Comparative Molecular Mechanisms of Biosynthesis of Naringenin and Related Chalcones in Actinobacteria and Plants: Relevance for the Obtention of Potent Bioactive Metabolites

Naringenin and its glycosylated derivative naringin are flavonoids that are synthesized by the phenylpropanoid pathway in plants. We found that naringenin is also formed by the actinobacterium Streptomyces clavuligerus, a well-known microorganism used to industrially produce clavulanic acid. The production of naringenin in S. clavuligerus involves a chalcone synthase that uses p-coumaric as a starter unit and a P450 monoxygenase, encoded by two adjacent genes (ncs-ncyP). The p-coumaric acid starter unit is formed by a tyrosine ammonia lyase encoded by an unlinked, tal, gene. Deletion and complementation studies demonstrate that these three genes are required for biosynthesis of naringenin in S. clavuligerus. Other actinobacteria chalcone synthases use caffeic acid, ferulic acid, sinapic acid or benzoic acid as starter units in the formation of different antibiotics and antitumor agents. The biosynthesis of naringenin is restricted to a few Streptomycess species and the encoding gene cluster is present also in some Saccharotrix and Kitasatospora species. Phylogenetic comparison of S. clavuligerus naringenin chalcone synthase with homologous proteins of other actinobacteria reveal that this protein is closely related to chalcone synthases that use malonyl-CoA as a starter unit for the formation of red-brown pigment. The function of the core enzymes in the pathway, such as the chalcone synthase and the tyrosine ammonia lyase, is conserved in plants and actinobacteria. However, S. clavuligerus use a P450 monooxygenase proposed to complete the cyclization step of the naringenin chalcone, whereas this reaction in plants is performed by a chalcone isomerase. Comparison of the plant and S. clavuligerus chalcone synthases indicates that they have not been transmitted between these organisms by a recent horizontal gene transfer phenomenon. We provide a comprehensive view of the molecular genetics and biochemistry of chalcone synthases and their impact on the development of antibacterial and antitumor compounds. These advances allow new bioactive compounds to be obtained using combinatorial strategies. In addition, processes of heterologous expression and bioconversion for the production of naringenin and naringenin-derived compounds in yeasts are described.

Orange-Brown Pigment Production from an Endophytic Fungus Aspergillus Sp. N11 and its Potential Pharmaceutical Applications

Endophytic fungi are the main source of natural compounds including pigments having various industrial applications. Present study describes the production of extracellular orange-brown pigment from an endophytic fungal isolate Aspergillus sp. N11from Teucrium stocksianum. The optimum conditions for pigment production from this isolate was investigated and results showed that highest yield was observed in Potato dextrose broth, at pH 5 and 30 ℃ under shaking condition at 150 rpm for 7-10 days. The pigment was extracted in ethyl acetate and purified using column chromatography. Three different pigments were purified (yellow, light brown and orange-brown) and characterized based on Thin layer chromatography and Fourier transform infrared spectroscopy. The antimicrobial activity of purified fragments showed maximum zone of inhibition of 40 mm against S. aureus while for P. aeruginosa maximum zone of 50 mm and maximum antifungal activity of 20 mm against C. albicans. The antioxidant potential of purified pigment obtained from Aspergillus sp. N11 indicates that maximum scavenging activity of 67%. The results showed that purified pigments are astaxanthins belonging to oxygen containing carotenoids. The purified astaxanthins showed antibacterial, antifungal and antioxidant activities indicating its potential to be utilized in pharmaceutical and food industries.

Malignant Melanoma With Neuroendocrine Differentiation: A Case Report and Literature Review

BackgroundMelanoma has a wide range of histologic variants and cytomorphologic features that make its diagnosis challenging. Melanoma can also rarely have neuroendocrine markers adding further diagnostic uncertainty particularly given that unrelated tumor types, such as prostate cancer, can also display focal neuroendocrine differentiations.Case presentationOur patient is a 74-year-old Caucasian man found to have a lung mass. Initial biopsy revealed typical microscopic morphology and neuroendocrine differentiation consistent with small cell carcinoma. Despite standard chemoradiation treatment, the patient continued to progress with new metastasis in the brain, liver and bone. Subsequent chest wall biopsy revealed golden-brown pigment associated with melanin. Further tumor immunohistochemistry revealed extensive neuroendocrine differentiation with CD56, synaptophysin, and INSM1, as well as strong immunoreactivity for melanocyte markers including SOX10, S100, PRAME, and MITF, consistent with metastatic melanoma with neuroendocrine differentiation. Genomic testing revealed increased tumor mutational burden and alterations in NF1, BRAF, CDKN2A/B, TERT. The patient was transitioned to checkpoint inhibitor therapy with nivolumab and ipilimumab and had resolution of his intracranial mass and decrease in size of other metastatic lesions.ConclusionOften the combination of anatomic findings such as a lung mass, typical microscopic morphology, and confirmation of neuroendocrine differentiation correctly identifies a patient with small cell carcinoma. However, in a patient who fails to respond to treatment, a broader immunohistochemical workup along with molecular testing with additional tissue may be warranted.

Highly Effective Protocol for Differentiation of Induced Pluripotent Stem Cells (iPS) into Melanin-Producing Cells

Melanin is a black/brown pigment present in abundance in human skin. Its main function is photo-protection of underlying tissues from harmful UV light. Natural sources of isolated human melanin are limited; thus, in vitro cultures of human cells may be a promising source of human melanin. Here, we present an innovative in vitro differentiation protocol of induced pluripotent stem cells (iPS) into melanin-producing cells, delivering highly pigmented cells in quantity and quality incomparably higher than any other methods previously described. Pigmented cells constitute over 90% of a terminally differentiated population and exhibit features characteristic for melanocytes, i.e., expression of specific markers such as MITF-M (microphthalmia-associated transcription factor isoform M), TRP-1 (tyrosinase-related protein 1), and TYR (tyrosinase) and accumulation of black pigment in organelles closely resembling melanosomes. Black pigment is unambiguously identified as melanin with features corresponding to those of melanin produced by typical melanocytes. The advantage of our method is that it does not require any sophisticated procedures and can be conducted in standard laboratory conditions. Moreover, our protocol is highly reproducible and optimized to generate high-purity melanin-producing cells from iPS cells; thus, it can serve as an unlimited source of human melanin for modeling human skin diseases. We speculate that FGF-8 might play an important role during differentiation processes toward pigmented cells.

First report of Stemphylium lycopersici causing leaf spot on Polygonatum kingianum in China

Polygonatum kingianum, a member of the Liliaceae, is valued in traditional medicine and as a vegetable food crop. In July 2019, more than 50% of P. kingianum growth was suppressed in several field nurseries in Simao, Mojiang, Jingdong and Lancang County, Puer City, China. At the early stage of infection, symptoms manifested as a small circular brown spot. As the lesion matured, the spot gradually enlarged, forming an oval to irregular lesion with reddish-brown and dark green borders. In serious cases, the leaves were withered, and became brittle with cracks. The infected plants were collected from six major fields. The tissues of diseased leaves were soaked in 75% ethanol for 10 s, 0.1% mercuric chloride for 2 min, rinsed with sterilized water, and placed on potato dextrose agar (PDA) at 25℃ for 7 days. On PDA, four strains were isolated, and the colony was gray to dark yellowish-brown, flocculent, regular with concentric growth rings. Strain PKLS06 produced a dark red to brown pigment in the agar medium. On lesions, the conidiophores were solitary or in fascicles, straight or slightly curved, brown, with a conical apex, with three to five septa. The conidiogenous cells were pale-brown and swollen at the apex. On PDA, spores were solitary, oblong, bluntly rounded or sometimes with a point at the apex, with two to five transverse septa and one to two longitudinal septa with contractions at the main transverse septum. Morphological characteristics were consistent with published descriptions of Stemphylium lycopersici (Kee et al. 2017; Xie et al. 2018). For molecular identification, rDNA internal transcribed spacer (ITS) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene were amplified and sequenced (ITS accessions: MW243098, MW243099, MW243100, MW243101; gpd accessions: MW246803, MW246804, MW246805, MW246806) using published primers (White et al. 1990; Câmara et al. 2002). A phylogenetic tree was developed by Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian inference (BI). These four isolates fall into the S. lycopersici clade with strong support and all isolates were distinguished clearly from other species. Pathogenicity tests were performed using these four isolates. Each isolate was cultured on PDA and shake-cultured in V-8 juice broth (Nasehi et al. 2014). Conidia were resuspended in sterilized water (1×106 conidia/mL) and inoculated on intact leaves with injury of 1-year-old P. kingianum. The plants were incubated at 25℃ with a 12 h photoperiod and 90% humidity. A small spot began to appear after 3 days, and symptoms were similar to the those observed in the nursery after 10 days. Interestingly, the pathogenicity of strain PKLS06 was relatively weaker. Control plants treated with sterile water showed no disease symptoms. Re-isolated strains had the same morphological characteristics and the same ITS and gpd sequences as the original isolates, thus fulfilling Koch’s postulates. S. lycopersici, an important pathogen, is widely distributed, and can cause a variety of plant diseases. It is noteworthy that the disease was observed on a plant in the Liliaceae, expanding the host range of S. lycopersici, which previously was reported to primarily infect plants in the Solanaceae. Based on the results presented above, P. kingianum is a new host plant of S. lycopersici in China. This disease is a threat for nursery production of P. kingianum, leading to a reduction in yields and economic losses. References Kee, Y. J., et al. 2017. Plant Disease 102 (2): 445–446 Xie, X. W., et al. 2019. Canadian Journal of Plant Pathology-Revue Canadienne De Phytopathologie 41 (1): 124–128 White, T. J., et al. 1990. PCR Protocols: A Guide to Methods and Applications PCR Protocols: A Guide to Methods and Applications 18: 315–322 Câmara M. P. S., et al. 2002. Mycologia 94 (4): 660–672 Nasehi A., et al. 2014. Archives of Phytopathology & Plant Protection, 47 (14): 1658-1665.

Gingival depigmentation with scalpel and diode laser

Melanin, a nonhemoglobin-derived brown pigment, is the most common of the endogenous pigments and is produced by melanocytes present in the basal layer of the epithelium. Gingival hyperpigmentation is caused by excessive deposition of melanin located in the basal and suprabasal cell layers of the epithelium. Pigmentation of gingiva not just has an impact on esthetics but also creates psychological negativity. Though a wide range of techniques are available to manage this condition. Depigmentation procedures such as scalpel surgery, gingivectomy with free gingival autografting, electrosurgery, cryosurgery, chemical agents such as 90% phenol and 95% alcohol, abrasion with diamond bur, Nd: YAG laser, semiconductor diode laser, and CO laser have been employed for removal of melanin hyper pigmentation. The following case report describes two different surgical depigmentation techniques scalpel technique and lasers. Better results of depigmentation were achieved with diode laser than conventional scalpel with respect to esthetics and less postoperative discomfort.

Melanin: Production from Cheese Bacteria, Chemical Characterization, and Biological Activities

Pigments are compounds of importance to several industries, for instance, the food industry, where they can be used as additives, color intensifiers, and antioxidants. As the current trend around the world is shifting to the use of eco-friendly commodities, demand for natural dyes is increasing. Melanins are pigments that are produced by several microorganisms. Pseudomonas putida ESACB 191, isolated from goat cheese rind, was described as a brown pigment producer. This strain produces a brown pigment via the synthetic Müeller-Hinton Broth. This brown compound was extracted, purified, analyzed by FTIR and mass spectrometry, and identified as eumelanin. The maximum productivity was 1.57 mg/L/h. The bioactivity of eumelanin was evaluated as the capacity for scavenging free radicals (antioxidant activity), EC50 74.0 ± 0.2 μg/mL, and as an acetylcholinesterase inhibitor, with IC50 575 ± 4 μg/mL. This bacterial eumelanin did not show cytotoxicity towards A375, HeLa Kyoto, HepG2, or Caco2 cell lines. The effect of melanin on cholesterol absorption and drug interaction was evaluated in order to understand the interaction of melanin present in the cheese rind when ingested by consumers. However, it had no effect either on cholesterol absorption through an intestinal simulated barrier formed by the Caco2 cell line or with the drug ezetimibe.

Enhancing the enzymatic browning inhibition capacity of Moringa oleifera seed extract via the Maillard reaction

The antioxidant and anti-browning activity of heated plant extracts have been attributed to the formation of Maillard reaction products (MRPs) via the Maillard reaction (MR). The inhibitory effect of heated Moringa oleifera (MO) seed extract on banana polyphenol oxidase (PPO) was investigated. The Plain MO seed extracts and those with added glucose and glycine (1.5 mM each) were heated at 100°C for 15, 30, 60 and 120 min. The pH and brown colour development decreased and increased significantly (P <0.05) with increased reaction time, respectively, with heated moringa glucose-glycine HMGGL for 120 min exhibiting the highest pH reduction (2.58) and darkest extracts at an L* value of 8.11. This phenomenon is associated with progression of the MR. With reference to enzymatic browning, heated MO seed extracts exhibited stronger inhibitory effect against banana PPO activity in vivo and in vitro than the unheated counterpart. Evident to this are the higher inhibition percentages and lower ΔE values. Among model systems, the highest in vitro browning inhibition was exhibited mostly by longer heating times of 60 and 120 min. Model system HMGGL 120 min proved to be superior at 96% inhibition, which was comparable to known synthetic commercial antioxidants such as ascorbic acid (AA) at 99%, as well as ethylenediaminetetraacetic acid (EDTA) and citric acid (CA), both at 100% inhibition. In vivo enzymatic browning inhibition followed a similar trend, where the brown pigment (melanin) intensified as shown by an increase in ΔE as the storage time increased from 0.5 to 24 h. The model system UMGGL exhibited highest inhibition of brown melanin (p <0.05). Although it was the best amongst other model systems, it was surpassed by synthetic antioxidants AA, EDTA and CA, which were ranked amongst the top three in inhibiting brown pigment formation in vivo. To further illustrate the effect of MR augmented MO seed extracts on enzyme activity inhibition, UMGGL 60 and 120 at 5 and 24 h storage surpassed the inhibitory effect of AA. At the said storage times, AA lost its inhibitory potential against pigment formation. This was due to oxidation of AA to form dehydroascorbic acid, which lacks inhibitory potential. This study proved that heating MO plant extracts increases their enzymatic browning inhibition potential, furthermore, the inhibitory capacity was heightened when reacted via the MR.

Pyomelanin Synthesis in Alternaria alternata Inhibits DHN-Melanin Synthesis and Decreases Cell Wall Chitin Content and Thickness

The genus Alternaria includes several of fungi that are darkly pigmented by DHN-melanin. These are pathogenic to plants but are also associated with human respiratory allergic diseases and with serious infections in immunocompromised individuals. The present work focuses on the alterations of the composition and structure of the hyphal cell wall of Alternaria alternata occuring under the catabolism of L-tyrosine and L-phenylalanine when cultured in minimal salt medium (MM). Under these growing conditions, we observed the released of a brown pigment into the culture medium. FTIR analysis demonstrates that the produced pigment is chemically identical to the pigment released when the fungus is grown in MM with homogentisate acid (HGA), the intermediate of pyomelanin, confirming that this pigment is pyomelanin. In contrast to other fungi that also synthesize pyomelanin under tyrosine metabolism, A. alternata inhibits DHN-melanin cell wall accumulation when pyomelanin is produced, and this is associated with reduced chitin cell wall content. When A. alternata is grown in MM containing L-phenylalanine, a L-tyrosine percursor, pyomelanin is synthesized but only at trace concentrations and A. alternata mycelia display an albino-like phenotype since DHN-melanin accumulation is inhibited. CmrA, the transcription regulator for the genes coding for the DHN-melanin pathway, is involved in the down-regulation of DHN-melanin synthesis when pyomelanin is being synthetized, since the CMRA gene and genes of the enzymes involved in DHN-melanin synthesis pathway showed a decreased expression. Other amino acids do not trigger pyomelanin synthesis and DHN-melanin accumulation in the cell wall is not affected. Transmission and scanning electron microscopy show that the cell wall structure and surface decorations are altered in L-tyrosine- and L-phenylalanine-grown fungi, depending on the pigment produced. In summary, growth in presence of L-tyrosine and L-phenylalanine leads to pigmentation and cell wall changes, which could be relevant to infection conditions where these amino acids are expected to be available.