Identification and characterization of a novel tyrosine aminotransferase gene (SmTAT3-2) promotes the biosynthesis of phenolic acids in Salvia miltiorrhiza Bunge.

Rosmarinic acid (RA) and salvianolic acid B (SAB) are main phenolic acids in Salvia miltiorrhiza Bunge have been widely used in the treatment of cardiovascular and cerebrovascular diseases due to their excellent pharmacological activity. RA is a precursor of SAB, and tyrosine transaminase (TAT, EC 2.6.1.5) is a crucial rate-limiting enzyme in their metabolism pathway. This study identified a novel TAT gene, SmTAT3-2, and found that it is a new transcript derived from unconventional splicing of SmTAT3. We used different substrates for enzymatic reaction with SmTAT1, SmTAT3 and SmTAT3-2. Subcellular localization of SmTAT1 and SmTAT3-2 was completed based on submicroscopic techniques. In addition, they were overexpressed and CRISPR/Cas9 gene edited in hairy roots of S. miltiorrhiza. Revealed SmTAT3-2 and SmTAT1 showed a stronger affinity for L-tyrosine than SmTAT3, localized in the cytoplasm, and promoted the synthesis of phenolic acid. In overexpressed SmTAT3-2 hairy roots, the content of RA and SAB was significantly increased by 2.53 and 3.38 fold, respectively, which was significantly higher than that of overexpressed SmTAT1 strain compared with EV strain. These findings provide a valuable key enzyme gene for the phenolic acids metabolism pathway and offer a theoretical basis for the clinical application.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to α-ketoglutarate to produce 4-hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

Role of the tyrosine aminotransferase AccTATN gene in the response to pesticide and heavy metal stress in Apis cerana cerana.

Tyrosine aminotransferase (TATN) is the first enzyme involved in the metabolic degradation of tyrosine, and it plays an important role in tyrosine detoxification and helps the body resist oxidative damage. However, the function of TATN in Apis cerana cerana (A. c. cerana) remains unclear. To explore the role of TATN in the response to pesticide and heavy metal stress in A. c. cerana, AccTATN was isolated and identified. AccTATN was highly expressed in the integument and the adult stage. Exposure to multiple pesticides and heavy metal stress upregulated AccTATN expression. RNA interference experiments showed that silencing AccTATN reduced the resistance of A. c. cerana to glyphosate and avermectins stress. The expression of antioxidant-related genes and the activity of antioxidant enzymes were reduced after AccTATN was silenced, leading to the accumulation of oxidative damage. Overexpression of the recombinant AccTATN protein in a prokaryotic system also confirmed its role in heavy metal stress and improved antioxidant capacity. Our study showed that AccTATN may promote resistance to pesticide and heavy metal stress by regulating the antioxidant capacity of A. c. cerana. This study provides a valuable theoretical basis for A. c. cerana conservation.

Knockout of Tyrosine Aminotransferase Gene by Homologous Recombination Arrests Growth and Disrupts Redox Homeostasis in Leishmania Parasite.

Tyrosine aminotransferase is a well-characterized enzyme in the Leishmania parasite, but the role of TAT in the parasite functioning remains largely unknown. In this study, we attempt to gain a better understanding of the enzyme's role in the parasite by gene knockout and overexpression of the TAT gene. The overexpression of TAT protein was well tolerated by the parasites in two independent repeats. Single knockout of TAT gene by homologous recombination, LdTAT+/- displayed distinct retardation in the proliferation rates and entered the death phase immediately. Morphology of LdTAT+/- parasites had important structural defects as they rounded up with elongated flagella. Gene regulation studies suggested the upregulation of key apoptotic and redox metabolism genes in LdTAT+/-. Moreover, LdTAT+/- cells accumulated higher ROS, thiols, intracellular Ca2+ concentrations, and mitochondrial membrane depolarization signifying the onset of apoptosis. Tocopherol levels were reduced by 50% in LdTAT+/- suggesting the involvement of TAT in tocopherol biosynthesis in the parasite. Overall, our results provide the first evidence that gene knockout of TAT results in apoptosis and that TAT is required for the survival and viability of Leishmania donovani.

Identification and biochemical characterisation of tyrosine aminotransferase from Anthoceros agrestis unveils the conceivable entry point into rosmarinic acid biosynthesis in hornworts.

MAIN CONCLUSION: Tyrosine aminotransferase (AaTAT) from the hornwort Anthoceros agrestis Paton (Anthocerotaceae) was amplified and expressed in E. coli. The active enzyme is able to accept a wide range of substrates with distinct preference for L-tyrosine, therefore, possibly catalysing the initial step in rosmarinic acid biosynthesis. The presence of rosmarinic acid (RA) in the hornwort A. agrestis is well known, and some attempts have been made to clarify the biosynthesis of this caffeic acid ester in lower plants. Parallel to the biosynthesis in vascular plants, the involvement of tyrosine aminotransferase (EC 2.6.1.5; TAT) as the initial step was assumed. The amplification of a nucleotide sequence putatively encoding AaTAT (Genbank MN922307) and expression in E. coli were successful. The enzyme proved to have a high acceptance of L-tyrosine (Km 0.53 mM) whilst slightly preferring 2-oxoglutarate over phenylpyruvate as co-substrate. Applying L-phenylalanine as a potential amino donor or using oxaloacetate or pyruvate as a replacement for 2-oxoglutarate as amino acceptor resulted in significantly lower catalytic efficiencies in each of these cases. To facilitate further substrate search, two methods were introduced, one using ninhydrin after thin-layer chromatography and the other using derivatisation with o-phthalaldehyde followed by HPLC or LC-MS analysis. Both methods proved to be well applicable and helped to confirm the acceptance of further aromatic and aliphatic amino acids. This work presents the first description of a heterologously expressed TAT from a hornwort (A. agrestis) and describes the possible entry into the biosynthesis of RA and other specialised compounds in a so far neglected representative of terrestrial plants and upcoming new model organism.

Production of rosmarinic acid and correlated gene expression in hairy root cultures of green and purple basil (Ocimum basilicum L.).

Rosmarinic acid (RA) is an active constituent of Ocimum basilicum. It has been shown that hairy root production (measured as dry weight) improves when green basil (O. basilicum "Cinnamon") is cultured under the light. In contrast, purple basil (O. basilicum "Purpurascens") shows greater hairy root production when cultured under dark conditions. The level of gene expression was highest in hairy roots of green basil under dark conditions for up to 1 week. Transcript levels were highest in hairy roots of purple basil under both dark and light conditions after 2 weeks of culturing. After 3 weeks of culture under light conditions, green basil had accumulated 1.9-fold higher RA content than that of purple basil, which in turn was fivefold higher than that of the natural roots (42.86 µg/mg). Tyrosine aminotransferase showed a higher transcript level when compared to the other phenylpropanoid pathway genes (phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, and coenzyme-A ligase) in both dark and light conditions and in all-time regimens. RA accumulation was higher in the cultured hairy roots of green basil than those of purple basil under both light and dark conditions.

Mapping N- and C-terminals of Leishmania donovani tyrosine aminotransferase by gene truncation strategy: a functional study using in vitro and in silico approaches.

Tyrosine aminotransferase (TAT) catalyzes the transamination of amino acids in Leishmania sp.. TAT from Leishmania donovani has been found to be extremely stable at extreme temperatures and pH conditions. This study was conceived to map the functions of the non-conserved N-terminal and conserved C-terminal domain of TAT. N-terminal (NTAT) and C-terminal (CTAT) domain of TAT was truncated and cloned into the pET28a(+) vector. The truncated proteins were expressed, purified, and biochemically characterized. The Km of NTAT and CTAT for the tyrosine-pyruvate pair was determined to be 3.468 ± 0.796 mM and 4.581 ± 0.627 mM, repectively. Temperature and pH stability studies found NTAT to be stable like TAT but CTAT was extremely susceptible to temperature and pH changes. Upon docking and simulation for 100 ns, NTAT had lower SASA values. From UV spectroscopic study, PLP bound better to CTAT than NTAT because of the reduced SASA of NTAT. The sensitivity of CTAT was reasoned when the urea denaturation studies showed two-state denaturation which differed from NTAT's and TAT's biphasic folding mechanism. From this study, the authors hypothesize that the N-terminal is responsible for PLP stabilization and C-terminal protects the active site from extreme conditions.

Metabolic Engineering of Saccharomyces cerevisiae for Rosmarinic Acid Production.

Rosmarinic acid is a hydroxycinnamic acid ester commonly found in the Boraginaceae and Lamiaceae plant families. It exhibits various biological activities, including antioxidant, anti-inflammatory, antibacterial, antiallergic, and antiviral properties. Rosmarinic acid is used as a food and cosmetic ingredient, and several pharmaceutical applications have been suggested as well. Rosmarinic acid is currently produced by extraction from plants or chemical synthesis; however, due to limited availability of the plant sources and the complexity of the chemical synthesis method, there is an increasing interest in producing this compound by microbial fermentation. In this study, we aimed to produce rosmarinic acid by engineered baker's yeast Saccharomyces cerevisiae. Multiple biosynthetic pathway variants, carrying only plant genes or a combination of plant and Escherichia coli genes, were implemented using a full factorial design of experiment. Through analysis of variances, the effect of each enzyme variant (factors), together with possible interactions between these factors, was assessed. The best pathway variant produced 2.95 ± 0.08 mg/L rosmarinic acid in mineral medium with glucose as the sole carbon source. Increasing the copy number of rosmarinic acid biosynthetic genes increased the titer to 5.93 ± 0.06 mg/L. The study shows the feasibility of producing rosmarinic acid by yeast fermentation.

Significant random signatures reveals new biomarker for breast cancer.

BACKGROUND: In 2012, Venet et al. proposed that at least in the case of breast cancer, most published signatures are not significantly more associated with outcome than randomly generated signatures. They suggested that nominal p-value is not a good estimator to show the significance of a signature. Therefore, one can reasonably postulate that some information might be present in such significant random signatures. METHODS: In this research, first we show that, using an empirical p-value, these published signatures are more significant than their nominal p-values. In other words, the proposed empirical p-value can be considered as a complimentary criterion for nominal p-value to distinguish random signatures from significant ones. Secondly, we develop a novel computational method to extract information that are embedded within significant random signatures. In our method, a score is assigned to each gene based on the number of times it appears in significant random signatures. Then, these scores are diffused through a protein-protein interaction network and a permutation procedure is used to determine the genes with significant scores. The genes with significant scores are considered as the set of significant genes. RESULTS: First, we applied our method on the breast cancer dataset NKI to achieve a set of significant genes in breast cancer considering significant random signatures. Secondly, prognostic performance of the computed set of significant genes is evaluated using DMFS and RFS datasets. We have observed that the top ranked genes from this set can successfully separate patients with poor prognosis from those with good prognosis. Finally, we investigated the expression pattern of TAT, the first gene reported in our set, in malignant breast cancer vs. adjacent normal tissue and mammospheres. CONCLUSION: Applying the method, we found a set of significant genes in breast cancer, including TAT, a gene that has never been reported as an important gene in breast cancer. Our results show that the expression of TAT is repressed in tumors suggesting that this gene could act as a tumor suppressor in breast cancer and could be used as a new biomarker.

Biochemical and structural characterization of tyrosine aminotransferase suggests broad substrate specificity and a two-state folding mechanism in Leishmania donovani.

Tyrosine aminotransferase (TAT) is an aminotransferase with broad substrate specificity that catalyzes the transamination of aromatic amino acids in Leishmania donovani and plays a crucial role in the survival and pathogenicity of the parasite. In this study, we have biochemically characterized tyrosine aminotransferase from Leishmania donovani using in vitro and in silico techniques. Leishmania donovani tyrosine aminotransferase (LdTAT) was cloned into the pET28a(+) vector and expressed in the BL21 strain of Escherichia coli. The Ni-NTA-purified protein was then characterized biochemically, and its various kinetic parameters were investigated. The apparent Km value for the tyrosine-pyruvate pair was determined to be 3.5 ± 0.9 mm, and Vmax was analyzed to be at 11.7 ± 1.5 µm·min.µg-1 . LdTAT was found to exhibit maximum activity at 50 °C and at a pH of 8.0. Cofactor identification for LdTAT showed that pyridoxal-5-phosphate (PLP) binds with a Km value of 23.59 ± 3.99 µm and that the phosphate group is vital for the activity of the enzyme. Sequence analysis revealed that S151, Y256, K286, and P291 are conserved residues and form hydrogen bonds with PLP. Urea-based denaturation studies revealed a biphasic folding mechanism involving N→X→D states. Molecular dynamic simulations of modeled LdTAT at various conditions were performed to understand enzyme behavior and interactions at the molecular level. The biochemical and structural divergence between host and parasite TAT suggests the LdTAT has evolved to utilize pyruvate rather than α-ketoglutarate as co-substrate. Furthermore, our data suggest that LdTAT may be a potential drug target due to its divergence in structure and substrate specificity from the host.

Tyrosine aminotransferase is involved in the oxidative stress response by metabolizing meta-tyrosine in Caenorhabditis elegans.

Under oxidative stress conditions, hydroxyl radicals can oxidize the phenyl ring of phenylalanine, producing the abnormal tyrosine isomer meta-tyrosine (m-tyrosine). m-Tyrosine levels are commonly used as a biomarker of oxidative stress, and its accumulation has recently been reported to adversely affect cells, suggesting a direct role for m-tyrosine in oxidative stress effects. We found that the Caenorhabditis elegans ortholog of tyrosine aminotransferase (TATN-1)-the first enzyme involved in the metabolic degradation of tyrosine-is up-regulated in response to oxidative stress and directly activated by the oxidative stress-responsive transcription factor SKN-1. Worms deficient in tyrosine aminotransferase activity displayed increased sensitivity to multiple sources of oxidative stress. Biochemical assays revealed that m-tyrosine is a substrate for TATN-1-mediated deamination, suggesting that TATN-1 also metabolizes m-tyrosine. Consistent with a toxic effect of m-tyrosine and a protective function of TATN-1, tatn-1 mutant worms exhibited delayed development, marked reduction in fertility, and shortened lifespan when exposed to m-tyrosine. A forward genetic screen identified a mutation in the previously uncharacterized gene F01D4.5-homologous with human transcription factor 20 (TCF20) and retinoic acid-induced 1 (RAI1)-that suppresses the adverse phenotypes observed in m-tyrosine-treated tatn-1 mutant worms. RNA-Seq analysis of F01D4.5 mutant worms disclosed a significant reduction in the expression of specific isoforms of genes encoding ribosomal proteins, suggesting that alterations in protein synthesis or ribosome structure could diminish the adverse effects of m-tyrosine. Our findings uncover a critical role for tyrosine aminotransferase in the oxidative stress response via m-tyrosine metabolism.

TAT1 and TAT2 tyrosine aminotransferases have both distinct and shared functions in tyrosine metabolism and degradation in Arabidopsis thaliana.

Plants produce various l-tyrosine (Tyr)-derived compounds that are critical for plant adaptation and have pharmaceutical or nutritional importance for human health. Tyrosine aminotransferases (TATs) catalyze the reversible reaction between Tyr and 4-hydroxyphenylpyruvate (HPP), representing the entry point in plants for both biosynthesis of various natural products and Tyr degradation in the recycling of energy and nutrients. To better understand the roles of TATs and how Tyr is metabolized in planta, here we characterized single and double loss-of-function mutants of TAT1 (At5g53970) and TAT2 (At5g36160) in the model plant Arabidopsis thaliana As reported previously, tat1 mutants exhibited elevated and decreased levels of Tyr and tocopherols, respectively. The tat2 mutation alone had no impact on Tyr and tocopherol levels, but a tat1 tat2 double mutant had increased Tyr accumulation and decreased tocopherol levels under high-light stress compared with the tat1 mutant. Relative to WT and the tat2 mutant, the tat1 mutant displayed increased vulnerability to continuous dark treatment, associated with an early drop in respiratory activity and sucrose depletion. During isotope-labeled Tyr feeding in the dark, we observed that the tat1 mutant exhibits much slower 13C incorporation into tocopherols, fumarate, and other tricarboxylic acid (TCA) cycle intermediates than WT and the tat2 mutant. These results indicate that TAT1 and TAT2 function together in tocopherol biosynthesis, with TAT2 having a lesser role, and that TAT1 plays the major role in Tyr degradation in planta Our study also highlights the importance of Tyr degradation under carbon starvation conditions during dark-induced senescence in plants.

Comprehensive genomic analysis of the TYROSINE AMINOTRANSFERASE (TAT) genes in apple (Malus domestica) allows the identification of MdTAT2 conferring tolerance to drought and osmotic stresses in plants.

Tyrosine aminotransferase (TAT, EC 2.6.1.5) is the first key enzyme that catalyzes the reversible interconversion of tyrosine and 4-hydroxyphenylpyruvate in the tyrosine-derived pathway for syntheses of important secondary metabolites and compounds. Although plant TAT genes have been proposed to be important in response to abiotic stress, there is little information about TAT genes in woody perennial tree species, especially in economic fruit trees. Based on TAT domain searching, sequence homology screening and phylogenetic analysis, we identified four TATs in apple genome. Then, we carried out a detailed phylogenetic analysis of TAT genes from multi-species, focusing on apple (Malus domestica). The result showed that the TAT family comprises three major classes corresponding to genes from angiosperms, mammals, and bacteria. Angiosperm TAT genes could be further divided into six subclasses. Analysis of intron-exon structure revealed that the typical TAT gene contains six introns and seven exons, with exons of similar size at each exon location. Promoter analysis showed that the 5'-flanking region of apple MdTATs contain multiple cis-acting elements including those implicated in light, biotic stress, abiotic stress, and hormone response. MdTATs were expressed to various levels in all apple structures and organs evaluated, and showed distinct expression patterns under water deficit stress. Ectopic expression of MdTAT2 in Arabidopsis or over-expression of MdTAT2 in apple callus tissue conferred enhanced tolerance to drought and osmotic stress. Collectively, these results suggest a role for TAT genes in drought and osmotic stresses and provide valuable information for further research of TAT genes and their function in plants.

Receptor/gene/protein-mediated signaling connects methylprednisolone exposure to metabolic and immune-related pharmacodynamic actions in liver.

A multiscale pharmacodynamic model was developed to characterize the receptor-mediated, transcriptomic, and proteomic determinants of corticosteroid (CS) effects on clinically relevant hepatic processes following a single dose of methylprednisolone (MPL) given to adrenalectomized (ADX) rats. The enhancement of tyrosine aminotransferase (TAT) mRNA, protein, and enzyme activity were simultaneously described. Mechanisms related to the effects of MPL on glucose homeostasis, including the regulation of CCAAT-enhancer binding protein-beta (C/EBPß) and phosphoenolpyruvate carboxykinase (PEPCK) as well as insulin dynamics were evaluated. The MPL-induced suppression of circulating lymphocytes was modeled by coupling its effect on cell trafficking with pharmacogenomic effects on cell apoptosis via the hepatic (STAT3-regulated) acute phase response. Transcriptomic and proteomic time-course profiles measured in steroid-treated rat liver were utilized to model the dynamics of mechanistically relevant gene products, which were linked to associated systemic end-points. While time-courses of TAT mRNA, protein, and activity were well described by transcription-mediated changes, additional post-transcriptional processes were included to explain the lack of correlation between PEPCK mRNA and protein. The immune response model quantitatively discerned the relative roles of cell trafficking versus gene-mediated lymphocyte apoptosis by MPL. This systems pharmacodynamic model provides insights into the contributions of selected molecular events occurring in liver and explores mechanistic hypotheses for the multi-factorial control of clinically relevant pharmacodynamic outcomes.

Comparative Analysis of Tocopherol Biosynthesis Genes and Its Transcriptional Regulation in Soybean Seeds.

Tocopherols composed of four isoforms (α, ß, γ, and δ) and its biosynthesis comprises of three pathways: methylerythritol 4-phosphate (MEP), shikimate (SK) and tocopherol-core pathways regulated by 25 enzymes. To understand pathway regulatory mechanism at transcriptional level, gene expression profile of tocopherol-biosynthesis genes in two soybean genotypes was carried out, the results showed significantly differential expression of 5 genes: 1-deoxy-d-xylulose-5-P-reductoisomerase (DXR), geranyl geranyl reductase (GGDR) from MEP, arogenate dehydrogenase (TyrA), tyrosine aminotransferase (TAT) from SK and γ-tocopherol methyl transferase 3 (γ-TMT3) from tocopherol-core pathways. Expression data were further analyzed for total tocopherol (T-toc) and α-tocopherol (α-toc) content by coregulation network and gene clustering approaches, the results showed least and strong association of γ-TMT3/tocopherol cyclase (TC) and DXR/DXS, respectively, with gene clusters of tocopherol biosynthesis suggested the specific role of γ-TMT3/TC in determining tocopherol accumulation and intricacy of DXR/DXS genes in coordinating precursor pathways toward tocopherol biosynthesis in soybean seeds. Thus, the present study provides insight into the major role of these genes regulating the tocopherol synthesis in soybean seeds.

JTP-117968, a novel selective glucocorticoid receptor modulator, exhibits improved transrepression/transactivation dissociation.

Classic glucocorticoids that have outstanding anti-inflammatory effects are still widely prescribed for the treatment of various inflammatory and autoimmune diseases. Conversely, glucocorticoids cause numerous unwanted side effects, particularly systemically dosed glucocorticoids. Therefore, selective glucocorticoid receptor modulator (SGRM), which maintains beneficial anti-inflammatory effects while reducing the occurrence of side effects, is one of the most anticipated drugs. However, there have been no SGRMs marketed to date. The assumption is that there are two major mechanisms of action of glucocorticoids via glucocorticoid receptors, transrepression (TR) and transactivation (TA). In general, the anti-inflammatory effects of glucocorticoids are mostly mediated through TR, while the side effects associated with glucocorticoids are largely caused by TA. We started to evaluate novel orally available SGRMs that maintain anti-inflammatory effects while minimizing adverse effects by favoring TR over TA. Based on this evaluation, we discovered JTP-117968, (4b'S,7'R,8a'S)-4b'-benzyl-7'-hydroxy-N-(2-methylpyridin-3-yl)-7'-(trifluoromethyl)-4b',6',7',8',8a',10'-hexahydro-5'H-spiro[cyclopropane-1,9'-phenanthrene]-2'-carboxamide, a non-steroidal SGRM. JTP-117968 has partial TR activity, but exhibits extremely low TA activity. The maximum TR efficacy of JTP-117968 was comparable to its structural analogue, PF-802, (4bS,7R,8aR)-4b-Benzyl-7-hydroxy-N-(2-methylpyridin-3-yl)-7-(trifluoromethyl)-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide, which is the active form of Fosdagrocorat that has been developed clinically as a first-in-class orally available SGRM. Remarkably, the TA activity of JTP-117968 was much weaker than PF-802 not only in in vitro assays, but also in in vivo mice experiments. These findings indicate that JTP-117968 exhibits improved TR/TA dissociation because the compound has significantly lower TA activity compared with an already reported SGRM. Therefore, JTP-117968 is expected to be a useful compound for evaluating ideal SGRMs in the future.

Identification and validation of reference genes for quantitative real-time PCR studies in long yellow daylily, Hemerocallis citrina Borani.

Gene expression analysis using reverse transcription quantitative real-time PCR (RT-qPCR) requires the use of reference gene(s) in the target species. The long yellow daylily, Hemerocallis citrina Baroni. is rich in beneficial secondary metabolites and is considered as a functional vegetable. It is widely cultivated and consumed in East Asian countries. However, reference genes for use in RT-qPCR in H. citrina are not available. In the present study, six potential reference genes, actin (ACT), AP-4 complex subunit (AP4), tubulin (TUB), ubiquitin (UBQ), 18S and 60S ribosomal RNA, were selected and their expression stability in different developmental stages, organs and accessions was evaluated using four statistical software packages (geNorm, NormFinder, BestKeeper, and RefFinder). For commercial flower buds of different landraces, the combination of 60S, TUB, and AP4 was appropriate whereas ACT and 60S was suitable for normalization of different organs. In addition, AP4 exhibited the most stable expression in flower buds among different developmental stages. UBQ was less stable than the other reference genes under the experimental conditions except under different organs was 18S. The relative expression levels of two genes, primary-amine oxidase (HcAOC3) and tyrosine aminotransferase (HcTAT) which play important roles in alkaloid biosynthesis were also examined in different organs of the 'Datong' landrace, which further confirmed the results of selected reference genes. This is the first report to evaluate the stability of reference genes in the long yellow daylily that can serve as a foundation for RT-qPCR analysis of gene expression in this species.

Tyrosinemia type II: Mutation update, 11 novel mutations and description of 5 independent subjects with a novel founder mutation.

BACKGROUND: Tyrosinemia type II, also known as Richner-Hanhart Syndrome, is an extremely rare autosomal recessive disorder, caused by mutations in the gene encoding hepatic cytosolic tyrosine aminotransferase, leading to the accumulation of tyrosine and its metabolites which cause ocular and skin lesions, that may be accompanied by neurological manifestations, mostly intellectual disability. AIMS: To update disease-causing mutations and current clinical knowledge of the disease. MATERIALS AND METHODS: Genetic and clinical information were obtained from a collection of both unreported and previously reported cases. RESULTS: We report 106 families, represented by 143 individuals, carrying a total of 36 genetic variants, 11 of them not previously known to be associated with the disease. Variants include 3 large deletions, 21 non-synonymous and 5 nonsense amino-acid changes, 5 frameshifts and 2 splice variants. We also report 5 patients from Gran Canaria, representing the largest known group of unrelated families sharing the same P406L mutation. CONCLUSIONS: Data analysis did not reveal a genotype-phenotype correlation, but stressed the need of early diagnosis: All patients improved the oculocutaneous lesions after dietary treatment but neurological symptoms prevailed. The discovery of founder mutations in isolated populations, and the benefits of early intervention, should increase diagnostic awareness in newborns.

Tyrosine Detoxification Is an Essential Trait in the Life History of Blood-Feeding Arthropods.

Blood-feeding arthropods are vectors of infectious diseases such as dengue, Zika, Chagas disease, and malaria [1], and vector control is essential to limiting disease spread. Because these arthropods ingest very large amounts of blood, a protein-rich meal, huge amounts of amino acids are produced during digestion. Previous work on Rhodnius prolixus, a vector of Chagas disease, showed that, among all amino acids, only tyrosine degradation enzymes were overexpressed in the midgut compared to other tissues [2]. Here we demonstrate that tyrosine detoxification is an essential trait in the life history of blood-sucking arthropods. We found that silencing Rhodnius tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD), the first two enzymes of the phenylalanine/tyrosine degradation pathway, caused the death of insects after a blood meal. This was confirmed by using the HPPD inhibitor mesotrione, which selectively killed hematophagous arthropods but did not affect non-hematophagous insects. In addition, mosquitoes and kissing bugs died after feeding on mice that had previously received a therapeutic effective oral dose (1 mg/kg) of nitisinone, another HPPD inhibitor used in humans for the treatment of tyrosinemia type I [3]. These findings indicate that HPPD (and TAT) can be a target for the selective control of blood-sucking disease vector populations. Because HPPD inhibitors are extensively used as herbicides and in medicine, these compounds may provide an alternative less toxic to humans and more environmentally friendly than the conventional neurotoxic insecticides that are currently used, with the ability to affect only hematophagous arthropods.