Alkaptonuria (AKU) is an ultra-rare inborn error of metabolism characterized by homogentisic acid (HGA) accumulation due to a deficient activity of the homogentisate 1.2-dioxygenase (HGD) enzyme. This leads to the production of dark pigments that are deposited onto connective tissues, a condition named 'ochronosis' and whose mechanisms are not completely clear. Recently, the potential role of hitherto unidentified proteins in the ochronotic process was hypothesized, and the presence of Serum Amyloid A (SAA) in alkaptonuric tissues was reported, allowing the classification of AKU as a novel secondary amyloidosis.

Alkaptonuria (AKU) is an ultra-rare disease caused by the deficient activity of homogentisate 1,2-dioxygenase enzyme, leading the accumulation of homogentisic acid (HGA) in connective tissues implicating the formation of a black pigmentation called "ochronosis." Although AKU is a multisystemic disease, the most affected tissue is the articular cartilage, which during the pathology appears to be highly damaged. In this study, a model of alkaptonuric chondrocytes and cartilage was realized to investigate the role of HGA in the alteration of the extracellular matrix (ECM). The AKU tissues lost its architecture composed of collagen, proteoglycans, and all the proteins that characterize the ECM. The cause of this alteration in AKU cartilage is attributed to a degeneration of the cytoskeletal network in chondrocytes caused by the accumulation of HGA. The three cytoskeletal proteins, actin, vimentin, and tubulin, were analyzed and a modification in their amount and disposition in AKU chondrocytes model was identified. Cytoskeleton is involved in many fundamental cellular processes; therefore, the aberration in this complex network is involved in the manifestation of AKU disease.

Macrophages are one of the most functionally-diverse cell types with roles in innate immunity, homeostasis and disease making them attractive targets for diagnostics and therapy. Photo- or optoacoustics could provide non-invasive, deep tissue imaging with high resolution and allow to visualize the spatiotemporal distribution of macrophages in vivo. However, present macrophage labels focus on synthetic nanomaterials, frequently limiting their ability to combine both host cell viability and functionality with strong signal generation. Here, we present a homogentisic acid-derived pigment (HDP) for biocompatible intracellular labeling of macrophages with strong optoacoustic contrast efficient enough to resolve single cells against a strong blood background. We study pigment formation during macrophage differentiation and activation, and utilize this labeling method to track migration of pro-inflammatory macrophages in vivo with whole-body imaging. We expand the sparse palette of macrophage labels for in vivo optoacoustic imaging and facilitate research on macrophage functionality and behavior.

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.

Strawberry tree (Arbutus unedo L.) honey (STH), also known as "bitter honey", is a traditional medicine widely used in the Mediterranean area. Regardless of geographical origin, it usually has a very high content of phenolic compounds and strong antioxidant capacity. Yet, little is still known about the effects of STH, its phenolic extract (STHE), and its main bioactive compound - homogentisic acid (HGA) - at the cell level. The aim of this study was to estimate total phenolic content, DPPH radical scavenging activity, and ferric reducing antioxidant power of STH made in Croatia and investigate cytotoxic and pro-oxidative effects of STH, STHE and HGA on three human cell lines: tongue squamous cell carcinoma (CAL 27), hepatocellular carcinoma (HepG2), and epithelial colorectal adenocarcinoma cells (Caco-2) cells. These substances were tested at four concentrations (0.5-5× average human daily intake of STH) and over 30 min and 1 and 2 h. Croatian STH had a total phenolic content of 1.67 g gallic acid equivalents (GAE) per kg of honey, DPPH radical scavenging activity of 2.96 mmol Trolox equivalents (TE) per kg of honey, and ferric reducing antioxidant power (FRAP) of 13.5 mmol Fe2+ per kg of honey. Our results show no clear and consistent time- or concentration-dependent cytotoxicity in any of the cell lines. ROS levels in all the three cell types at almost all exposure times were not significantly higher than control. The most important observation is that the tested substances have low cytotoxicity and high biocompatibility, regardless of concentration, which is a good starting point for further research of their biological effects in other models.

Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype. We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a -/- mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy. Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA. This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.

Bacillus thuringiensis formulation losing its activity under field conditions due to UV radiation and photoprotection of B. thuringiensis based on melanin has attracted the attention of researchers for many years. Here, a single amino acid substitution (G272E) in homogentisate 1,2-dioxygenase was found to be responsible for pigment overproduction in B. thuringiensis BMB181, a derivative of BMB171. Disrupting the gene encoding homogentisate dioxygenase in BMB171 induced the accumulation of the homogentisic acid and provoked an increased pigment formation. To gain insights into homogentisate 1,2-dioxygenase in B. thuringiensis, we constructed a total of 14 mutations with a single amino acid substitution, and six of the mutant proteins were found to affect the melanin production when substituted by alanine. This study provides a new way to construct pigment-overproducing strains by impairing the homogentisate dioxygenase with a single mutation in B. thuringiensis, and the findings will facilitate a better understanding of this enzyme.

Several studies have shown that catecholamines can inhibit the fibrillation of alpha-synuclein (alpha-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of alpha-Syn, due to non-covalent binding of DOPAC to alpha-Syn monomer. Intriguingly, the presence of alpha-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of alpha-Syn, probably due to the H(2)O(2) production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the alpha-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the alpha-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of alpha-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify alpha-Syn covalently, probably due to the less steric hindrance in the Michael addition.

P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling.

The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Alkaptonuria, caused by a deficiency of homogentisate 1,2-dioxygenase, results in the accumulation of homogentisic acid (2,5-dihydroxyphenylacetic acid, HGA) in the urine. Alkaptonuria is suspected when the urine changes color after it is left to stand at room temperature for several hours to days; oxidation of homogentisic acid to benzoquinone acetic acid underlies this color change, which is accelerated by the addition of alkali. In an attempt to develop a facile screening test for alkaptonuria, we added alkali to urine samples obtained from patients with alkaptonuria and measured the absorbance spectra in the visible light region.

Metabolic disorder alkaptonuria is an autosomal recessive disorder caused by mutations in the HGD gene, and a deficiency HGD enzyme activity results in an accumulation of homogentisic acid (HGA), ochronosis, and destruction of connective tissue.

The homogentisic acid-lowering therapy nitisinone is being evaluated for the treatment of alkaptonuria (AKU) at the National Centre for AKU. Beyond hypertyrosinemia, the wider metabolic consequences of its use are largely unknown. The aim of this work was to evaluate the impact of nitisinone on the serum metabolome of patients with AKU after 12 and 24 months of treatment.

Alkaptonuria is an inherited disease that is caused by homogenticate accumulation. Deficiency or mutation in Homogentisate 1,2 dioxygenase gene (chromosome 3q21-q23) leads to production of incorrectly folded or truncated enzyme. Several studies indicated that competitive inhibitors of Homogentisate 1,2 dioxygenase like Nitisinone could be used for Alkaptonuria treatment. Therefore, it is of interest to design better inhibitors of the enzyme. We used subset 3_p.0.5 from Zinc database as the virtual screening library by PyRx software relaying on Lamarckian genetics algorithm. Top 10 hits with more efficient binding affinity were analyzed and hit No#5 and No# 7 was selected for further design. In Lig No#5, we decreased the hydrophobicity by adding oxygen in the hydrophobic tail of the molecule at positions C5 and C10. The new compound of (2Z, 5Z, 8Z)-6,9-Dihydroxy-2-(2-hydroxy-5-oxo-1,3-cyclohexadien-1-yl)-2,5,8-decatrienoic acid satisfied Lipinski rules as well as PhysChem and FafDrugs filters. Moreover, the modified version of Lig No# 7 with the IUPAC name of [2-(Carboxymethyl)-3,5-dihydroxyphenyl] acetic acid satisfies the Lipisnki, FafDrugs and Physchem.

Melanin-producing Cryptococcus and Aspergillus are highly invasive and can suppress or escape the immune system of the host. Since non-melanin-producing strains do not affect the immune system, melanin may play a role in immune system suppression. Artificial melanin synthesized using conventional methods is insoluble, making structural and functional analysis of this chemical difficult. In this study, we describe a melanin solubilization method based on polymerization of homogentisic acid (solubilizing component) and an equivalent amount of L-DOPA in the presence of laccase. In addition, we investigated the effect of melanin on the immune system. Homogentisic acid and L-DOPA mixed melanin (HALD), the synthetic solubilized melanin, did not exert a cytotoxic effect on mouse macrophages. HALD suppressed cytokine and reactive oxygen species production by macrophages when they were stimulated by fungal components. HALD also suppressed the phagocytosis of fungal components by macrophages. These results suggest that HALD can suppress the function of macrophages without causing cytotoxicity.

Alkaptonuria, a rare autosomal recessive metabolic disorder caused by deficiency in homogentisate 1,2-dioxygenase activity, leads to accumulation of oxidised homogentisic acid in cartilage and collagenous structures present in all organs and tissues, especially joints and heart, causing a pigmentation called ochronosis. A secondary amyloidosis is associated with AKU. Here we report a study of an aortic valve from an AKU patient.

This study was designed to unravel the role of Lactobacillus rhamnosus in the bioconversion of cranberry proanthocyanidins and cytotoxicity of resulting metabolites to hepatocellular carcinoma HepG2 cells. Crude (CR) and flavonol+dihydrochalcone- (FL+DHC-), anthocyanin- (AN-), proanthocyanidin- (PR-), and phenolic acid+catechin- (PA+C-) rich fractions were subjected to fermentation with L. rhamnosus at 37°C for 12, 24, and 48 h under anaerobic conditions. The major metabolites produced by bioconversion of polyphenols were 4-hydroxyphenylacetic acid, 3-(4-hydroxyphenyl)propionic acid, hydrocinnamic acid, catechol, and pyrogallol. Furthermore, cytotoxicity of the biotransformed extracts was compared to their parent extracts using human hepatocellular carcinoma HepG2 cells. The results showed that PR-biotransformed extract completely inhibited HepG2 cell proliferation in a dose- and time-dependent manner with IC50 values of 47.8 and 20.1 μg/mL at 24 and 48 h, respectively. An insight into the molecular mechanisms involved revealed that the cytotoxic effects of PR at 24 h incubation were mitochondria-controlled and not by proapoptotic caspase-3/7 dependent. The present findings suggest that the application of a bioconversion process using probiotic bacteria can enhance the pharmacological activities of cranberry proanthocyanidins by generating additional biologically active metabolites.

Natural and bioactive products have been of great interest due to their benefit as health foods and drugs to prevent various diseases. The aim of this study is to efficiently reuse marine chitinous materials (CMs), abundant and low-cost fishery by-products, for the bio-synthesis, isolation, and identification of antioxidant compounds possessing some other beneficial bioactivities. Paenibacillus sp. was used to convert CMs to antioxidants. Among various tested CMs, squid pen powder (SPP) gave the best results when used as the sole carbon/nitrogen source. Fermented SPP (FSPP) had comparable antioxidant activity (IC50 = 124 µg/mL) to that of α-tocopherol (IC50 = 30 µg/mL). The antioxidant productivity increased 1.83-fold after culture optimization. The use of multiple techniques, including Diaion, silica, and preparative HPLC columns coupled with a bioassay resulted in the isolation of two major antioxidants characterized as exopolysaccharides and homogentisic acid. These isolated compounds showed great maximum activity and low IC50 values (96%, 30 µg/mL and 99%, 5.4 µg/mL, respectively) which were comparable to that of α-tocopherol (95%, 24 µg/mL). The crude sample, fractions, and isolated compounds also demonstrated α-glucosidase inhibition and anti⁻inflammatory activity. Notably, homogentisic acid was found as a non-sugar-based moiety α-glucosidase inhibitor which show much higher inhibition (IC50 = 215 µg/mL) than that of acarbose (IC50 = 1324 µg/mL) and also possessed acceptable anti⁻inflammatory activity (IC50 = 9.8 µg/mL). The results highlighted the value of using seafood processing by-products, like squid pens, for the production of several compounds possessing multi-benefit bioactivities and no cytotoxicity.

Alkaptonuria (AKU) is an ultra-rare disease developed from the lack of homogentisic acid oxidase activity, causing homogentisic acid (HGA) accumulation that produces a HGA-melanin ochronotic pigment, of unknown composition. There is no therapy for AKU. Our aim was to verify if AKU implied a secondary amyloidosis. Congo Red, Thioflavin-T staining and TEM were performed to assess amyloid presence in AKU specimens (cartilage, synovia, periumbelical fat, salivary gland) and in HGA-treated human chondrocytes and cartilage. SAA and SAP deposition was examined using immunofluorescence and their levels were evaluated in the patients' plasma by ELISA. 2D electrophoresis was undertaken in AKU cells to evaluate the levels of proteins involved in amyloidogenesis. AKU osteoarticular tissues contained SAA-amyloid in 7/7 patients. Ochronotic pigment and amyloid co-localized in AKU osteoarticular tissues. SAA and SAP composition of the deposits assessed secondary type of amyloidosis. High levels of SAA and SAP were found in AKU patients' plasma. Systemic amyloidosis was assessed by Congo Red staining of patients' abdominal fat and salivary gland. AKU is the second pathology after Parkinson's disease where amyloid is associated with a form of melanin. Aberrant expression of proteins involved in amyloidogenesis has been found in AKU cells. Our findings on alkaptonuria as a novel type II AA amyloidosis open new important perspectives for its therapy, since methotrexate treatment proved to significantly reduce in vitro HGA-induced A-amyloid aggregates.

Cava is a sparkling wine obtained by a secondary fermentation in its own bottle. Grape skin contains several compounds, such as polyphenols, which act like natural protectors and provide flavor and color to the wines. In this paper, a previously optimized method based on reversed phase high performance liquid chromatography (HPLC) with ultraviolet/visible (UV/Vis) detection has been applied to determine polyphenols in cava wines. Compounds have been separated in a C18 core-shell column using 0.1% formic acid aqueous solution and methanol as the components of the mobile phase. Chromatograms have been recorded at 280, 310 and 370 nm to gain information on the composition of benzoic acids, hidroxycinnamic acids and flavonoids, respectively. HPLC-UV/vis data consisting of compositional profiles of relevant analytes has been exploited to characterize cava wines produced from different base wine blends using chemometrics. Other oenological variables, such as vintage, aging or malolatic fermentation, have been fixed over all the samples to avoid their influence on the description. Principal component analysis and other statistic methods have been used to extract of the underlying information, providing an excellent discrimination of samples according to grape varieties and coupages.