Search

Alkaptonuria (AKU) is a rare tyrosine metabolism disorder caused by homogentisate 1,2-dioxygenase (HGD) mutations and homogentisic acid (HGA) accumulation. In this study, we investigated the genotype-phenotype relationship in AKU patients with a novel HGD gene mutation from a Chinese Hani family.Routine clinical examination and laboratory evaluation were performed, urine alkalinization test and urinary gas chromatography-mass spectrometry were used to assess HGA. Gene sequencing was utilized to study the defining features of AKU. NetGene2-2.42 and BDGP software was used to predict protein structure online. Flow cytometry and RT-PCR were used to analyze HGD proteins and HGD mRNA, respectively.Two pediatric patients fulfilled diagnostic criteria for AKU with eddish-brown or black diapers and urine HGA testing. Sequencing testing revealed that all members of this family had a novel samesense mutation c.15G A at the edge of exon 1 of the HGD. By flow cytometry, the expression of HGD protein in the pediatric patients' peripheral blood mononuclear cells was barely expressed. NetGene2-2.42 and BDGP software showed that the mutation reduced the score of the 5' splice donor site and disrupted its normal splicing, and the RT-PCR product also demonstrated that the defect in the HGD protein was due to the lack of the first exon containing the start codon ATG after the mutation.The novel mutation c.15G A in HGD is associated with the AKU phenotype. It may affect the splicing of exon 1, leading to exon skipping, which impairs the structure and function of the protein.

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in the body. Affected individuals lack enough functional levels of an enzyme required to breakdown HGA. Mutations in the HGD gene cause AKU and they are responsible for deficient levels of functional homogentisate 1,2-dioxygenase (HGD), which, in turn, leads to excess levels of HGA. Although HGA is rapidly cleared from the body by the kidneys, in the long term it starts accumulating in various tissues, especially cartilage. Over time (rarely before adulthood), it eventually changes the color of affected tissue to slate blue or black. Here we report a comprehensive mutation analysis of 111 pathogenic and 190 non-pathogenic HGD missense mutations using protein structural information. Using our comprehensive suite of graph-based signature methods, mCSM complemented with sequence-based tools, we studied the functional and molecular consequences of each mutation on protein stability, interaction and evolutionary conservation. The scores generated from the structure and sequence-based tools were used to train a supervised machine learning algorithm with 84% accuracy. The empirical classifier was used to generate the variant phenotype for novel HGD missense mutations. All this information is deployed as a user friendly freely available web server called HGDiscovery (http://biosig.unimelb.edu.au/hgdiscovery/).

Ochronosis is a syndrome characterized by bluish black discoloration due to the deposition of polymerized products of homogentisic acid (HGA) in the connective tissues. The endogenous variety (alkaptonuria), is a rare autosomal recessive metabolic disorder. The disorder is manifested by deficiency of the enzyme homogentisate 1,2-dioxygenase. The characteristic of the condition is a triad of pigmentation of skin, cartilage, and sclera; ochronotic arthropathies and homogentisic aciduria (resulting in darkening of urine). More rarely, it may affect the breast. This rare and interesting case of a woman with ochronosis of both breasts and chest wall, prompted us to write this case report.

Background p53 immunostaining is routinely used as a surrogate marker for TP53 mutational status. In urine cytology, p53 immunocytochemistry is reportedly useful in detecting urothelial carcinoma cells as well as in improving the detection sensitivity and specificity. However, to the best of our knowledge, p53 expression in repair/reactive renal tubular cells (RRTCs) from urine cytologic specimens has not been assessed to date. Methods We evaluated the immunoexpression of p53 and homogentisate 1,2‐dioxygenase (HGD) antibody, a renal tubular cells marker, in RRTCs using voided urine and renal biopsy samples from 80 patients who were histologically diagnosed with glomerular disease. Results Repair/reactive renal tubular cells were detected in 68 (68/80, 85%) samples at a mean count of 141.1 cells per sample (range, 5–4220). Immunocytochemical analysis found p53‐positive RRTCs in all the samples (68/68, 100%) with an average p53 positivity rate of RRTCs per sample at 47.7% (range, 3.8%–96.5%). Of the 68 p53‐positive RRTC samples, 38 (55.9%) included cells that were HGD positive for cytoplasm. Similarly, renal biopsy analysis revealed p53‐positive RRTCs in all the specimens (68/68, 100%). All 68 (100%) cases showed RRTCs that were positive for both p53 and HGD. Conclusion To avoid false positives of p53 immunocytochemistry, cytologists must consider the fact that RRTCs from patients with glomerular disease are positive for p53., Although the existence and cytological features of repair/reactive renal tubular cells in urine are not yet widely well known, these cells cytomorphologically mimic cancer cells. This study found that repair/reactive renal tubular cells are positive for p53 immunocytochemistry, which is crucial information for helping to avoid false positives in urine cytology.

Alkaptonuria is characterized by the accumulation of homogentisic acid (HGA), part of which is excreted in the urine but the excess HGA forms a dark brown ochronotic pigment that deposits in the connective tissue (ochronosis), eventually leading to early-onset severe arthropathy. We analyzed a cohort of 48 Russian AKU families by sequencing all 14 exons (including flanking intronic sequences) of the homogentisate 1,2-dioxygenase gene (HGD) and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. Nine novel likely pathogenic HGD variants were identified, which have not been reported previously in any other country. Recently, Bychkov et al. [1] reported on the variant spectrum in another cohort of 49 Russian AKU patients. Here we summarize complete data from both cohorts that include 82 Russian AKU families. Taken together, 31 different HGD variants were found in these patients, of which 14 are novel and found only in Russia. The most common variant was c.481G>A (p.(Gly161Arg)), present in almost 54% of all AKU alleles.

The emergence and spread of antibiotic resistance pose serious environmental and health challenges. Attention has been drawn to phage therapy as an alternative approach to combat antibiotic resistance with immense potential. However, one of the obstacles to phage therapy is phage resistance, and it can be acquired through genetic mutations, followed by consequences of phenotypic variations. Therefore, understanding the mechanisms underlying phage-host interactions will provide us with greater detail on how to optimize phage therapy. In this study, three lytic phages (phipa2, phipa4, and phipa10) were isolated to investigate phage resistance and the potential fitness trade-offs in Pseudomonas aeruginosa. Specifically, in phage-resistant mutants phipa2-R and phipa4-R, mutations in conferring resistance occurred in genes

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

Staple grains with low levels of provitamin A carotenoids contribute to the global prevalence of vitamin A deficiency and therefore are the main targets for provitamin A biofortification. However, carotenoid stability during both seed maturation and postharvest storage is a serious concern for the full benefits of carotenoid biofortified grains. In this study, we utilized Arabidopsis as a model to establish carotenoid biofortification strategies in seeds. We discovered that manipulation of carotenoid biosynthetic activity by seed-specific expression of Phytoene synthase (PSY) increases both provitamin A and total carotenoid levels but the increased carotenoids are prone to degradation during seed maturation and storage, consistent with previous studies of provitamin A biofortified grains. In contrast, stacking with Orange (ORHis), a gene that initiates chromoplast biogenesis, dramatically enhances provitamin A and total carotenoid content and stability. Up to 65- and 10-fold increases of β-carotene and total carotenoids, respectively, with provitamin A carotenoids composing over 63% were observed in the seeds containing ORHis and PSY. Co-expression of Homogentisate geranylgeranyl transferase (HGGT) with ORHis and PSY further increases carotenoid accumulation and stability during seed maturation and storage. Moreover, knocking-out of β-carotene hydroxylase 2 (BCH2) by CRISPR/Cas9 not only potentially facilitates β-carotene accumulation but also minimizes the negative effect of carotenoid over production on seed germination. Our findings provide new insights into various processes on carotenoid accumulation and stability in seeds and establish a multiplexed strategy to simultaneously target carotenoid biosynthesis, turnover, and stable storage for carotenoid biofortification in crop seeds.

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.

Alkaptonuria (AKU) is a multisystemic autosomal recessive disease due to deficiency of enzyme homogentisate dioxygenase leading to accumulation of homogentisic acid. Urine becoming dark on oxygenation or alkalinization is the first symptom but is often ignored. The patient usually presents with pigment deposition in connective tissue and cartilage after the third decade. This pigment deposition not only alters the aesthetics but also leads to alteration in the activity of different tissues due to inflammation and subsequent fibrosis or calcification. We are presenting an adult male who was asymptomatic till 5 years back when he started having backache for which he was taking analgesics off and was diagnosed to be having AKU at the age of 58 years.

Alkaptonuria (AKU) is a rare metabolic disease which is inherited as an autosomal recessive trait. It is characterized by the accumulation of homogentisic acid over time in various tissues of the body particularly connective tissues. This genetic disease is caused by mutation of the Homogentisate 1,2-dioxygenase ( HGD ) gene which encodes for enzyme essential for the catabolism of phenylalanine and tyrosine. The aim of the present study is to investigate variant types in Jordanian patients with alkaptonuria. Genomic DNA was extracted from whole blood samples of the participated AKU family members (n = 23). The 14 exons of HGD gene for the proband were amplified using specific PCR primers. The sequenced data were analysed and the pathogenicity of the identified variants were predicted using the online bioinformatics programs: PolyPhen2, SIFT and Mutation taster. The analysis showed that the proband was compound heterozygous for the missense mutations A122V and R225C found within E6 and E10, respectively. R225C variant is novel and the genotyping of the family members indicated that HGDA122V and HGDR225C alleles were fully segregated. Moreover, the cousins of the proband who are AKU patients inherited the homozygous pattern of the novel mutation. This study extends the pathogenic mutations spectrum of the HGD gene. It identified the novel mutation R225C and at the same time confirmed the high prevalence of the founder mutation A122V in Jordanian AKU patients.

Kidney renal clear cell carcinoma (KIRC) with poor prognosis is the main histological subtype of renal cell carcinoma, accounting for more than 80% of patients. Most patients are diagnosed at an advanced stage due to being asymptomatic early on. Advanced KIRC has an extremely poor prognosis due to its inherent resistance to radiotherapy and chemotherapy. Therefore, a comprehensive understanding of the molecular mechanisms of KIRC and the development of effective early diagnostic and therapeutic strategies is urgently needed. In this study, we aimed to identify the prognosis-related biomarker and analyzed its relationship with tumor progression. Metabolic changes are an important feature of kidney cancer, where the reduction of fumarate allows us to target the tyrosine metabolic pathway. The homogentisate 1,2-dioxygenase (HGD) and glutathione S-transferase zeta 1 (GSTZ1) related with prognosis of KIRC was identified through bioinformatics analysis based on The Cancer Genome Atlas (TCGA) databases. Mechanistically, we found that decreased HGD and GSTZ1 promote aerobic glycolysis in KIRC, coordinate the balance of amino acid metabolism and energy metabolism in tumor cells, and ultimately activate the tumor cell cycle and tumor progression. In summary, we identified the tyrosine metabolizing enzymes HGD and GSTZ1 as biomarkers of KIRC, which will further the understanding of the tumor metabolism profile, provide novel strategies and theoretical support for diagnosing and treating KIRC and as referential for future clinical research.

Ochronosis is the black discoloration of connective tissues seen with alkaptonuria, a metabolic disorder. Alkaptonuria is a rare autosomal recessive metabolic disorder caused by the lack of homogentisic acid oxidase enzyme. Alkaptonuria causes degenerative changes in cartilage, intervertebral disc and other tissues. The patients operated due to lumbar disc herniation in alkatonuria are very few. In this article a case of ochronosis in which the patient was determined after lumbar discectomy is presented.

Based on the structures of isoxaflutole (IFT) and N-isobutyl-N-(4-chloro-benzyl)-4-chloro-2-pentenamide, a series of N-benzyl-5-cyclopropyl-isoxazole-4-carboxamides was designed by connecting their pharmacophores (i.e., a multitarget drug design strategy). A total of 27 N-benzyl-5-cyclopropyl-isoxazole-4-carboxamides were prepared from 5-cyclopropylisoxazole-4-carboxylic acid and substituted benzylamines, and their structures were confirmed by NMR and MS. Laboratory bioassays indicated that I-26 showed 100% inhibition against Portulaca oleracea and Abutilon theophrasti at a concentration of 10 mg/L, better than the positive control butachlor (50% inhibition for both weeds). A strong growth inhibition was observed, but a typical bleaching phenomenon of IFT could not be observed in the Petri dish assay. I-05 displayed excellent postemergence herbicidal activity against Echinochloa crusgalli and A. theophrasti at a rate of 150 g/ha, and bleaching symptoms were observed in the leaves of treated weeds. The bleaching effect of Chlamydomonas reinhardtii treated by I-05 could be reversed by adding homogentisate. Enzymatic bioassays indicated that I-05 could not inhibit 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, but II-05, an isoxazole ring-opening product of I-05, could inhibit HPPD activity with an EC50 value of 1.05 μM, similar to that of mesotrione (with an EC50 value of 1.35 μM). Detailed discussion about observed herbicidal symptoms is provided in the Results and Discussion section. This investigation provided a proof-of-concept foundation that a multitarget drug design strategy could be applied in agrochemical research.

Vitamin E is a fat-soluble compound with powerful antioxidative and anticancer activity, which constitutes eight isoforms, namely α-, β-, γ-, δ-tocopherol and α-, β-, γ-, δ-tocotrienol. With the wide use of vitamin E in food, medicine, cosmetics and other fields, the demand of vitamin E in market is huge. Currently in the market, vitamin E is about 80% from chemical synthesis and 20% from extraction of oil distillates derived from plants or seeds. However, chemical synthesis of vitamin E suffered from the use of toxic catalysts, making the process environmentally unfriendly and unsustainable. In addition, the chemical synthesis technologies mainly synthesized the racemate of vitamin E and the enantiomerically pure vitamin E is mostly extracted from limited agricultural resources. Recently, using microorganisms to heterologously express metabolic pathways originated from plants is an attractive strategy due to the environmentally friendly, economic and easily scalable. Moreover, the progress of synthetic biology and metabolic engineering enabled the genetically tractable microorganisms such as Escherichia coli and Saccharomyces cerevisiae to successfully produce many natural compounds. As the natural pathways and related enzymes to synthesize vitamin E have been found in the last decades, it is feasible to engineer microorganisms to produce vitamin E. δ-tocotrienol, one isoform of vitamin E, was de novo heterologously synthesized in Escherichia coli and Saccharomyces cerevisiae with the production of 15 μg/(g cdw) and the titer of 4.10 mg/L (1.3 mg/(g cdw)), respectively. In this review, we firstly summarized the natural pathways and the recent progress on the enhanced biosynthesis of Vitamin E. Then the pathways and the recent advances on metabolically engineering strategies for the enhanced key intimidates (homogentisate and geranylgeranyl diphosphate) in industrial model microorganism was summarized. Specifically, we reviewed the advances on the shikimate pathway and the 2-C-methyl-d-erythritol-4-phosphate (MEP) and mevalonic acid (MVA) pathway in different chassis. Finally, we summarized the present situation and prospected the potential of biosynthesis of vitamin E in microorganisms. Based on the current researches on the improving production of vitamin E in microorganisms, in addition to enhance the supply of the precursors (homogentisate and geranylgeranyl diphosphate) by metabolic engineering, more attention needs to be paid to improve the enzyme activities of the key enzymes like homogentisate phytyl transferase (HPT) and tocopherol/tocotrienol cyclase (TC), which restricted the conversion rate of the precursors to the target product vitamin E. In the future, biotechnology such as genetic engineering, metabolic engineering and synthetic biology can be combined with the natural pathway of vitamin E to biosynthesize specific monomer or mixture of vitamin E in different model microorganisms. To further improve the production of vitamin E in the engineered microorganism, diverse genes and enzymes from multiple species involved in the biosynthesis of vitamin E needs to be characterized and functionalized. Enzyme engineering could also be used to improve the enzyme activities and consequently enhance the production of vitamin E.

Alkaptonuria (AKU) is an autosomal recessive disorder, which is caused by a site-specific mutation(s) and thus, impaired the function of Homogentisate-1, 2-dioxygenase (HGD), an essential enzyme for the catabolism of phenylalanine and tyrosine. Among frameshift, intronic, splice-site and missense mutations, the latter has been the most common form of genetic variations for the disease. How do the acquired mutations in HGD correlate with the disease? Systematic staged-screening of some sixty-five mutations, which are known to have a relation with the disease, by GVGD, SIFT, SNAP, PANTHER, SDM, PHD-SNP, Meta-SNP, Pmut and Mutpred methods, showed that mutations

BACKGROUND: Aclonifen is a unique diphenyl ether herbicide. Despite its structural similarities to known inhibitors of the protoporphyrinogen oxidase (e.g. acifluorfen, bifenox or oxadiazon), which result in leaf necrosis, aclonifen causes a different phenotype that is described as bleaching. This also is reflected by the Herbicide Resistance Action Committee (HRAC) classification that categorizes aclonifen as an inhibitor of pigment biosynthesis with an unknown target. RESULTS: A comprehensive Arabidopsis thaliana RNAseq dataset comprising 49 different inhibitor treatments and covering 40 known target pathways was used to predict the aclonifen mode of action (MoA) by a random forest classifier. The classifier predicts for aclonifen a MoA within the carotenoid biosynthesis pathway similar to the reference compound norflurazon that inhibits the phytoene desaturase. Upon aclonifen treatment, the phytoene desaturation reaction is disturbed, resulting in a characteristic phytoene accumulation in vivo. However, direct enzyme inhibition by the herbicide was excluded for known herbicidal targets such as phytoene desaturase, 4‐hydroxyphenylpyruvate dioxygenase and homogentisate solanesyltransferase. Eventually, the solanesyl diphosphate synthase (SPS), providing one of the two homogentisate solanesyltransferase substrate molecules, could be identified as the molecular target of aclonifen. Inhibition was confirmed using biochemical activity assays for the A. thaliana SPSs 1 and 2. Furthermore, a Chlamydomonas reinhardtii homolog was used for co‐crystallization of the enzyme–inhibitor complex, showing that one inhibitor molecule binds at the interface between two protein monomers. CONCLUSION: Solanesyl diphosphate synthase was identified as the target of aclonifen, representing a novel mode of action for herbicides. © 2020 Society of Chemical Industry

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.

BACKGROUND: Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. METHODS: Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700μmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. RESULTS: Elevated tyrosine (813μmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3μmol/L, 274.8μmol/L and 144.3μmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3μmol/L, 530.2μmol/L and 656.2μmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (p=0.002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine

Alkaptonuria is an ultra-rare autosomal recessive disorder of tyrosine metabolism, whereby the homogentisate 1,2-dioxygenase (HGD) enzyme is deficient, causing an elevation of its substrate homogentisic acid (HGA). Overtime, elevated HGA causes connective tissue ochronosis, leading to a severe and early onset osteoarthropathy. The use of HGD deficient mouse models in this metabolic bone disease have provided the opportunity to investigate AKU pathophysiology and potential treatments. An ENU mutagenesis AKU mouse model (BALB/c Hgd−/−) provided the means to explore the onset of pigmentation in cartilage and treatment of AKU with nitisinone, an inhibitor of the upstream enzyme forming HGA. This work provided evidence that nitisinone could not only lower circulating HGA, but could also prevent ochronosis and halt disease progression, leading to its off-label use at the National Alkaptonuria Centre (Liverpool, UK) and its subsequent testing in human clinical trials (DevelopAKUre). Recently, a new targeted AKU mouse model (Hgd tm1a−/−, C57BL/6) has been established, offering a LacZ reporter gene for localising gene expression and LoxP and FRT sites that enabled generation of an inducible and liver-specific HGD knockout model (Hgd tm1d MxCre+/−). This conditional model determined the importance of the liver as a target organ for future gene/enzyme replacement therapies in AKU. The contribution of AKU mouse models has clearly accelerated the treatment and knowledge of this rare disease, and will continue to be used.

The last 15 years have been the most fruitful in the history of research on the metabolic disorder alkaptonuria (AKU). AKU is caused by a deficiency of homogentisate dioxygenase (HGD), the enzyme involved in metabolism of tyrosine, and is characterized by the presence of dark ochronotic pigment in the connective tissue that is formed, due to high levels of circulating homogentisic acid. Almost 120 years ago, Sir Archibald Garrod used AKU to illustrate the concept of Mendelian inheritance in man. In January 2019, the phase III clinical study SONIA 2 was completed, which tested the effectiveness and safety of nitisinone in the treatment of AKU. Results were positive, and they will serve as the basis for the application for registration of nitisinone for treatment of AKU at the European Medicines Agency. Therefore, AKU might become a rare disease for which a cure will be found by 2020. We understand the natural history of the disease and the process of ochronosis much more, but at the same time there are still unanswered questions. One of them is the issue of the factors influencing the varying severity of the disease, since our recent genotype-phenotype study did not show that differences in residual homogentisic acid activity caused by the different mutations was responsible. Although nitisinone has proved to arrest the process of ochronosis, it has some unwanted effects and does not cure the disease completely. As such, enzyme replacement or gene therapy might become a new focus of AKU research, for which a novel suitable mouse model of AKU is available already. We believe that the story of AKU is also a story of effective collaboration between scientists and patients that might serve as an example for other rare diseases.

Scientific reports 12, 19452 (2022). doi:10.1038/s41598-022-23702-y, Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Objectives Alkaptonuria is a rare autosomal recessive genetic disorder resulting from the deficiency of homogentisate 1,2 dioxygenase (HGD), the third enzyme in the tyrosine degradation pathway. Homogentisic acid produced in excess oxidizes into ochronotic pigment polymer. Accumulation of this pigment in various tissues leads to systemic disease. Methods Clinical, laboratory, molecular findings and treatment characteristics of 35 patients followed up in Ege University Pediatric Nutrition, and Metabolism Department with the diagnosis of alkaptonuria were evaluated retrospectively. Results Twenty-four males (68.57%) and 11 females (31.42%) with a confirmed diagnosis of alkaptonuria from 32 different families were included in the study. We identified 11 different genetic variants; six of these were novel. c.1033C>T, c.676G>A, c.664G>A, c.731_734del, c.1009G>T, c.859_862delins ATAC were not previously reported in the literature. 24 (68.57%) patients only adhered to a low-protein diet in our study group. Seven (20%) patients initiated a low protein diet and NTBC therapy. Mean urinary HGA decreased by 88.7% with nitisinone. No statistical changes were detected in urinary HGA excretion with the low protein diet group. Conclusions In our study, alkaptonuria patients were diagnosed at different ages, from infancy to adulthood, and progressed with other systemic involvement in the follow-up. Since the initial period is asymptomatic, giving potentially effective treatment from an early age is under discussion. Raising disease awareness is very important in reducing disease mortality and morbidity rates.

Alkaptonuria is a rare autosomal recessive inherited disorder of tyrosine metabolism, which causes ochronosis, arthropathy, cardiac valvular calcification, and urolithiasis. The epidemiology of alkaptonuria in East Asia is not clear. In this study, patients diagnosed with alkaptonuria from January 2010 to June 2020 were reviewed. Their clinical and molecular features were further compared with those of patients from other countries. Three patients were found to have alkaptonuria. Mutation analyses of the homogentisate 1,2-dioxygenase gene (HGD) showed four novel variants c.16-2063 A > C, p.(Thr196Ile), p.(Gly344AspfsTer25), and p.(Gly362Arg) in six mutated alleles (83.3%). RNA sequencing revealed that c.16-2063 A > C activates a cryptic exon, causing protein truncation p.(Tyr5_Ile6insValTer17). A literature search identified another 6 patients with alkaptonuria in East Asia; including our cases, 13 of the 18 mutated alleles have not been reported elsewhere in the world. Alkaptonuria is rare in Taiwan and East Asia, with HGD variants being mostly novel and private.

Sweet corn is consistently one of the most highly consumed vegetables in the U.S., providing a valuable opportunity to increase nutrient intake through biofortification. Significant variation for carotenoid (provitamin A, lutein, zeaxanthin) and tocochromanol (vitamin E, antioxidants) levels is present in temperate sweet corn germplasm, yet previous genome-wide association studies (GWAS) of these traits have been limited by low statistical power and mapping resolution. Here, we employed a high-quality transcriptomic dataset collected from fresh sweet corn kernels to conduct transcriptome-wide association studies (TWAS) and transcriptome prediction studies for 39 carotenoid and tocochromanol traits. In agreement with previous GWAS findings, TWAS detected significant associations for four causal genes,β-carotene hydroxylase (crtRB1),lycopene epsilon cyclase(lcyE),γ-tocopherol methyltransferase(vte4), andhomogentisate geranylgeranyltransferase(hggt1) on a transcriptome-wide level. Pathway-level analysis revealed additional associations fordeoxy-xylulose synthase2(dxs2),diphosphocytidyl methyl erythritol synthase2(dmes2),cytidine methyl kinase1(cmk1), andgeranylgeranyl hydrogenase1(ggh1), of which,dmes2, cmk1, andggh1have not previously been identified through maize association studies. Evaluation of prediction models incorporating genome-wide markers and transcriptome-wide abundances revealed a trait-dependent benefit to the inclusion of both genomic and transcriptomic data over solely genomic data, but both transcriptome- and genome-wide datasets outperformeda prioricandidate gene-targeted prediction models for most traits. Altogether, this study represents an important step towards understanding the role of regulatory variation in the accumulation of vitamins in fresh sweet corn kernels.Core IdeasTranscriptomic data aid the study of vitamin levels in fresh sweet corn kernels.crtRB1, lcyE, dxs2, dmes2, andcmk1were associated with carotenoid traits.vte4, hggt1, andggh1were associated with tocochromanol traits.Transcriptomic data boosted predictive ability over genomic data alone for some traits.Joint transcriptome- and genome-wide models achieved the highest predictive abilities.

Increasing demands for efficient and versatile chemical reactions have prompted innovations in enzyme engineering. A major challenge in engineering α-ketoglutarate-dependent oxygenases is to develop a rational strategy which can be widely used for directly evolving the desired mutant to generate new products. Herein, we report a strategy for rational redesign of a model enzyme, 4-hydroxyphenylpyruvate dioxygenase (HPPD), based on quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamic simulations. This strategy enriched our understanding of the HPPD catalytic reaction pathway and led to the discovery of a series of HPPD mutants producing hydroxyphenylacetate (HPA) as the alternative product other than the native product homogentisate. The predicted HPPD-Fe(IV)═O-HPA intermediate was further confirmed by the crystal structure of Arabidopsis thaliana HPPD/S267W complexed with HPA. These findings not only provide a good understanding of the structure-function relationship of HPPD but also demonstrate a generally applicable platform for the development of biocatalysts.

The genus Alternaria includes several of fungi that are darkly pigmented by DHNmelanin. 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. This study was partly supported by the FEDER funds through the Operational Programme Competitiveness Factors-COMPETE and national funds by FCT-Foundation for Science and Technology under the strategic projects UIDB/00285/2020 and UID/NEU/04539/2013 the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme under project CENTRO-01-0145-FEDER-000012 and project CENTRO-01-0145-FEDER-022095:ViraVector, and through the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation and Portuguese national funds via FCT—Fundação para a Ciência e a Tecnologia, under project UIDB/04539/2020, and the European Regional Development Fund (ERDF), through the Centro 2020 Regional Operational Programme: project CENTRO-01-0145-FEDER- 000012-HealthyAging 2020, the COMPETE 2020—Operational Programme for Competitiveness and Internationalisation, and the Portuguese national funds via FCT—Fundação para a Ciência e a Tecnologia, I.P.: project POCI-01-0145-FEDER- 007440. IF thank the Foundation for Science and Technology (FCT, Portugal) and FEDER under Program PT2020 for financial support to CIMO (UID/AGR/00690/2013), LB (SFRH/BPD/107855/2015) and MD (SFRH/BD/84485/2012) grants. To POCI-01-0145-FEDER-006984 (LA LSRE-LCM), funded by ERDF, through POCI-COMPETE2020 and FCT. AC was supported in part by 5R01HL059842, 5R01AI033774, 5R37AI033142, and 5R01AI052733. info:eu-repo/semantics/publishedVersion

Alkaptonuria is a rare autosomal recessive metabolic disorder due to mutation in enzyme homogentisate 1,2-dioxygenase resulting in accumulation of homogentisic acid. The homogentisate 1,2-dioxygenase (HGD) gene has been mapped to chromosome 3q21- q23 and comprises 14 exons. A wide variety of causative mutations has been reported. Here, we are presenting a case report of a 2-year-old male child with a history of dark black-brown spots of urine on diaper with c.674G>C (p.Arg225Pro) mutation at exon 10 of HGD gene. The observed variant had a minor allele frequency of 0.0200% and 0.0004% in 1000 genomes and gnomAD database, respectively.

Background Cyclopyrimorate is a highly effective bleaching herbicide discovered by Mitsui Chemicals Agro, Inc. The target site was recently reported to be homogentisate solanesyltransferase (HST) in the plastoquinone (PQ) biosynthesis pathway on the basis of the number of intermediates in cyclopyrimorate-treated plants and in vitro HST assays. Here, the target site of cyclopyrimorate was further explored using both in vivo and in vitro experiments. Results The cyclopyrimorate-dependent bleaching effect on Arabidopsis thaliana was reversed by decyl PQ, suggesting that this symptom is attributable to the inhibition of PQ biosynthesis. Furthermore, homogentisate (HGA), a substrate of HST, weakly reversed the bleaching effect of cyclopyrimorate in a dose-dependent manner. We expected that the weak reversal by HGA was due to competitive inhibition by cyclopyrimorate or des-morpholinocarbonyl cyclopyrimorate (DMC), a metabolite of cyclopyrimorate in plants that exhibit higher HST-inhibitory activity as compared to cyclopyrimorate. Kinetic analysis was therefore conducted using DMC. DMC inhibited HST competitively with respect to HGA, and was a mixed non-competitive inhibitor with respect to the other substrate, farnesyl diphosphate. Moreover, neither cyclopyrimorate nor DMC inhibited 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanesyl-1,4-benzoquinone methyltransferase, which is located downstream of HST in the PQ biosynthesis pathway. Conclusions The target site of cyclopyrimorate and DMC is HST, which is a novel target site for commercial herbicides. © 2019 Society of Chemical Industry.

Elaeis guineensis is a tropical oil crop and has the highest oil yield per unit area. Palm oil has high palmitic acid content and is also rich in vitamins, including vitamin E. We conducted genome-wide association studies in a diversity panel of 161 E. guineensis accessions to identify single-nucleotide polymorphisms (SNPs) linked with vitamin E and validated candidate genes in these marker-associated intervals. Based on the SNPs reported in our previous research, 47 SNP markers were detected to be significantly associated with the variation of tocopherol and tocotrienol content at a cutoff P value of 6.3 × 10-7. A total of 656 candidate genes in the flanking regions of the 47 SNPs were identified, followed by pathway enrichment analysis. Of these candidate genes, EgHGGT (homogentisate geranylgeranyl transferase) involved in the biosynthesis of tocotrienols had a higher expression level in the mesocarp compared to other tissues. Expression of the EgHGGT gene was positively correlated with the variation in α-tocotrienol content. Induced overexpression of the gene in Arabidopsis caused a significant increase in vitamin E content and production of α-tocotrienols compared to wild Arabidopsis.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

p-Hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27, HPPD) is a potential target for herbicide discovery, which catalyzes the conversion of p-hydroxyphenylpyruvate to homogentisate. Triketone HPPD inhibitors have been widely applied to weed control for many years. To discover potential HPPD inhibitors with novel scaffold, a virtual screening procedure combining pharmacophore model, molecular docking and molecular dynamics (MD) simulation was carried out. The crystal complex (PDB: 6ISD ) with sulcotrione was selected to establish the receptor-ligand pharmacophore model (CBP). The verified CBP model with key features and interactions was employed to seek structures with potential inhibitory from ZINC database. The best-fit compounds from pharmacophore screening (2050 compounds) were subsequently subjected to molecular docking with CDOCKER method to identify the binding affinity of inhibitors with amino acid residues in active pocket. Six compounds were obtained as the final hits with the Fe(Ш) coordination and π-π interaction with Phe381 and Phe424. MD results demonstrated that Phe424 made great contributions to binding affinities of all the systems. This work provided powerful insights into the design and development of novel HPPD inhibitor in silico techniques. ZINC000255329531 was identified as the most promising candidate due to its high HPPD inhibition effect (ΔGbind = 25.66 kcal mol−1), in comparison with the commercial triketone HPPD herbicide sulcotrione (ΔGbind = 18.94 kcal mol−1).

BACKGROUND Identification of unknown chemical entities is a major challenge in metabolomics. To address this challenge, we developed a comprehensive targeted profiling strategy, combining 3 complementary liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) techniques and in-house accurate mass retention time (AMRT) databases established from commercial standards. This strategy was used to evaluate the effect of nitisinone on the urinary metabolome of patients and mice with alkaptonuria (AKU). Because hypertyrosinemia is a known consequence of nitisinone therapy, we investigated the wider metabolic consequences beyond hypertyrosinemia. METHODS A total of 619 standards (molecular weight, 45–1354 Da) covering a range of primary metabolic pathways were analyzed using 3 liquid chromatography methods—2 reversed phase and 1 normal phase—coupled to QTOF-MS. Separate AMRT databases were generated for the 3 methods, comprising chemical name, formula, theoretical accurate mass, and measured retention time. Databases were used to identify chemical entities acquired from nontargeted analysis of AKU urine: match window theoretical accurate mass ±10 ppm and retention time ±0.3 min. RESULTS Application of the AMRT databases to data acquired from analysis of urine from 25 patients with AKU (pretreatment and after 3, 12, and 24 months on nitisinone) and 18 HGD−/− mice (pretreatment and after 1 week on nitisinone) revealed 31 previously unreported statistically significant changes in metabolite patterns and abundance, indicating alterations to tyrosine, tryptophan, and purine metabolism after nitisinone administration. CONCLUSIONS The comprehensive targeted profiling strategy described here has the potential of enabling discovery of novel pathways associated with pathogenesis and management of AKU.

Alkaptonuria (AKU) is a rare metabolic disorder caused by a deficient enzyme in the tyrosine degradation pathway, homogentisate 1,2-dioxygenase (HGD). In 172 AKU patients from 39 countries, we identified 28 novel variants of the HGD gene, which include three larger genomic deletions within this gene discovered via self-designed multiplex ligation-dependent probe amplification (MLPA) probes. In addition, using a reporter minigene assay, we provide evidence that three of eight tested variants potentially affecting splicing cause exon skipping or cryptic splice-site activation. Extensive bioinformatics analysis of novel missense variants, and of the entire HGD monomer, confirmed mCSM as an effective computational tool for evaluating possible enzyme inactivation mechanisms. For the first time for AKU, a genotype–phenotype correlation study was performed for the three most frequent HGD variants identified in the Suitability Of Nitisinone in Alkaptonuria 2 (SONIA2) study. We found a small but statistically significant difference in urinary homogentisic acid (HGA) excretion, corrected for dietary protein intake, between variants leading to 1% or >30% residual HGD activity. There was, interestingly, no difference in serum levels or absolute urinary excretion of HGA, or clinical symptoms, indicating that protein intake is more important than differences in HGD variants for the amounts of HGA that accumulate in the body of AKU patients.

Bacillus thuringiensis BMB181 (Bt BMB181), with high melanin production, is an ideal candidate for industrial scale production of light-stable insecticides. However, its melanogenic pathways remain unclear. In the present study, we demonstrated that Bt BMB181 failed to produce melanin after treatment with mesotrione, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase in the homogentisic acid pathway of melanin synthesis. Heterologous expression experiments suggested that homogentisate-1,2-dioxygenase (HmgA) in Bt BMB171 functions normally, yet HmgA in Bt BMB181 had lost its activity, at least partly. Using the CRISPR-Cas9 system, the hmgA gene in Bt BMB171 was knocked out and the mutant strain gained the ability to produce melanin. Furthermore, the complemented strain reverted to the wild-type phenotype. In addition, overexpression of its own hmgA gene in Bt BMB181 also resulted in failure to produce the pigment. BLAST results indicated that the amino acid alteration (G272E) in HmgA of Bt BMB181 was caused by a single point mutation (815G→ A). The enzyme activity of purified HmgA171 was more than 10-fold higher than that of HmgA181. Finally, we determined that the mutation in hmgA was responsible for melanin accumulation in Bt BMB181. Our results provided new insights into the synthesis and regulation of melanin production in B.thuringiensis and will promote its future industrial application.

The self-sufficient cytochrome P450 RhF and its homologues belonging to the CYP116B subfamily have attracted considerable attention due to the potential for biotechnological applications based in their ability to catalyse an array of challenging oxidative reactions without requiring additional protein partners. In this work, we showed for the first time that a CYP116B self-sufficient cytochrome P450 encoded by the ohpA gene harboured by Cupriavidus pinatubonensis JMP134, a β-proteobacterium model for biodegradative pathways, catalyses the conversion of 2-hydroxyphenylacetic acid (2-HPA) into homogentisate. Mutational analysis and HPLC metabolite detection in strain JMP134 showed that 2-HPA is degraded through the well-known homogentisate pathway requiring a 2-HPA 5-hydroxylase activity provided by OhpA, which was additionally supported by heterologous expression and enzyme assays. The ohpA gene belongs to an operon including also ohpT, coding for a substrate-binding subunit of a putative transporter, whose expression is driven by an inducible promoter responsive to 2-HPA in presence of a predicted OhpR transcriptional regulator. OhpA homologues can be found in several genera belonging to Actinobacteria and α-, β- and γ-proteobacteria lineages indicating a widespread distribution of 2-HPA catabolism via homogentisate route. These results provide first time evidence for the natural function of members of the CYP116B self-sufficient oxygenases and represent a significant input to support novel kinetic and structural studies to develop cytochrome P450-based biocatalytic processes. This work was funded by FONDECYT 1201741, ANID PIA/Anillo ACT172128, and ANID PIA/BASAL FB0002 grants, the InGEMICS-CM (S2017/BMD-3691) Project of the Comunidad de Madrid – European Structural and Investment Funds – (FSE, FECER), and the L318-07 Project supported by the Fund of Scientific and Technological Equipment, year 2018, Universidad Tecnológica Metropolitana. Peerreview

Alkaptonuria (AKU), a rare genetic disorder, is characterized by the accumulation of homogentisic acid (HGA) in organs due to a deficiency in functional levels of the enzyme homogentisate 1,2-dioxygenase (HGD), required for the breakdown of HGA, because of mutations in the HGD gene. Over time, HGA accumulation causes the formation of the ochronotic pigment, a dark deposit that leads to tissue degeneration and organ malfunction. Such behaviour can be observed also in vitro for HGA solutions or HGA-containing biofluids (e.g. urine from AKU patients) upon alkalinisation, although a comparison at the molecular level between the laboratory and the physiological conditions is lacking. Indeed, independently from the conditions, such process is usually explained with the formation of 1,4-benzoquinone acetic acid (BQA) as the product of HGA chemical oxidation, mostly based on structural similarity between HGA and hydroquinone that is known to be oxidized to the corresponding para-benzoquinone. To test such correlation, a comprehensive, comparative investigation on HGA and BQA chemical behaviours was carried out by a combined approach of spectroscopic techniques (UV spectrometry, Nuclear Magnetic Resonance, Electron Paramagnetic Resonance, Dynamic Light Scattering) under acid/base titration both in solution and in biofluids. New insights on the process leading from HGA to ochronotic pigment have been obtained, spotting out the central role of radical species as intermediates not reported so far. Such evidence opens the way for molecular investigation of HGA fate in cells and tissue aiming to find new targets for Alkaptonuria therapy.

Alkaptonuria (AKU) is an ultra-rare genetic disease caused by a deficient activity of the enzyme homogentisate 1,2-dioxygenase (HGD) leading to the accumulation of homogentisic acid (HGA) on connective tissues. Even though AKU is a multi-systemic disease, osteoarticular cartilage is the most affected system and the most damaged tissue by the disease. In chondrocytes, HGA causes oxidative stress dysfunctions, which induce a series of not fully characterized cellular responses. In this study, we used a human chondrocytic cell line as an AKU model to evaluate, for the first time, the effect of HGA on autophagy, the main homeostasis system in articular cartilage. Cells responded timely to HGA treatment with an increase in autophagy as a mechanism of protection. In a chronic state, HGA-induced oxidative stress decreased autophagy, and chondrocytes, unable to restore balance, activated the chondroptosis pathway. This decrease in autophagy also correlated with the accumulation of ochronotic pigment, a hallmark of AKU. Our data suggest new perspectives for understanding AKU and a mechanistic model that rationalizes the damaging role of HGA.

The zona pellucida domain protein Dusky (Dy) plays a vital role in wing morphogenesis in insects, but little information on its function has been reported. In this study, we found that dy regulated wing cell size, larval and pupal duration, and the metabolism of amino acid and 20-hydroxyecdysone in Tribolium castaneum. Using RNA-seq, 413 differentially expressed genes were identified between physiological buffer-injected and dy-double-stranded RNA-treated larvae, including 88 downregulated genes and 325 upregulated genes. Among these genes, dy knockdown increased CYP18A1 expression to elevate the 26-hydroxylation of 20-hydroxyecdysone, which ultimately led to growth defects in wing cells. Silencing of dy upregulated the transcription of genes encoding tyrosine aminotransferase, 4-hydroxyphenylpyruvate dioxygenase, homogentisate 1, 2-dioxygenase, and Pale to promote the catabolism of tyrosine and phenylalanine, which eventually reduced amino acid content. Furthermore, dy knockdown upregulated 4E-BP expression, and 4E-BP silencing partially phenocopied dy RNA interference-mediated wing morphogenesis. These results suggest that Dy controls 20-hydroxyecdysone and amino acid metabolism to regulate wing morphogenesis in the insect.

Rice nutrition including vitamin and amylose contents become important aspect for many people around the world. Rice with high amylose content (low glycemic index) is good for those with Diabetes mellitus. Tocotrienol, one precursor of Vitamin-E biosynthesis is catalyzed by enzymes encoded HGGT, while amylose biosynthesis is catalyzed by enzymes encoded GBSSI. The objective of this study was to find rice varieties with high tocotrienol and/or amylose content based on the expression of HGGT and GBSSI among eight Banyuwangi local rice varieties. Relative expression of HGGT and GBSSI was measured by qRT-PCR and analyzed using 2ΔCt method. Statistical analysis resulted in the significantly different of HGGT and GBSSI relative expression among samples. Relative expression of HGGT from the highest to the lowest were demonstrated by Hitam Melik, Hitam Pekat, Blambangan A3, Merah Bali, Blambangan A2, Berlian, Janur Kuning, and SOJ A3, respectively; while relative expression of GBSSI from the highest to the lowest were demonstrated by Hitam Melik, Hitam Pekat, SOJ A3, Janur Kuning, Berlian, Merah Bali, Blambangan A3, and Blambangan A2, respectively. Based on this research we conclude that Hitam Melik potentially produces higher tocotrienol and lower glycemic index than other studied varieties.

Ochronosis and alkaptonuria are manifestations of the same condition—a rare autosomal recessive disorder resulting from a constitutional lack of homogentisate 1,2-dioxygenase (HGD) with the consequent accumulation of homogentisic acid (HGA). In ochronosis, HGA undergoes autoxidation as well as enzymatic oxidation to form an ochronotic pigment that accumulates in cartilage and connective tissues. In the beginning, there is homogentisic aciduria and pigmentation of cartilages and other connective tissues. In later years, generalized osteoarthritis of the spine and large joints, termed ochronotic arthropathy, develops. The diagnosis is confirmed by quantitative measurement of HGA in urine and mutation analysis of the HGD gene. One of the differential diagnoses for the skin findings is exogenous ochronosis, a limited hyperpigmentation of skin caused by some chemicals. As for the lumbar spine findings, there can be radiographic similarities with ankylosing spondylitis (AS) including reduced intervertebral disc spaces and loss of lumbar lordosis; however, ochronosis will spare the sacroiliac joint, and the lumbar spine will show dense, wafer-like disk calcification with a vacuum disc phenomenon and broad syndesmophytes. Here, we present a case of a patient with probable ochronosis that was treated many years as ankylosing spondylitis without response, and we provide a review of the current literature on ochronosis pathogenesis, diagnosis, and treatment.

Background. 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. Methods. We clinically evaluated 18 alkaptonuria patients (age range, 3 to 60 years) from four unrelated families. Furthermore, 11 out of 18 alkaptonuria patients and 7 unaffected members were enrolled for molecular investigations by utilizing Sanger sequencing to identify variants of the 14 exons of HGD gene. Results. We found that the seven patients from the 4 unrelated families carried a recurrent pathogenic missense variant (c.365C>T, p. Ala122Val) in exon 6 of HGD gene. The variant was fully segregated with the disease in affected family members while the other unaffected family members were heterozygous carriers for this variant. Additionally, the clinical features were fully predicted with alkaptonuria disorder. Conclusion. In this study, we confirmed that the most common variants in Jordanian AKU patients was c.365C>T, p. Ala122Val in exon 6 of HGD gene. Additionally, we correlated the clinical and genetic features of AKU patients at various ages (3-60 years).