Pyomelanin produced by Streptomyces sp. ZL-24 and its protective effects against SH-SY5Y cells injury induced by hydrogen peroxide.

A soluble melanin pigment produced by Streptomyces sp. ZL-24 was purified and named StrSM. The elemental analysis of StrSM showed it consists of carbon, hydrogen, and oxygen. The spectrum analysis, including ultraviolet-visible absorption spectrum, Fourier-transform infrared spectrum, and pyrolysis-gas chromatography-mass spectrometry, indicated that StrSM might be pyomelanin. High performance liquid chromatography and liquid chromatography-mass spectra analysis of intermediate metabolite showed the presence of homogentisic acid (HGA). Moreover, the enzyme 4-hydroxyphenylpyruvate dioxygenase, involved in HGA biosynthesis, showed high activity during melanin production. Subsequently, a tyrosinase gene (melC2) and hydroxyphenylpyruvate dioxygenase gene double mutant demonstrated StrSM is pyomelanin. In vitro bioactivity assay showed that StrSM had excellent protective capability against SH-SY5Y cell oxidative injury. To our knowledge, the results firstly provide comprehensive data on Streptomyces pyomelanin identification and a promising candidate compound to treat oxidative injury of neurocytes.

Production, purification, and characterization of Streptomyces sp. strain MPPS2 extracellular pyomelanin pigment.

Melanins are natural pigments widely distributed in nature from bacteria to humans. These complex, negatively charged, amorphous, high molecular mass natural biopolymers have many different bioactive properties such as antimicrobial, antiviral, antioxidant, liver protective effects, etc. In this study, some chemical and physical properties of the purified extracellular pyomelanin pigment were investigated via XRD (X-ray diffraction), FT-IR (Fourier transform infrared), and 1H NMR (Nuclear magnetic resonance). Additionally, the melanin pigment producer Streptomyces sp. strain MPPS2 was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and 16S rDNA sequence analysis. 16S rDNA nucleotide sequence analysis result was deposited in NCBI GenBank® under accession number MT825616.

Identification of the pigment and its role in UV resistance in Paecilomyces variotii, a Chernobyl isolate, using genetic manipulation strategies.

Fungi produce secondary metabolites that are not directly involved in their growth, but often contribute to their adaptation to extreme environmental stimuli and enable their survival. Conidial pigment or melanin is one of the secondary metabolites produced naturally by a polyketide synthesis (PKS) gene cluster in several filamentous fungi and is known to protect these fungi from extreme radiation conditions. Several pigmented or melanized fungi have been shown to grow under extreme radiation conditions at the Chernobyl nuclear accident site. Some of these fungi, including Paecilomyces variotii, were observed to grow towards the source of radiation. Therefore, in this study, we wanted to identify if the pigment produced by P. variotii, contributes to providing protection against radiation condition. We first identified the PKS gene responsible for synthesis of pigment in P. variotii and confirmed its role in providing protection against UV irradiation through CRISPR-Cas9 mediated gene deletion. This is the first report that describes the use of CRISPR methodology to create gene deletions in P. variotii. Further, we showed that the pigment produced by this fungus, was not inhibited by DHN-melanin pathway inhibitors, indicating that the fungus does not produce melanin. We then identified the pigment synthesized by the PKS gene of P. variotii, as a naptho-pyrone Ywa1, by heterologously expressing the gene in Aspergillus nidulans. The results obtained will further aid in understanding the mechanistic basis of radiation resistance.

Integrated metabolomics and transcriptomics reveal the anti-aging effect of melanin from Sepiella maindroni ink (MSMI) on D-galactose-induced aging mice.

Sepiella maindroni ink, a flavoring and coloring agent in food, has attracted considerable attention due to its various pharmacological activities. Our previous study showed that the melanin of Sepiella maindroni ink (MSMI) can alleviate oxidative damage and delay aging in D-galactose(D-gal)-induced aging mice. This study aimed to reveal the possible mechanisms of the anti-aging effect of MSMI. In this article, a comprehensive analysis of gas chromatography-mass spectrometry (GC-MS)-based metabolomics and microarray-based transcriptomics revealed that 221 mRNAs were differentially expressed and 46 metabolites were significantly changed in the anti-aging progress of MSMI. Integrated analysis of transcript and metabolic profiles indicated that MSMI mainly altered carbohydrate metabolism, lipid metabolism, and insulin signaling pathway. MSMI achieved anti-aging effects not only by reducing oxidative damage and sorbitol toxicity but also by regulating lipid metabolism, improving insulin sensitivity, and reducing the formation of advanced glycation end products (AGEs). Moreover, our findings firstly demonstrated that MSMI could increase the expression of interferon-induced proteins and might be a potential antiviral compound.

Biodegradation of bio-sourced and synthetic organic electronic materials towards green organic electronics.

Ubiquitous use of electronic devices has led to an unprecedented increase in related waste as well as the worldwide depletion of reserves of key chemical elements required in their manufacturing. The use of biodegradable and abundant organic (carbon-based) electronic materials can contribute to alleviate the environmental impact of the electronic industry. The pigment eumelanin is a bio-sourced candidate for environmentally benign (green) organic electronics. The biodegradation of eumelanin extracted from cuttlefish ink is studied both at 25 °C (mesophilic conditions) and 58 °C (thermophilic conditions) following ASTM D5338 and comparatively evaluated with the biodegradation of two synthetic organic electronic materials, namely copper (II) phthalocyanine (Cu-Pc) and polyphenylene sulfide (PPS). Eumelanin biodegradation reaches 4.1% (25 °C) in 97 days and 37% (58 °C) in 98 days, and residual material is found to be without phytotoxic effects. The two synthetic materials, Cu-Pc and PPS, do not biodegrade; Cu-Pc brings about the inhibition of microbial respiration in the compost. PPS appears to be potentially phytotoxic. Finally, some considerations regarding the biodegradation test as well as the disambiguation of "biodegradability" and "bioresorbability" are highlighted.

Medicinal Mushrooms against Influenza Viruses.

This review provides results obtained by scientists from different countries on the antiviral activity of medicinal mushrooms against influenza viruses that can cause pandemics. Currently, the search for antiviral compounds is relevant in connection with the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Medicinal mushrooms contain biologically active compounds (polysaccharides, proteins, terpenes, melanins, etc.) that exhibit an antiviral effect. The authors present the work carried out at the State Research Center of Virology and Biotechnology Vector in Russia, whose mission is to protect the population from biological threats. The research center possesses a collection of numerous pathogenic viruses, which allowed screening of water extracts, polysaccharides, and melanins from fruit bodies and fungal cultures. The results of investigations on different subtypes of influenza virus are presented, and special attention is paid to Inonotus obliquus (chaga mushroom). Compounds produced from this mushroom are characterized by the widest range of antiviral activity. Comparative data are presented on the antiviral activity of melanin from natural I. obliquus and submerged biomass of an effective strain isolated in culture against the pandemic strain of influenza virus A/California/07/09 (H1N1 pdm09).

Melanin pigments extracted from horsehair as antibacterial agents.

Here we present the important findings related to biologically derived pigments for potential use as antibacterial agents. Melanin biopigments extracted from Equus ferus hair exhibit a homogeneous elliptical microstructure with highly ordered semicrystalline features. Spectroscopic analysis indicates that melanin contains a high degree of redox active catechol groups, which can produce reactive oxygen species. The antibacterial activity of melanins was tested by incubating Escherichia coli and Staphylococcus aureus with melanins. The results showed 100% bacterial growth inhibition within 4 h. This finding suggests that melanin pigments may serve as naturally occurring antibacterial agents with unique redox chemistry and reactive oxygen species generation capability.

Obtaining chitin, chitosan and their melanin complexes from insects.

Interest in insects as a source of valuable biologically active substances has significantly increased over the past few years. Insects serve as an alternative source of chitin, which forms up to 40% of their exoskeleton. Chitosan, a deacetylated derivative of chitin, attracts the attention of scientists due to its unique properties (sorption, antimicrobial, film-forming, wound healing). Furthermore, some insect species are unique and can be used to obtain chitin- and chitosan-melanin complexes in the later stages of ontogenesis. Due to the synergistic effect, chitosan and melanin can enhance each other's biological activity, providing a wide range of potential applications.

Comparison of physicochemical and biochemical properties of natural and arginine-modified melanin from medicinal mushroom Ganoderma lucidum.

Melanin is a hydrophobic biomolecule produced widely in fungi. Compared with other fungi, health benefits have been associated with medicinal mushrooms, which may provide an excellent source of natural melanin. Nevertheless, the hydrophobicity of melanin may limit its applications. Consequently, the present study was carried out on isolation of melanin from the medicinal mushroom Ganoderma lucidum (GLM) and modification with arginine to improve its solubility. The physicochemical and biochemical properties of melanin were evaluated including structural characterization, solubility, stability, antioxidant activities, and inhibitory effect on pancreatic lipase activity. Arginine-modified melanin showed better solubility, higher color value, stronger antioxidant activity, and stronger inhibitory effect on pancreatic lipase activity in vitro than GLM. In addition, both have good stability in the dark and natural light. These results opened possibilities for providing an excellent source of natural melanin in health food or food additives fields.

Biological activities of melanin pigment extracted from Bombyx mori gut-associated yeast Cryptococcus rajasthanensis KY627764.

Melanin pigment has been produced and extracted from a wide variety of living forms ranging from microorganisms to higher organisms. Owing to the therapeutic nature of the pigment, various microbial populations have been explored for its production. Hence, we isolated a melanin producing yeast from the insect Bombyx mori gut microflora and identified it as Cryptococcus rajasthanensis based on the molecular characterization. The isolated yeast produced enhanced melanin pigment when cultured in the minimal L-tyrosine broth as compared to the Saboraud medium. The pigment was extracted and characterized as melanin based on UV-Visible spectroscopy, FTIR (Fourier-transform infrared) spectroscopy and 1H NMR (Nuclear magnetic resonance). The melanin pigment was evaluated as a potent bioactive molecule with bioactivity like antimicrobial, antioxidant, anti-inflammatory, and anticancer activity that describes the therapeutic nature of the extracted melanin pigment. Distinct from the biologically active role the melanin pigment isolated from the yeast, the Cryptococcus extract also exhibited killer toxin activity against the pathogenic yeast Candida albicans.

Multidisciplinary characterization of melanin pigments from the black fungus Cryomyces antarcticus.

Melanin is a natural pigment present in almost all biological groups, and is composed of indolic polymers and characterized by black-brown colorization. Furthermore, it is one of the pigments produced by extremophiles including those living in the Antarctic desert, and is mainly involved in their protection from high UV radiation, desiccation, salinity and oxidation. Previous studies have shown that melanized species have an increased capability to survive high level of radiation compared with the non-melanized counterpart. Understanding the molecular composition of fungal melanin could help to understand this peculiar capability. Here, we aimed to characterize the melanin pigment extracted from the Antarctic black fungus Cryomyces antarcticus, which is a good test model for radioprotection researches, by studying its chemical properties and spectral data. Our results demonstrated that, in spite of having a specific type of melanin as the majority of fungi, the fungus possesses the ability to produce both 1,8-dihydroxynaphthalene (DHN) and L 3-4 dihydroxyphenylalanine (L-DOPA) melanins, opening interesting scenarios for the protection role against radiation. Researches on fungal melanin have a huge application in different fields, including radioprotection, bioremediation, and biomedical applications. KEY POINTS: • Isolation and characterization by multidisciplinary approaches of fungal melanins. • Discovery that pathways for producing DOPA and DHN are both active even in its extreme habitat. • Hypothesis supporting the possibility of using melanin pigment for radioprotection.

Revisiting the role of Dcc in visual system development with a novel eye clearing method.

The Deleted in Colorectal Carcinoma (Dcc) receptor plays a critical role in optic nerve development. Whilst Dcc is expressed postnatally in the eye, its function remains unknown as Dcc knockouts die at birth. To circumvent this drawback, we generated an eye-specific Dcc mutant. To study the organization of the retina and visual projections in these mice, we also established EyeDISCO, a novel tissue clearing protocol that removes melanin allowing 3D imaging of whole eyes and visual pathways. We show that in the absence of Dcc, some ganglion cell axons stalled at the optic disc, whereas others perforated the retina, separating photoreceptors from the retinal pigment epithelium. A subset of visual axons entered the CNS, but these projections are perturbed. Moreover, Dcc-deficient retinas displayed a massive postnatal loss of retinal ganglion cells and a large fraction of photoreceptors. Thus, Dcc is essential for the development and maintenance of the retina.

Green pyomelanin-mediated synthesis of gold nanoparticles: modelling and design, physico-chemical and biological characteristics.

BACKGROUND: Synthesis of nanoparticles (NPs) and their incorporation in materials are amongst the most studied topics in chemistry, physics and material science. Gold NPs have applications in medicine due to their antibacterial and anticancer activities, in biomedical imaging and diagnostic test. Despite chemical synthesis of NPs are well characterized and controlled, they rely on the utilization of harsh chemical conditions and organic solvent and generate toxic residues. Therefore, greener and more sustainable alternative methods for NPs synthesis have been developed recently. These methods use microorganisms, mainly yeast or yeast cell extract. NPs synthesis with culture supernatants are most of the time the preferred method since it facilitates the purification scheme for the recovery of the NPs. Extraction of NPs, formed within the cells or cell-wall, is laborious, time-consuming and are not cost effective. The bioactivities of NPs, namely antimicrobial and anticancer, are known to be related to NPs shape, size and size distribution. RESULTS: Herein, we reported on the green synthesis of gold nanoparticles (AuNPs) mediated by pyomelanin purified from the yeast Yarrowia lipolytica. A three levels four factorial Box-Behnken Design (BBD) was used to evaluate the influence of temperature, pH, gold salt and pyomelanin concentration on the nanoparticle size distribution. Based on the BBD, a quadratic model was established and was applied to predict the experimental parameters that yield to AuNPs with specific size. The synthesized nanoparticles with median size value of 104 nm were of nanocrystalline structure, mostly polygonal or spherical. They exhibited a high colloidal stability with zeta potential of - 28.96 mV and a moderate polydispersity index of 0.267. The absence of cytotoxicity of the AuNPs was investigated on two mammalian cell lines, namely mouse fibroblasts (NIH3T3) and human osteosarcoma cells (U2OS). Cell viability was only reduced at AuNPs concentration higher than 160 µg/mL. Moreover, they did not affect on the cell morphology. CONCLUSION: Our results indicate that different process parameters affect significantly nanoparticles size however with the mathematical model it is possible to define the size of AuNPs. Moreover, this melanin-based gold nanoparticles showed neither cytotoxicity effect nor altered cell morphology.

ASSESSMENT OF RADIOPROTECTIVE ACTION OF BASIDIOMYCOTIC MELANIN PIGMENTS ON THE HEMATOPOIETIC SYSTEM OF Balb/C MICE UNDER EXPOSURE TO IONIZING RADIATION IN SUBLETHAL DOSE. / OTsINKA RADIOPROTEKTORNOÏ DIÏ MELANINOVYKh PIGMENTIV BAZYDIOMITsETIV NA GEMOPOETYChNU SYSTEMU MYShEY̆ Balb/C PRY OPROMINENNI IONIZUIuChOIu RADIATsIIeIu U SUBLETAL'NIY̆ DOZI.

OBJECTIVE: Assessment of radioprotective action of basidiomycotic melanin pigments on hematopoietic stem and progenitor bone marrow cells of Balb/C mice in case of exposure to ionizing radiation in sublethal dose. MATERIALS AND METHODS: Using original method of cultivation in gel diffusion chambers in vivo of bone marrow cells of Balb/C mice we investigated the colony-forming efficiency of hematopoietic progenitor cells of the ani- mals, which were exposed to ionizing radiation action in sublethal dose, in case of treatment with melanin pig- ments solution of basidiomycotic fungi as radioprotector. RESULTS AND CONCLUSIONS: Investigation of functional activity of bone marrow progenitor cells of Balb/C mice allowed assessing their hematopoiesis state in case of ionizing radiation action, as well as in case of previous treat- ment of the animals with the solution of melanin pigments as radioprotector. It was determined that under the influence of ionizing radiation the colony-forming activity of mice bone marrow has decreased comparing to con- trol. Solution of melanin pigments was able to enhance the functional activity of bone marrow of irradiated ani- mals. Obtained results of radioprotective action of basidiomycotic melanin pigments solution on irradiated stem cells and their descendants (progenitor cells) may become the evidence for development of the protective means for human organism from the injuring action of ionizing radiation.

Quantitation of eumelanin and pheomelanin markers in diverse biological samples by HPLC-UV-MS following solid-phase extraction.

Eumelanin and pheomelanin are well known and common pigments found in nature. However, their complex polymer structure and high thermostability complicate their direct chemical identification. A widely used analytical method is indirect determination using HPLC with UV detection of both types of melanin by their most abundant oxidation products: pyrrole-2,3-dicarboxylic acid (PDCA), pyrrole-2,3,5-tricarboxylic acid (PTCA), thiazole-4,5-dicarboxylic acid (TDCA), and thiazole-2,4,5-tricarboxylic acid (TTCA). An increasing interest in pigmentation in biological research led us to develop a highly sensitive and selective method to identify and quantify these melanin markers in diverse biological samples with complex matrices. By introducing solid-phase extraction (SPE, reversed-phase) following alkaline oxidation we could significantly decrease background signals while maintaining recoveries greater than 70%. Our HPLC-UV-MS method allows for confident peak identification via exact mass information in corresponding UV signals used for quantitation. In addition to synthetic melanin and Sepia officinalis ink as reference compounds eumelanin markers were detected in brown human hair and a brown bivalve shell (Mytilus edulis). Brown feathers from the common chicken (Gallus g. domesticus) yielded all four eumelanin and pheomelanin markers. The present method can be easily adapted for a wide range of future studies on biological samples with unknown melanin content.

Antioxidative Properties of Melanins and Ommochromes from Black Soldier Fly Hermetia illucens.

A comparative study of melanin and ommochrome-containing samples, isolated from the black soldier fly (BSF) by enzymatic hydrolysis, alkaline and acid alcohol extraction or by acid hydrolysis, was carried out. Melanin was isolated both as a melanin-chitin complex and as a water-soluble melanin. Acid hydrolysis followed by delipidization yielded a more concentrated melanin sample, the electron spin resonance (ESR) signal of which was 2.6 × 1018 spin/g. The ommochromes were extracted from the BSF eyes with acid methanol. The antiradical activity of BSF melanins and ommochromes was determined by the method of quenching of luminol chemiluminescence. It has been shown that delipidization of water-soluble melanin increases its antioxidant properties. A comparison of the antioxidant activity of BSF melanins and ommochromes in relation to photoinduced lipid peroxidation was carried out. The ESR characteristics of native and oxidized melanins and ommochromes were studied. It is assumed that H. illucens adult flies can be a useful source of natural pigments with antioxidant properties.

From Extraction to Advanced Analytical Methods: The Challenges of Melanin Analysis.

The generic term "melanin" describes a black pigment of biological origin, although some melanins can be brown or even yellow. The pigment is characterized as a heterogenic polymer of phenolic or indolic nature, and the classification of eu-, pheo- and allo- melanin is broadly accepted. This classification is based on the chemical composition of the monomer subunit structure of the pigment. Due to the high heterogeneity of melanins, their analytical characterization can be a challenging task. In the present work, we synthesized the current information about the analytical methods which can be applied in melanin analysis workflow, from extraction and purification to high-throughput methods, such as matrix-assisted laser desorption/ionization mass-spectrometry or pyrolysis gas chromatography. Our thorough comparative evaluation of analytical data published so far on melanin analysis has proven to be a difficult task in terms of finding equivalent results, even when the same matrix was used. Moreover, we emphasize the importance of prior knowledge of melanin types and properties in order to select a valid experimental design using analytical methods that are able to deliver reliable results and draw consistent conclusions.

Melanins of Inonotus Obliquus: Bifidogenic and Antioxidant Properties.

Extracts and melanins from Inonotus obliquus are widely used in medicine due to their high antioxidant properties. This study is dedicated to define the influence of the physicochemical and antioxidant properties of Inonotus obliquus melanins and their bifidogenic effects on Bifidobacterium bifidum 1 and Bifidobacterium animalis subsp. lactis. For this purpose, melanins precipitated from Inonotus obliquus aqueous extracts, obtained by a few methods, and separated melanin fractions by organic solvents were used. For the melanin physicochemical properties analysis spectrophotometry, electron paramagnetic resonance (EPR) spectroscopy and dynamic light scattering methods were applied. Melanins and their fractions difference in particle size and charge, antioxidant properties, and redox potential were revealed. It was shown that the redox potential, the size of melanin particles and the z-potential had maximum influence on bifidobacteria growth. The greatest activating effect on bifidobacteria was established by using melanin isolated from aqueous microwave extracts in concentrations of 10-13, 10-10, 10-5 g/cm3. The use of this melanin with antioxidant activity 0.67 ± 0.06 mg/g (expressed as ascorbic acid equivalent), and with redox potential -5.51 ± 2.22 mV as a prebiotic allowed the growth of Bifidobacterium bifidum 1 s to increase by 1.4 times in comparison with ascorbic acid by 24 h of cultivation.

Comparison of protocols for removal of melanin from genomic DNA to optimize PCR amplification of DNA purified from highly pigmented lesions.

Melanin is produced by melanocytes and protects against DNA damage by ultraviolet light. Unfortunately, the melanin protein present in melanoma tumor cells is often co-purified during DNA extraction, and this contamination may inhibit subsequent PCR methods, which directly impacts research applications and the molecular diagnostic tests needed for targeted therapeutics. There are presently no described purification protocols that efficiently remove melanin from genomic DNA. In this study, we compare six different methods for melanin removal from genomic DNA: Agarose Gel Electrophoresis, 1mg Chelex®-100, Chelex®-100 5%, centrifugation, OneStep™ PCR Inhibitor Removal Kit and centrifugation plus OneStep™ PCR Inhibitor Removal Kit. Each comparison was made using 16 formalin-fixed paraffin-embedded (FFPE) and 11 fresh cell line samples. All samples were initially tested using the multiplex PCR reaction for GAPDH gene that generates different sized amplified products: 100, 200, 300 and 400 base pairs, which could be inhibited by the addition of exogenous melanin. Six purification protocols were then applied, and all samples that amplified at least one GAPDH fragment were sequenced to analyze the presence of the BRAF V600E mutation. The efficiencies of amplification decreased for larger sized fragments in all methods. Our comparisons showed that centrifugation combined with the OneStep™ PCR Inhibitor Removal Kit was superior to all other methods for successful BRAF sequencing with 100% (100bp), 75% (200bp), 50% (300bp), and 31.3% (400bp) amplification efficiencies for the different amplicon sizes. In conclusion, this genomic DNA extraction method is highly efficient for successful PCR when tumor samples are contaminated with melanin.

Determination of eumelanin and pheomelanin in melanomas using solid-phase extraction and high performance liquid chromatography-diode array detection (HPLC-DAD) analysis.

Determination of eumelanin and pheomelanin in melanomas that exhibit different pigmentation was carried using a solid-phase extraction (SPE) preparation method based on weak anion exchange chemistry. This extraction significantly enhanced the chromatographic profile obtained by reverse phase high performance liquid chromatography-diode array detection (RP-HPLC-DAD). The SPE method was developed using aqueous standards of melanin markers: thiazole-2,4,5-tricarboxylic acid (TTCA), thiazole-4,5-dicarboxylic acid (TDCA), pyrrole-2,3-dicarboxylic acid (PDCA) and pyrrole-2,3,5-tricarboxylic acid (PTCA) and non-pigmented cell lines spiked with those markers. An excellent average recovery, above 90%, was obtained for the four markers with a relative standard deviation below 7%. We have also optimized the stationary phase and the mobile phase (phosphate concentration and pH) to improve sensitivity and to reduce the analysis time. Elution of the four markers is achieved in 5 min and total analysis of biological samples is completed in 15 min. The quantification limits for TDCA, TTCA, PDCA and PTCA are 60, 50, 47 and 48 ng/mL respectively. Furthermore, DAD detection improves the marker identification in complex matrices through the analysis of UV spectra. We have successfully applied this method to melanoma tumors and cells. Murine B16BL6 tumor are highly pigmented with mostly eumelanin (98.1% of eumelanin) while human SK-MEL-3 tumor contain about 30% pheomelanin. B16BL6 and B16F10 are eumelanic cells lines and NHEM melanocytes contain about 24% of pheomelanin.