Towards in vivo melanin radicals detection in melanomas by electron paramagnetic resonance (EPR) spectroscopy: a proof-of-concept study

Melanoma is the most aggressive skin tumour type. Although complete cure can be achieved when the whole tumour is resected, prognostic dramatically drops when melanoma cells reach deeper tissues and lymph nodes. Hence, there is an urgent need to develop accurate tools allowing (i) discriminating benign naevi from malignant tumours and (ii) being able to characterise melanoma infiltration. For that purpose, we exploited the paramagnetic properties of melanin by using electron paramagnetic resonance (EPR) spectroscopy to measure the melanin content in pigmented (B16F10 cancer cells) and non-pigmented melanomas (WM2664 cancer cells) inoculated intradermally in nude mice. Specifically, we took advantage of a new clinical EPR device (1?GHz), which provides sensitive measurements of radical species in vivo. Results showed that the melanin-specific EPR signal increased with tumour growth in pigmented tumours, whereas no EPR signal could be detected in achromic melanomas. These data plead for the development of new EPR spectrometers/imagers with an improved in-depth resolution for the detection of invasive melanomas.     

Suppressive effect of spinach extract on the formation of melanin in B16 mouse melanoma cells

A non-dialyzable extract of fresh spinach leaves exhibited a strong antioxidant activity towards oxidation of linoleic acid and suppressed the melanin formation of a mouse melanoma cell line, B16 melanoma 4A5, without any significant effect on the proliferation of cells.     

Characteristics of Bremsstrahlung emissions of 177Lu, 188Re,and 90Y for SPECT/CT quantification in radionuclide therapy

PurposeBeta particles emitted by radioisotopes used in targeted radionuclide therapies (TRT) create Bremsstrahlung (BRS) which may affect SPECT quantification when imaging these isotopes. The purpose of the current study was to investigate the characteristics of Bremsstrahlung produced in tissue by three β-emitting radioisotopes used in TRT.MethodsMonte Carlo simulations of ¹⁷⁷Lu, ¹⁸⁸Re, and ⁹⁰Y sources placed in water filled cylinders were performed. BRS yields, mean energies and energy spectra for (a) all photons generated in the decays, (b) photons that were not absorbed and leave the cylinder, and (c) photons detected by the camera were analyzed. Next, the results of simulations were compared with those from experiments performed on a clinical SPECT camera using same acquisition conditions and phantom configurations as in simulations.ResultsSimulations reproduced relatively well the shapes of the measured spectra, except for ⁹⁰Y which showed an overestimation in the low energy range. Detailed analysis of the results allowed us to suggest best collimators and imaging conditions for each of the investigated isotopes. Finally, our simulations confirmed that the BRS contribution to the energy spectra in quantitative imaging of ¹⁷⁷Lu and ¹⁸⁸Re could be ignored.ConclusionsFor ¹⁷⁷Lu and ¹⁸⁸Re, BRS contributes only marginally to the total spectra recorded by the camera. Our analysis shows that MELP and HE collimators are the best for imaging these two isotopes. For ⁹⁰Y, HE collimator should be used.     

Cell-intrinsic melanin fails to protect melanocytes from ultraviolet-mutagenesis in the absence of epidermal melanin

Melanin is a free-radical scavenger, antioxidant, and broadband absorber of ultraviolet (UV) radiation which protects the skin from environmental carcinogenesis. However, melanin synthesis and UV-induced reactive melanin species are also implicated in melanocyte genotoxicity. Here, we attempted to reconcile these disparate functions of melanin using a UVB-sensitive, NRAS-mutant mouse model, TpN. We crossed TpN mice heterozygous for an inactivating mutation in Tyrosinase to produce albino and black littermates on a C57BL/6J background. These animals were then exposed to a single UVB dose on postnatal day three when keratinocytes in the skin have yet to be melanized. Approximately one-third (35%) of black mice were protected from UVB-accelerated tumor formation. However, melanoma growth rates, tumor mutational burdens, and gene expression profiles were similar in melanomas from black and albino mice. Skin from albino mice contained more cyclobutane pyrimidine dimer (CPD) positive cells than black mice 1-h post-irradiation. However, this trend gradually reversed over time with CPDs becoming more prominent in black than albino melanocytes at 48 h. These results show that in the absence of epidermal pigmentation, melanocytic melanin limits the tumorigenic effects of acute UV exposure but fails to protect melanocytes from UVB-induced mutagenesis.     

Effects of melanin upon susceptibility of Cryptococcus to antifungals

Melanin is a recognized virulence factor in Cryptococcus neoformans; several pathogenetic mechanisms have been suggested. We studied melanin as an antifungal resistance factor. The growth of laccase-active strains of C. neoformans and C. albidus in L-DOPA resulted in the production of black pigment. The formal minimal inhibitory concentrations (MICs) of amphotericin B and fluconazole were not changed by melanization. However, when we examined those wells which contained inhibited cells, we found live cells only in wells containing melanized C. neoformans. In contrast, melanization did not protect C. albidus from killing by amphotericin B. In an amphotericin B time-kill study of C. neoformans, significantly more melanized cells than non-melanized survived for the first few hours. Fluorescence microscopy and flow cytometry analyses showed that fewer melanized cells were stained with the fluorescent dye MitoRed. Incubation of MitoRed (the model) or amphotericin B with melanin extracted from C. neoformans decreased the free concentrations of these substances. Fluconazole, in contrast, was not removed from solution by melanin. This suggests that neoformans cryptococcal melanin deposited amphotericin B in the cell wall binds, reducing its effective concentrations.     

Environmental correlates of internal coloration in frogs vary throughout space and lineages

Internal organs of ectotherms have melanin‐containing cells that confer different degrees of coloration to them. Previous experimental studies analyzed their developmental origin, role in immunity, and hormonal regulation. For example, melanin increases with ultraviolet radiation (UV) and temperature in frogs and fish. However, little is known about how environmental variables influence the amount of coloration on organs among amphibian species over a large spatial extent. Here, we tested how climatic variables (temperature, UV, and photoperiod) influence the coloration of internal organs of anurans. We recorded the level of melanin pigmentation using four categories on 12 internal organs and structures of 388 specimens from 43 species belonging to six anuran families. Then, we tested which climatic variables had the highest covariation with the pigmentation on each organ after controlling for spatial autocorrelation in climatic variables and phylogenetic signal in organ coloration using the extended version of the RLQ ordination. Coloration in all organs was correlated with the phylogeny. However, the coloration of different organs was affected by different variables. Specifically, the coloration of the heart, kidneys, and rectum of hylids, Rhinella schneideri, some Leptodactylus, and Proceratophrys strongly covaried with temperature and photoperiod, whereas that of the testicle, lumbar parietal peritoneum, lungs, and mesenterium of Leiuperinae, Hylodidae, Adenomera, and most Leptodactylus had highest covariation with UV‐B and temperature. Our results support the notion that melanin pigmentation on the surface of organs of amphibians has an adaptive function conferred by the protective functions of the pigment. But most importantly, internal melanin seems to respond differently to climatic variables depending on the lineage and locality in which species occur.     

异齿裂腹鱼仔鱼的黑色素分布

本文运用组织学方法对异齿裂腹鱼仔鱼发育过程中除眼睛以外的黑色素分布进行研究.结果 表明:异齿裂腹鱼孵化出膜后,黑色素在各个组织器官的出现顺序为:脑颅外膜→围心腔、背部皮肤→腹腔内膜、脊髓腔.其中,出膜5d时脑颅外膜处出现黑色素;7d时围心腔和背部皮肤出现黑色素;10d时腹腔内膜、脊髓腔出现黑色素.异齿裂腹鱼出膜10 d...     

Characterization of melanin in human iridal and choroidal melanocytes from eyes with various colored irides

Variance in iris color is related to the incidence of several important ocular diseases, including uveal melanoma and age-related macular degeneration. The purposes of this study were to determine the quantity and the types of melanin in cultured human uveal melanocytes in relation to the iris color. Sixty-one cell cultures of pure uveal melanocytes were isolated from donor eyes with various iris colors. The amount of eumelanin (EM) and pheomelanin (PM) of these cells was measured by chemical degradation and microanalytical high-performance liquid chromatography (HPLC) methods. The total amount of melanin was measured by both microanalytical methods and spectrophotometry. Total melanin content, measured by HPLC and spectrophotometry, correlated well with r = 0.872 (P < 0.0001). The quantity and type of melanin in iridal and choroidal melanocytes showed no significant difference (P > 0.05). When cells became senescent, the levels of EM, PM and total melanin were significantly increased. In both growing and senescent melanocytes, the quantity and type of melanin were closely correlated to the iris color. In cells from eyes with dark-colored irides (dark brown and brown), the amount of EM, the ratio of EM/PM and total melanin were significantly greater than that from eyes with light-colored irides (hazel, green, yellow-brown and blue) (P < 0.0001). The quantity of PM in uveal melanocytes from eyes with light-colored irides was slightly greater than that from dark-colored irides, although not statistically significant (P > 0.05). The present study shows that iris color is determined by both the quantity and the type of melanin in uveal melanocytes. These results suggest a possibility that uveal melanin in eyes with dark-colored irides is eumelanic at the surface and acts as an antioxidant while that in eyes with light-colored irides exposes pheomelanic core and behaves as a pro-oxidant.     

The biosynthesis of melanin in Alternaria

A melanin which is insoluble in strong alkali has been isolated from Alternaria mycelium. Alkali fusion of the pigment produced p-hydroxybenzoa     

真菌黑色素及其与巨噬细胞免疫研究进展

真菌黑色素在真菌抵抗外界理化损伤及宿主的免疫杀伤过程中起重要作用,是真菌重要的毒力因子。巨噬细胞是抗真菌固有免疫的一线细胞,探讨真菌黑色素与巨噬细胞的相互作用,可为阐明产黑色素真菌感染的发病机制奠定基础。本文重点从黑色素的生物学特性、真菌黑色素对巨噬细胞免疫的影响两个方面进行阐述。     

Photodynamic therapy-induced killing is enhanced in depigmented metastatic melanoma cells

The resistance of pigmented human melanomas over their unpigmented counterparts to a number of therapies has suggested that the presence of intracellular melanin plays a role in rendering these cells less susceptible to cell death, probably through the ability of this pigment to act as an intracellular antioxidant, thus neutralizing chemotherapeutic-induced ROS (reactive oxygen species). PDT (photodynamic therapy) was recently suggested as an attractive, adjunctive therapy owing to its cellular specificity and limited side effects. In the present study, we propose that first depigmenting melanomas with a reversible TYR (tyrosinase) inhibitor such as PTU (phenylthiourea) increases their susceptibility to HYP-PDT (hypericin-mediated PDT). Pigmented [UCT Mel-1 (University of Cape Town melanoma cell line 1)] and unpigmented (A375) melanomas were first characterized with respect to their TYR activities and melanin quantities and then treated with a TYR inhibitor for 48 h. Cell viability assays after treatment with 3 μM HYP-PDT showed a significant increase in cell death in depigmented melanomas compared with untreated melanomas that returned to the level of untreated melanoma cells on removing the TYR inhibitor. The present study supports the hypothesis that combining the inhibition of melanogenesis with PDT should be explored as a valid therapeutic target for the management of advanced melanoma.     

食用菌黑色素代谢的研究进展

黑色素的代谢对食用菌的生长发育具有至关重要的影响,黑色素的代谢异常导致了食用菌白色新品种的出现。对食用菌黑色素代谢的影响因子进行了综述,对黑色素代谢异常与食用菌白化菌株形成的关系进行了阐述,并介绍了食用菌黑色素的形成途径,对黑色素合成过程中的关键酶类及其抑制剂的研究进行了简要概括,主要包括酪氨酸酶、漆酶和聚酮合成酶的研究。并展望了食用菌黑色素代谢今后主要的研究方向,旨在为白色食用菌的进一步开发和利用提供参考。     

Assessment of radionuclide impurities in [18F]fluoromethylcholine ([18F]FMCH)

Purpose[¹⁸F]Fluoromethylcholine ([¹⁸F]FMCH) is a radiopharmaceutical used in positron emission tomography (PET) imaging for the study of prostate, breast, and brain tumors. It is usually synthesized in cyclotron facilities where ¹⁸F is produced by proton irradiation of [¹⁸O]H₂O through ¹⁸O(p,n)¹⁸F reaction. Due to the activation of target materials, the bombardment causes unwanted radionuclidic impurities in [¹⁸O]H₂O, that need to be removed during the radiopharmaceutical synthesis. Thus, the aim of this study is to quantify the radionuclide impurities in the ¹⁸F production process and in the synthesized [¹⁸F]FMCH, demonstrating the radionuclidic purity of this radiopharmaceutical.MethodsLong-lived radionuclide impurities were experimentally assessed using high-resolution gamma and liquid scintillation spectrometries, while short-lived impurities were monitored analyzing the decay curve of the irradiated [¹⁸O]H₂O with an activity calibrator. As spectrometric radionuclide library, a Geant4 Monte Carlo simulation of the ¹⁸F-target assembly was previously performed.Results³H, ^52,54Mn, ^56,57,58Co, ^95m,96Tc, ¹⁰⁹Cd, and ¹⁸⁴Re were found in the irradiated [¹⁸O]H₂O, but no radionuclide was found in the non-irradiated [¹⁸O]H₂O neither in the final [¹⁸F]FMCH solution with an activity concentration greater than the minimum detectable activity concentration. A total impurity activity <6.2 kBq was measured in the irradiated [¹⁸O]H₂O, whereas a [¹⁸F]FMCH radionuclide purity >99.9999998% was estimated. Finally, the decay curve of the irradiated [¹⁸O]H₂O revealed a very low maximum of ¹³N activity (<0.03% of ¹⁸F) even immediately after the end of bombardment.ConclusionsThis study demonstrated the radionuclidic purity of [¹⁸F]FMCH according to the EU Pharmacopeia.     

植物病原真菌1,8-间苯二酚黑色素研究进展

黑色素是一种广泛分布于生物体中的酚类聚合物疏水色素,分为1,8-间苯二酚(1,8-dihydroxynaphthalene,DHN)黑色素和3,4-二羟基苯丙氨酸(3,4-dihydroxyphenylalanine,L-DOPA)黑色素两种,其中DHN黑色素多存在于子囊菌门的植物病原真菌中。基因组和转录组技术的发展及功能基因组研究的深入,使DHN黑色素合成途径上关键基因在不同病原真菌中被鉴定,而且黑色素与真菌抗逆、发育和致病的关系受到越来越多的关注。本文阐述了DHN黑色素合成途径及其在真菌抗辐射与抗极端温度中的作用,以及黑色素对真菌侵染和细胞发育的影响,旨在加深人们对黑色素介导真菌与环境和寄主协同进化的认识,这对黑色素的基础研究和开发利用具有重要意义。     

Correlations between melanin pigmentation and element concentration in feathers of White-tailed Eagles (Haliaeetus albicilla)

Summary The element concentration of moult feathers of White-tailed Eagles was investigated. Using the 2- MeV Hamburg proton microprobe we tried to differentiate between elements incorporated into the feather via the food chain and those which are deposited externally onto the feather vane. Regarding incorporated elements, special attention has been given to a possible correlation between element concentration and feather pigmentation. Concerning the elements detected in this work (S, K, Ca, Ti, Mn, Fe, Cu, Zn, Hg, Pb), calcium, manganese and zinc show a considerable enhancement within the pigmented feather as compared with pigment-free feather sections. On the other hand, no differences were found in concentrations for sulfur, titanium, iron, copper, mercury and lead. Our findings therefore imply a special enrichment of Ca, Mn and Zn within melanin, the source of the feather's pigmentation. The possible role of these elements with regard to melanin formation is discussed.

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Melaninpigmentation und Elementkonzentration in Federn des Seeadlers (Haliaeetus albicilla)

Zusammenfassung Es wurden die Elementkonzentrationen in Mauserfedern von Seeadlern untersucht. Mit Hilfe der Hamburger 2-MeV-Protonenmikrosonde, die die Analyse von Probendetails im Mikrometerbereich ermöglicht, versuchen wir zwischen Elementen zu unterscheiden, die vom Adler mit der Nahrung inkorporiert, verstoffwechselt und während der Mauser in die Feder eingelagert wurden und solchen, die während der etwa einjährigen Verweilzeit der Feder im Gefieder extern aus der Atmosphäre aufgelagert wurden.Im Hinblick auf die eingelagerten Elemente untersuchen wir mögliche Zusammenhänge zwischen der Elementkonzentration und der Federpigmentierung. Von den nachgewiesenen Elementen (Schwefel, Kalium, Calcium, Titan, Mangan, Eisen, Kupfer, Zink, Quecksilber und Blei) zeigen Calcium, Mangan und Zink eine erhebliche Anreicherung in der pigmentierten Feder im Vergleich zu pigmentfreien Federausschnitten. Dagegen wurden keine signifikanten Unterschiede der Elementkonzentrationen von Schwefel, Titan, Eisen, Kupfer, Quecksilber und Blei festgestellt. Die gefundenen Ergebnisse bedeuten eine erhebliche Anreicherung von Calcium, Mangan und Zink im Melanin, das für die Federpigmentierung verantwortlich ist. Die mögliche Funktion dieser Elemente im Hinblick auf die Melaninbildung wird diskutiert.

     

Neoadjuvant BRAF‐targeted therapy in regionally advanced and oligometastatic melanoma

Current management of locoregional and oligometastatic melanoma is typically with surgery; however, some patients are unable to undergo resection due to location/size of their tumors and/or the anticipated morbidity of the surgery. While there are currently no established guidelines for neoadjuvant therapy in melanoma, neoadjuvant BRAF‐targeted therapy may make resection more feasible. A retrospective analysis was conducted of 23 patients with BRAFV600‐mutant, stage III/IV melanoma treated with BRAF‐targeted therapy prior to surgery, with no adjuvant treatment. Surgical specimens, preoperative imaging, and clinical outcomes were evaluated. Results: Ten of 23 patients (44%) attained a pathologic complete response (pCR), with no correlation between RECIST response based on preoperative imaging and pathologic response. After a median of 43‐month follow‐up, only 1 patient (10%) with a pCR recurred, while 8 of 13 (62%) patients without a pCR recurred. Patients with a pCR had significantly improved relapse‐free (RFS) and overall survival (OS) compared to patients with residual tumor. Neoadjuvant BRAF‐targeted therapy is associated with a high pCR rate in patients with stage III‐IV melanoma, which may correlate with improved RFS and OS.     

The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana

The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes (TH and DDC), in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.     

Fonsecaea monophora色素毒力性研究

目的探讨黑色素是否为Fonsecaea monophora的一个重要毒力因子。方法从Fonsecaea monophora的分生孢子突变株(CBS122845)传代接种产生白色突变株(CBS 125149)。透射电子显微镜(TEM)下观察到黑色素是位于分生孢子细胞壁表面上的电子致密颗粒。通过碱-酸法提取来自两个不同菌株的细胞壁色素颗粒。建立不同菌株或色素颗粒与活化巨噬细胞(RAW264.7)共培养体系,通过实时荧光相对定量PCR检测i-NOS基因的表达,格里斯法检测一氧化氮(NO)的表达结果,ELISA检测IL-12、TNF-α、IL-10的表达结果。结果色素型分生孢子和其色素颗粒能够降低巨噬细胞诱导型一氧化氮合酶(I-NOS)基因的表达和抑制一氧化氮的合成(P<0.05)。提高Th2细胞因子表达,同时抑制Th1细胞因子表达(P<0.05)。结论黑色素可能是Fonsecaea monophora逃避巨噬细胞对其氧化应激的重要机制。同时黑色素下调Th1免疫应答,可能利于真菌的持续感染。