH enzyme loading unit (U), several orders of magnitude higher than the values reported previously. These observations corroborated that the last electron donor facing to electrode was controlled to be in close proximity without electron-transfer intermediates and the native affinity for glucose was preserved. The design and construction of the site-specific "sticky-ended" proteins without loss of catalytic activity could be applied to other redox enzymes having a buried active site.

Selective Transformation of CO2 to CO at a Single Nickel Center.

Carbon dioxide conversion mediated by transition metal complexes continues to attract much attention because of its future potential utilization as a nontoxic and inexpensive C1 source for the chemical industry. Given the presence of nickel in natural systems that allow for extremely efficient catalysis, albeit in an Fe cluster arrangement, studies that focus on selective CO2 conversion with synthetic nickel species are currently of considerable interest in our group. In this Account, the selective conversion of CO2 to carbon monoxide occurring at a single nickel center is discussed. The chemistry is based on a series of related nickel pincer complexes with attention to the uniqueness of the coordination geometry, which is crucial in allowing for particular reactivity toward CO2. Our research is inspired by the efficient enzymatic CO2 catalysis occurring at the active site of carbon monoxide dehydrogenase. Since the binding and reactivity toward CO2 are controlled in part by the geometry of a L3Ni scaffold, we have explored the chemistry of low-valent nickel supported by PPMeP and PNP ligands, in which a pseudotetrahedral or square-planar geometry is accommodated. Two isolated nickel-CO2 adducts, (PPMeP)Ni(η2-CO2-κ C) (2) and {Na(12-C-4)2}{(PNP)Ni(η1-CO2-κ C)} (7), clearly demonstrate that the geometry of the nickel ion is crucial in the binding of CO2 and its level of activation. In the case of a square-planar nickel center supported by a PNP ligand, a series of bimetallic metallacarboxylate Ni-µ-CO2-κ C, O-M species (M = H, Na, Ni, Fe) were synthesized, and their structural features and reactivity were studied. Protonation cleaves the C-O bond, resulting in the formation of a nickel(II) monocarbonyl complex. By sequential reduction, the corresponding mono- and zero-valent Ni-CO species were produced. The reactivities of three nickel carbonyl species toward various iodoalkanes and CO2 were explored to address whether their corresponding reactivities could be controlled by the number of valence d electrons. In particular, a (PNP)Ni(0)-CO species (13) shows immediate reactivity toward CO2 but displays multiple product formation. By incorporation of a -CMe2- bridging unit, a structurally rigidified acriPNP ligand was newly designed and produced. This ligand modification was successful in preparing the T-shaped nickel(I) metalloradical species 9 exhibiting open-shell reactivity due to the sterically exposed nickel center possessing a half-filled d x2- y2 orbital. More importantly, the selective addition of CO2 to a nickel(0)-CO species was enabled to afford a nickel(II)-carboxylate species (22) with the expulsion of CO(g). Finally, the (acriPNP)Ni system provides a synthetic cycle in the study of the selective conversion of CO2 to CO that involves two-electron reduction of Ni-CO followed by the direct addition of CO2 to release the coordinated CO ligand.

σ-Complexation as a strategy for designing copper-based light emitters.

Strongly emissive cuprous complexes containing a stable σ-SiH-Cu motif were prepared. These complexes serve as a proof of principle that σ-complexation can be utilized as a design principle for engineering responsive light emitting materials. Their excited state lifetimes were found to be long (∼20 µs) with high quantum yields (φ = 0.40-0.59).

Phosphinite-Ni(0) mediated formation of a phosphide-Ni(II)-OCOOMe species via uncommon metal-ligand cooperation.

Reversible transformations are observed between a phosphide-nickel(II) alkoxide and a phosphinite-nickel(0) species via a P-O bond formation coupled with a 2-e(-) redox change at the nickel center. In the forward reaction, the nickel(0) dinitrogen species (PP(OMe)P)Ni(N2) (2) and {(PP(OMe)P)Ni}2(µ-N2) (3) were formed from the reaction of (PPP)NiCl (1) with a methoxy anion. In the backward reaction, a (PPP)Ni(II) moiety was regenerated from the CO2 reaction of 3 with the concomitant formation of a methyl carbonate ligand in (PPP)Ni(OCOOMe) (7). Thus, unanticipated metal-ligand cooperation involving a phosphide based ligand is reported.

Formation of a nickel carbon dioxide adduct and its transformation mediated by a Lewis acid.

An uncommon nickel dinitrogen adduct and its tendency toward CO2 binding are investigated using a (PP(Me)P)Ni scaffold. (PP(Me)P)Ni(N2) (1) and {(PP(Me)P)Ni}2(µ-N2) (2) were prepared and their treatment with CO2 revealed the formation of (PP(Me)P)Ni(η(2)-CO2) (3). This is a new type of CO2 binding for a zero-valent nickel center supported by three donor ligands, reminiscent of the CODH active site environment. Clear unique structural differences in 3 are evident when compared with previous 4-coordinate Ni-CO2 adducts. Compound 3 when treated with B(C6F5)3 gives the Lewis acid-base adduct (PP(Me)P)Ni{COOB(C6F5)3} (4) possessing a Ni-µ-CO2-κ(2)C,O-B moiety.

20-O-ß-D-glucopyranosyl-20(S)-protopanaxadiol-fortified ginseng extract attenuates the development of atopic dermatitis-like symptoms in NC/Nga mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng and ginsenosides are frequently used in the treatment of chronic inflammatory diseases. Recently, 20-O-ß-d-glucopyranosyl-20(S)-protopanaxadiol (GPD), the main metabolite of ginsenosides, was reported to have both anti-allergic and anti-pruritic effects. The immunomodulatory effects of GPD-fortified ginseng extract (GFGE) on atopic dermatitis (AD)-like symptoms in mice were investigated. This study was designed to investigate the preventive effect of GFGE on AD-like symptoms. MATERIALS AND METHODS: The effects of orally administered GFGE on Dermatophagoides farinae body extract (DFE)-induced AD-like symptoms in NC/Nga mice were assessed by analyzing dermatitis score, ear thickness, scratching time, skin histological changes, and serum level of macrophage-derived chemokine (MDC). In addition, splenocytes were isolated from the mice and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies to produce cytokines. RESULTS: Oral administration of GFGE significantly attenuated DFE-induced increases in dermatitis score, ear thickness, scratching time, and severity of skin lesions in NC/Nga mice. GFGE treatment also reduced level of MDC in serum, infiltration of eosinophils and mast cells in skin, and production of cytokines in splenocytes. CONCLUSIONS: These results suggest that GFGE might ameliorate DFE-induced AD-like symptoms and be an alternative therapeutic agent for the prevention of AD.