Melanin-like Pigment Research Articles

The Production of Melanin Pigments From Some Pathogenic Fungi in Response to L-Tyrosine Under Laboratory Conditions

Background: This study aims to evaluate response of plant pathogenic fungi to the amino acid L-tyrosine through the production of melanin. Methods: Potato dextrose broth (PDB) and PDB with 1% L-tyrosine were used for current evaluation. Melanin was produced, extracted and characterized from pathogenic fungi that infected rice in Iraq. Results: Results indicated that L-tyrosine was used as a source of the precursor by fungi belonging to Bipolaris. spicifera R15, Alternaria. alternata R18, Exserohilum. rostratum R19, Alternaria. alternate R20, and Alternaria. tenuissima R23, also produced higher levels of condensed black pigment. While, fungi like Nigrospora oryzae R9, Curvularia lunata R7, C lunata R21 and A tenuissima R24 were used more than one pathway for melanin production. UV transmittance increased significantly from 600 nm to 300 nm whereas absorbance decreased at 260 and 280 nm for all of the studied fungi. Analysis of FT-IR spectra and related assignment group of resolved that melanin extracted from B. spicifera R15 (3408.22), A. alternate R20 (3392.79), and A. tenuissima R24 (3387). Wavenumbers 2924 cm−1 and 2854 cm−1 were found in E. rostratum R19, A. alternative R20, and A. tenuissima R24 melanin extracts, respectively. Meanwhile, B. spicifera R15 revealed a single peak at 2929 cm−1. 3007.02 cm−1, 3072.6 cm−1, 3074.53 cm−1, 2920.3 cm−1, 2704.2 cm−1, and 1861.31 cm−1 are also assigned wavenumbers. Conclusion: Melanin-like pigments are produced via L-tyrosine-dependent and independent pathways. All colors extracted from fungal hyphae were morphologically and chemically identical to melanin. A UV scan and FT-IR analysis revealed differences in the physical appearance of building units and the structure of the melanin pigment produced by plant pathogenic fungi.

Bioinspired Three-Component System to Prepare Full-Color Functional Biomimetic Pigments.

Nature provides a great source of inspiration for the development of sustainable materials with excellent properties, among which melanin with optical, electronic, and radiation protection properties are considered to be promising coloring materials. However, compared to chemical pigments, the single color, complex oxidation process, and poor solubility of natural melanin strongly limit their further applications. Here, we introduce a series of melanin-like polymeric pigments with amino acid-encoded physicochemical properties by a simple three-component reaction system. Our protocol enables artificial control of the chromophore structures through the rational design of the substrates and dopants, thereby combining the safety and functionality of biopigments with the color richness of chemical dyes. Similar to the photoprotective effect of natural melanin, the polymeric pigments showed excellent antioxidant activity in reducing free radicals and have the advantages of iridescent color, strong tinting strength, stability, and affordability. Furthermore, due to their ability to dye substrates, these biomimetic are expected to become new low-cost bioactive chromophores and find various biochemical applications such as in clothing and hair dyeing, food addition, and anticounterfeiting detection.

Mutation of hmgA, encoding homogentisate 1,2-dioxygenase, is responsible for pyomelanin production but does not impact the virulence of Burkholderia cenocepacia in a chronic granulomatous disease mouse lung infection.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals; pigment production has been reported to enable Bcc strains to overcome the host cell oxidative burst. In this work, we investigated the role of pyomelanin in resistance to oxidative stress and virulence in strains J2315 and K56-2, two epidemic CF isolates belonging to the Burkholderia cenocepacia ET-12 lineage. We previously reported that a single amino acid change from glycine to arginine at residue 378 in homogentisate 1,2-dioxygenase (HmgA) affects the pigment production phenotype: pigmented J2315 has an arginine at position 378, while non-pigmented K56-2 has a glycine at this position. Herein, we performed allelic exchange to generate isogenic non-pigmented and pigmented strains of J2315 and K56-2, respectively, and tested these to determine whether pyomelanin contributes to the protection against oxidative stress in vitro as well as in a respiratory infection in CGD mice in vivo. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist oxidative stress with H2O2 and NO in vitro and did not change the virulence and infection outcome in CGD mice in vivo suggesting that other factors besides pyomelanin are contributing to the pathophysiology of these strains.IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria that are often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals and overcoming the host cell oxidative burst. We investigated the role of pyomelanin in Burkholderia cenocepacia strains J2315 (pigmented) and K56-2 (non-pigmented) and performed allelic exchange to generate isogenic non-pigmented and pigmented strains, respectively. Our results indicate that the altered pigment phenotype does not significantly impact these strains' ability to resist H2O2 or NO in vitro and did not alter the outcome of a respiratory infection in CGD mice in vivo. These results suggest that pyomelanin may not always constitute a virulence factor and suggest that other features are contributing to the pathophysiology of these strains.

Site-specific fabrication of a melanin-like pigment through spatially confined progressive assembly on an initiator-loaded template

Melanin-like nanomaterials have emerged in surface biofunctionalization in a material-independent manner due to their versatile adhesion arising from their catechol-rich structures. However, the unique adhesive properties of these materials ironically raise difficulties in their site-specific fabrication. Here, we report a method for site-specific fabrication and patterning of melanin-like pigments, using progressive assembly on an initiator-loaded template (PAINT), different from conventional lithographical methods. In this method, the local progressive assembly could be naturally induced on the given surface pretreated with initiators mediating oxidation of the catecholic precursor, as the intermediates generated from the precursors during the progressive assembly possess sufficient intrinsic underwater adhesion for localization without diffusion into solution. The pigment fabricated by PAINT showed efficient NIR-to-heat conversion properties, which can be useful in biomedical applications such as the disinfection of medical devices and cancer therapies.

Identification and Biosynthesis of DHN-melanin Related Pigments in the Pathogenic Fungi Monilinia laxa, M. fructicola, and M. fructigena

Monilinia is the causal agent of brown rot in stone fruit. The three main species that cause this disease are Monilinia laxa, M. fructicola, and M. fructigena, and their infection capacity is influenced by environmental factors (i.e., light, temperature, and humidity). To tolerate stressful environmental conditions, fungi can produce secondary metabolites. Particularly, melanin-like pigments can contribute to survival in unfavorable conditions. In many fungi, this pigment is due to the accumulation of 1,8-dihydroxynaphthalene melanin (DHN). In this study, we have identified for the first time the genes involved in the DHN pathway in the three main Monilinia spp. and we have proved their capacity to synthetize melanin-like pigments, both in synthetic medium and in nectarines at three stages of brown rot development. The expression of all the biosynthetic and regulatory genes of the DHN-melanin pathway has also been determined under both in vitro and in vivo conditions. Finally, we have analyzed the role of three genes involved in fungi survival and detoxification, and we have proved that there exists a close relationship between the synthesis of these pigments and the activation of the SSP1 gene. Overall, these results deeply describe the importance of DHN-melanin in the three main species of Monilinia: M. laxa, M. fructicola, and M. fructigena.

Isolation and characterization of blackish-brown BY2-melanin accumulated in cultured tobacco BY-2 cells.

The tobacco BY-2 cell line is one of the most utilized plant cell lines. After long-term culture, the cells turn brown to black, but the causal pigment is unknown. We successfully isolated a blackish-brown pigment from BY-2 cells cultured for 3 weeks. Morphological and spectroscopic analyses indicated that the pigment had similar features to a melanin-like substance reported previously. Furthermore, physicochemical analyses revealed that this pigment possessed most of the properties of melanin-like pigments. In addition, the high nitrogen content suggested that it differed from common plant melanins classified as allomelanins, suggesting a novel eumelanin-like pigment: "BY2-melanin". This is the first example showing that eumelanin-like pigments are produced in the cultures of plant cells for which the accumulation of melanin has not been reported. This tobacco BY-2 cell culture technique may represent a customizable and sustainable alternative to conventional melanin production platforms, with significant potential for industrial and pharmacological applications.

SEASONAL OCCURRENCE OF NEOECHINORHYNCHUS EMYDIS (PHYLUM: ACANTHOCEPHALA) IN THE FRESHWATER SNAIL, PLANORBELLA CF. P. TRIVOLVIS, FROM OKLAHOMA.

The acanthocephalan Neoechinorhynchus emydis has a complex life cycle and infects turtle, ostracod, and snail hosts. However, little information is available on the seasonal distribution or the effects of N. emydis on freshwater snail hosts. To address this, we examined the seasonal distribution and melanization of acanthocephalans in Planorbella cf. Planorbella trivolvis snails from a single location in north-central Oklahoma. Seasonally, prevalence of N. emydis was 0% during the winter, increased to 50% during the summer, and declined to 17% in the fall. Mean abundance exhibited more variation but generally followed a similar pattern as prevalence. More important, all acanthocephalans located within the head/foot region of snail hosts contained melaninlike pigment surrounding each worm, suggesting that snails were mounting an immunological reaction to infections with N. emydis. Snail shell diameter was greatest during the fall and decreased during the winter, indicating that larger or older snails were dying during the winter. However, because field-collected snails were commonly infected with trematodes, and snail size varied significantly with season, it was unclear whether the observed seasonal dynamics of acanthocephalan infections were a result of snail mortality resulting from snail age, parasitic infections, or a combination of factors. To control for these factors, we exposed laboratory-reared Planorbella cf. P. trivolvis snails to naturally infected ostracods in field cages for 5-wk intervals during the winter, spring, and summer. Data from snail-cage infections were consistent with the seasonal field survey such that N. emydis infections were highest in the summer (20%) and lowest (0%) in the winter, suggesting that snails were not ingesting infected ostracods during the winter. However, fewer of our laboratory-reared snails survived in field cages during winter than during spring and summer, suggesting that snails may die more often during harsh winter conditions. Finally, we conducted a laboratory survival experiment by testing the life span and egg production of field-collected snails of various sizes that were naturally infected with acanthocephalans or trematodes or both. Our snail-survival experiment indicated that snail size but not infection status with acanthocephalans or trematodes affected snail survival, with larger snails surviving a shorter amount of time than smaller snails. In addition, snails infected with trematodes laid significantly fewer eggs compared with uninfected snails or snails infected with acanthocephalans. However, we found no significant difference in the number of eggs laid by acanthocephalan-infected and uninfected snails. Although other abiotic factors still need evaluation, we suggest that the occurrence of acanthocephalans in snails throughout the year may be partially influenced by the abundance of infected ostracods that snails may be ingesting and snail population fluctuations during the year.

Ochronosis – a rare metabolic disease

Alkaptonuria is a rare disorder, an autosomal recessive condition with genetic determinism and hereditary transmission, having a prevalence of 1 per 1 million population in USA. The pathogenesis includes the deficiency of the homogentisate 1,2-dioxygenase (HGD) enzyme, an intermediary enzyme in phenylalanine and tyrosine catabolism. Mutations in HGD gene leads to deficient levels of functional HGD and an excess of homogentisic acid (HGA). Although HGA is rapidly excreted by the kidneys, it slowly accumulates in various tissues. Due to HGA oxidase deficiency, HGA turns into melanin-like pigment which determines: alkaptonuria, accumulation in the connective tissues, in the joints, or can make cardiovascular and genitourinary deposits. The chronic accumulation of HGA destroys the affected tissue, leading to the characteristic black-blue color and to clinical symptoms of alkaptonuria. The aim of this paper is to investigate the particularities of rheumatic manifestations in a rare metabolic disease and to support the correct diagnosis. A 58-year-old male patient was admitted to our clinic in 2019 for bilateral knee and left shoulder pain. In 2008 he was diagnosed with polyarticular ochronosis having dorsal and lumbar pain, mixed scapulohumeral pain, lumbar intervertebral disk calcifications; at that time, a diagnosis of ankylosing spondylitis or Forestier disease was excluded. At the current admission, the patient has been thoroughly reassessed to obtain a proper diagnosis and to determine the severity of the disease. The ochronotic axial damage caused important differential diagnosis problems with ankylosing spondylitis. Pigment deposition in the eyes, ears and skin does not cause problems to patients, but cardiovascular and genitourinary deposition leads to important complications. Kinetotherapy and NSAIDs are beneficial for pain symptoms. There is no specific medication for stopping the disease progression. Conclusions. Ochronosis is a rare disease which can cause a lot of problems regarding a proper diagnosis and treatment. When differential diagnosis with AS is difficult, the HLA-B27 genotyping is recommended. Final diagnosis is based on qualitative and quantitative urinary tests. The treatment includes only symptomatic drugs such as NSAIDs and kinetotherapy to improve joint mobility and muscle toning.

Pyomelanin-Producing Brevundimonas vitisensis sp. nov., Isolated From Grape (Vitis vinifera L.).

A novel endophytic bacterial strain, designated GR-TSA-9T, was isolated from surface-sterilized grape (Vitis vinifera L.). 16S rRNA gene sequence analyses showed that the isolate was grouped within the genus Brevundimonas, displaying the highest similarity with Brevundimonas lenta DS-18T (97.9%) and Brevundimonas kwangchunensis KSL-102T (97.8%) and less than 97.5% similarity with other members of Brevundimonas. The strain GR-TSA-9T was a gram negative, rod shaped, facultatively anaerobic, catalase and oxidase positive, and motile bacterium. Its growth occurred at 10–37°C (optimally 25–30°C), at pH 7.0–8.0, and in NaCl 0–1% (optimally 0%). It contained ubiquinone-10 as a respiratory quinone, and the major cellular fatty acids (>10% of the total) were C16:0 (14.2%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c, 65.6%). The polar lipids present in the strain were phosphoglycolipids, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol, and unidentified lipids (L1, L2, and L4). The strain had one 2,976,716bp circular chromosome with a G+C content of 66.4%. The digital DNA–DNA hybridization value between strain GR-TSA-9T and B. lenta DS-18T was 20.9%, while the average nucleotide identity value was 76.7%. In addition, the dDDH and ANI values to other members in this genus, whose genome sequences are available, are less than 21.1 and 77.6%. Genome annotation predicted the presence of some gene clusters related to tyrosine degradation and pyomelanin formation. Strain GR-TSA-9T produced a brown melanin-like pigment in the presence of L-tyrosine-containing media. The highest pigment production (0.19g/L) was observed in tryptic soy broth with 1.0mg/ml L-tyrosine at 25°C for 6days of culture. Biophysical characterization by ultraviolet (UV)–visible spectroscopy, Fourier-transform infrared spectroscopy, and electrospray ionization mass spectrometry confirmed that the pigment was pyomelanin. Additionally, melanized GR-TSA-9T cells could protect the cells against UVC exposure. The phylogenetic, genomic, phenotypic, and chemotaxonomic features indicated that strain GR-TSA-9T represents a novel melanin-producing species of Brevundimonas. The type strain was GR-TSA-9T (KCTC 82386T=CGMCC 1.18820T).

Pulmonary Phaehyphomycosis in Retroviral Disease Patient

Pulmonary phaeohyphomycosis is a rare infection in the lung caused by black fungi containing a cytoplasmic melanin-like pigment. A 42-year-old man with underlying retroviral disease on HAART was investigated for having constitutional symptoms. Despite undetectable viral load and a high CD4 count, he was found to have unexplained significant loss of weight and appetite over a period of 6 months. Clinical examination revealed a cachexic man with multiple inguinal lymphadenopathies. Excisional biopsy of the inguinal lymph node revealed reactive follicular hyperplasia. CT Thorax, Abdomen and Pelvis was arranged to look for occult malignancy or infection and he was found to have multiple non-enhancing subcentimeter lung nodules mainly at the lateral segment of the right middle lobe of his lung. The largest nodule measured about 0.8 x 1.5 x 0.5 (AP x W x CC), with some nodules having an irregular margin with no extension into the adjacent bronchi. Bronchoscopy was done and demonstrated a black patch at the right intermedius, lateral segment of the middle lobe which did not disappear upon bronchial flush or wash. Histopathological examination found focal areas of blackish pigment and the bronchial alveolar lavage sent for fungal culture grew Cladosporium species. The patient was treated with oral Itraconazole with marked clinical improvement. This case highlights an unusual black fungi infection in the lung that stands out not only for its rarity and it's responsiveness to treatment, but also the susceptibility of an RVD positive patient to this infection despite having suppressed viral load and normal CD4 count.International Journal of Human and Health Sciences Supplementary Issue-2: 2021 Page: S17

Oxidative stress in the skin: Impact and related protection.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Colorful Pigments for Hair Dyeing Based on Enzymatic Oxidation of Tyrosine Derivatives.

Melanin exists widely in nature and can afford a variety of colors from black to brown and red according to chemical structure differences and specific mixtures. Inspired by nature, this work reports that tyrosine derivatives with different protecting groups at its N- or C-terminal can be enzymatically oxidized into melanin-like pigments with a wide range of colors. The emergence of colorful pigments can be attributed to the incomplete enzymatic oxidation and polymerization caused by the chemical premodification of the tyrosine molecule. The pigments can be deposited on the surface of the hair to obtain a series of colorful and saturated hair dye effects. Moreover, after the pigments were coated on the hair, we can further deposit silver nanoparticles through in situ reduction, making these coatings have anti-inflammatory and antibacterial potential, thereby expanding their potential use for people with low immunity or those who work in hospitals. This work proposes a green and effective way to synthesize colorful pigments with great potential applications in the hair dying and cosmetic industries.

Candida glabrata produces a melanin-like pigment that protects against stress conditions encountered during parasitism.

Aim: Melanin has been linked to pathogenesis in several fungi. They often produce melanin-like pigments in the presence of L-dihydroxyphenylalanine (L-DOPA), but this is poorly studied in Candida glabrata. Methods & materials:C. glabrata was grown in minimal medium with or without L-DOPA supplementation and submitted to a chemical treatment with denaturant and hot acid. Results:C. glabrata turned black when grown in the presence of L-DOPA, whereas cells grown without L-DOPA supplementation remained white. Biophysical properties demonstrated that the pigment was melanin. Melanized C. glabrata cells were effectively protected from azoles and amphotericin B, incubation at 42°Cand macrophage killing. Conclusion: In the presence of L-DOPA, C. glabrata produces melanin, increases antifungal resistance and enhances host survival.

Chemical Composition and Ultraviolet Absorption Activity of an Aqueous Alkali Extract from the Fruiting Bodies of the Tinder Conk Mushroom, Fomes fomentarius (Agaricomycetes).

White rot mushroom Fomes fomentarius is a medicinal fungus with great potential to be explored. This work focused on the chemical composition of a basic aqueous extract from F. fomentarius fruiting bodies. The extract was mostly composed of phenolics, carbohydrates, minerals, and crude fat with a low amount of proteins and chitin. One-third of the total carbohydrates were in the form of beta-glucans with minor amounts of alpha-glucans. The most valuable essential part of the extract was composed of an acid-resistant ultraviolet (UV)-absorbing mixture of phenolic compounds such as melanins, lignins, and humic acids. These compounds, also referred to as melanin-like pigments, provided for the high antioxidant activity of the extract measured in vitro. Moderate sun-protective capacity was observed with regard to UVB rays and also expected in the UVA range. Quantification of melanin-like pigments in the F. fomentarius extract was possible either gravimetrically as acid-insoluble residue or spectrophotometrically in the UV region. Melanin estimation, based on nitrogen measurements, offered misleading results due to the presence of nitrogen-free melanins along with other nitrogen-containing compounds such as proteins and chitin. F. fomentarius water-soluble basic extract, containing beta-glucans and rich in melanin-like substances, could be used, for example, for topical skin application to prevent cell damage caused by excessive UV exposure or cytotoxic free radicals. The bioactive potential, safety, and further applications of the F. fomentarius extract are currently being investigated.

Homogentisic acid affects human osteoblastic functionality by oxidative stress and alteration of the Wnt/β-catenin signaling pathway.

Alkaptonuria (AKU) is a rare disease correlated with deficiency of the enzyme homogentisate 1,2 dioxygenase, which causes homogentisic acid (HGA) accumulation. HGA is subjected to oxidation/polymerization reactions, leading to the production of a peculiar melanin-like pigmentation (ochronosis) after chronic inflammation, which is considered as a triggering event for the generation of oxidative stress. Clinical manifestations of AKU are urine darkening, sclera pigmentation, early severe osteoarthropathy, and cardiovascular and renal complication. Despite major clinical manifestations of AKU being observed in the bones and skeleton, the molecular and functional parameters are so far unknown in AKU. In the present study, we used human osteoblasts supplemented with HGA as a AKU cellular model. We observed marked oxidative stress, and for the first time, we were able to correlate HGA deposition with an impairment in the Wnt/β-catenin signaling pathway, opening a range of possible therapeutic strategies for a disease still lacking a known cure.

Presentation of 14 alkaptonuria patients from Turkey.

Background Alkaptonuria (OMIM: 203500) is an inborn error of metabolism due to homogentisate 1,2-dioxygenase homogentisic acid 1,2 dioxygenase (HGD) enzyme deficiency. Due to the enzyme deficiency, homogentisic acid cannot be converted to maleylacetoacetate and it accumulates in body fluids. Increased homogentisic acid is converted to benzoquinones, the resulting benzoquinones are converted to melanin-like pigments, and these pigments are deposited in collagen - this process is called ochronosis. In patients with alkaptonuria, the urine is darkened, which is misinterpreted as hematuria, the incidences of renal stones, arthritis and cardiac valve calcification are increased, and spontaneous tendon ruptures, prostatitis and prostate stones can be encountered. The present study aimed to evaluate the HGD gene mutations in 14 patients with alkaptonuria. Methods Fourteen patients diagnosed with alkaptonuria and followed up from 1990 to 2014 were retrospectively evaluated. Their demographic, clinical and treatment-related data were retrieved from hospital files. For mutation analysis, genomic DNAs of the patients were isolated from their peripheral blood samples. Variations in the HGD gene were scanned on the HGD-mutation database (http://hgddatabase.cvtisr.sk). Results Among 14 patients, the female/male ratio was 1/1 and the median age was 9 years (range, 6-59 years). All patients were symptomatic at their first visit and the most common symptom was dark urine (71%) followed by arthralgia. Independent of the urinary homogentisic acid concentrations, patients with the presenting symptom of arthralgia were elder. Nine different mutations including p.Ser59AlafsX52, p.Gly161Arg, p.Asn219Ser, p.Gly251Asp, p.Pro274Leu, p.Arg330Ser, p.Gly372Ala, c.656_657insAATCAA and a novel mutation of p.Val316Ile were detected. All of the pediatric-age patients (n = 13) were treated with ascorbic acid at a dose of 250-1000 mg/day. Conclusions Nine different HGD gene mutations with a novel one, p.Val316Ile, were detected. The most common mutation was p.Ser59AlafsX52 for the HGD gene followed by p.Gly161Arg and p.asn219Ser, which can be considered specific to the Turkish population.

Characterization of a nontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica.

In this study, we report on the ability of the yeast Yarrowia lipolytica W29 to produce an extracellular melanin-like brown pigment at high yield (0.5 mg/ml) in culture medium supplemented with L-tyrosine. This pigment has been characterized as pyomelanin and its synthesis was found to occur by the so-called HGA-melanin pathway. The purified pyomelanin was found embedded with antioxidant properties as it exhibited a radical scavenging activity toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 of 230 μg/ml. It was also characterized as noncytotoxic toward two mammalian cell lines, namely the mouse fibroblast NIH3T3 and human keratinocytes HaCaT. When blended with different commercial sunscreens, the purified pyomelanin increased significantly the sun protection factor (SPF) value, highlighting its potential utilization as UV-filter in cosmetic preparations.

A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors

Hereditary tyrosinemia type 1 (HT1) and alkaptonuria (AKU) are inherited metabolic disorders caused by defective enzymes involved in tyrosine catabolism. Nitisinone, an ex-herbicide and member of the β-triketone family, is therapeutically applied to prevent accumulation of toxic metabolites in patients by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD). Here, we developed a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of human HPD inhibitors in a high-throughput and a robust fashion. The principle of our screening system is based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in E. coli and subsequent production of a soluble melanin-like pigment. With the aim to optimise the assay, we tested different E. coli strains, expression and reaction temperatures, and time-points for supplementing the substrate. We found that in our assay the addition of prototypical β-triketone HPD inhibitors decreases pigment production in a dose-dependent manner with increasing inhibitor concentrations. In addition, plate uniformity, signal variability and spatial uniformity assessment showed that we have developed a robust high-throughput screening assay that is simple to use, cost-effective and enables identification and evaluation of novel therapeutic human HPD inhibitors for the treatment of tyrosine-related metabolic disorders.

Acorn-related acquired pseudomelanosis in Calabrian black pigs

BackgroundMelanosis of lymph nodes in black pigs has generally been related to regression of congenital melanoma and, occasionally, to ingestion of acorns. The aim of this manuscript is to confirm the hypothesis of a possible acquired acorn-related pseudomelanosis in the Nero Calabrese pig, a swine breed belonging to the group of Italian native breeds and whose coverage area corresponds to the region of Calabria, southern Italy. This pig is characterized by slow-growing subjects, producing, however, high quality meat suitable for the production of sausages and fine hams. The study was carried out on 142 normally slaughtered pigs. All organs were examined. Lymph nodes and intestine (jejunum) were sampled. Histochemistry was performed on deparaffinized histological sections to identify the cell types involved and to characterize the pigment stored. To further confirm the pigmentation disorder, immunohistochemistry was carried out. Total phenolic substances were identified in acorns through the use of a biochemical reaction.ResultsLymph node pigmentation appears directly related to acorn ingestion, with a higher incidence in the group which was 70% natural fed (acorn of Quercus virgiliana). Moreover, findings obtained revealed how different amounts of phenolic substrates present in Q. virgiliana and Q. ilex acorns can influence the incidence of such exogenous pigmentation.ConclusionThe findings obtained in this study confirm the acquired nature of the melanin-like pigmentation detected in lymph nodes from acorn-fed swine. Acquired pigmentation must be differentiated from true melanosis as well as from melanosis related to tumor regression of congenital melanoma. This thesaurismosis can be proposed as a marker of wellbeing and quality, confirming that the pigs have been bred and fed in natural conditions.

Serum Oxidative-Antioxidative Status in Patients With Alkaptonuria

BackgroundAlkaptonuria (AKU) is a rare genetic disease associated with the deposition of melanin-like pigments (ochronosis) in connective tissues. However, data regarding the effect of oxidative stress products on disease pathogenesis are limited. The purpose of this study was to investigate oxidative stress and related factors in patients with alkaptonuria and compare the findings with those in healthy control subjects.MethodsThe study sample comprised of 21 AKU patients and 19 age- and sex-matched healthy controls. Serum samples were obtained to detect the total antioxidative capacity (TAC), and oxidation degradation products of thiobarbituric acid-reactive substances, protein carbonyls, advanced oxidation protein products, and homogentisic acid levels in urine were determined.ResultsSerum TAC, oxidation degradation products of thiobarbituric acid-reactive substances, and protein carbonyl levels in the AKU group were higher than those measured for the control subjects, and the difference was statistically significant (P < 0.05). Moreover, a positive correlation was found between the patient’s serum protein carbonyl, patient’s age and AKU severity score (r = 0.492 and 0.746, respectively; P < 0.05). Furthermore, the protein carbonyl serum levels can be used to predict the disease severity score in alkaptonuria patients (P < 0.05).ConclusionsIn sum, the study results provide further support for the role of oxidation in the pathogenesis of alkaptonuria, suggesting presence of a more complex relationship than what has been previously assumed. Thus, further studies are needed to clarify these conflicting results.