A Simple Substitution on Thyroid Hormones Remarkably Alters the Regioselectivity of Deiodination by a Deiodinase Mimic.

The regioselective deiodinations of L-thyroxine (T4) play key roles in the thyroid hormone homeostasis. These reactions are catalyzed by three isoforms of the selenoenzymes, iodothyronine deiodinases (Dio1, Dio2 and Dio3), which are highly homologous in nature. Dio1 mediates 5'- or 5-deiodinations of T4 to produce T3 and rT3, respectively. In contrast, Dio2 and Dio3 are selective to 5'- or 5-deiodination to produce T3 and rT3, respectively. Understanding of the regioselectivity of deiodination at the molecular level is important as abnormal levels of thyroid hormone have been implicated in various clinical conditions, such as hypoxia, myocardial infarction, neuronal ischemia and cancer. In this paper, we report that the electronic properties of the iodine atoms in thyroxine (T4) can be modulated through a simple substitution in the 4'-phenolic moiety. This leads to the change in the regioselectivity of deiodination by different small molecule mimics of Dio enzymes. By using this chemical approach, we also show that the substitution of a strong electron withdrawing group facilitates the removal of all four iodine atoms in the T4 derivative. Theoretical investigations on the hydrogen bonded adducts of T4 with imidazole indicate that the charge on the iodine atoms depend on the nature of hydrogen bond between the -OH group of T4 and the imidazole moiety. While the imidazole can act as either hydrogen bond acceptor (HBA) or hydrogen bond donor (HBD), the protonated imidazole acts exclusively as HBD in T4-imidazole complex. These studies support the earlier observations that the histidine residue at the active sites of the deiodinases play an important role not only in the substrate binding, but also in altering the regioselectivity of the deiodination reactions.

Modeling Type-1 Iodothyronine Deiodinase with Peptide-Based Aliphatic Diselenides: Potential Role of Highly Conserved His and Cys Residues as a General Acid Catalyst.

Type-1 iodothyronine deiodinase (ID-1) catalyzes the reductive elimination of 5'-I and 5-I on the phenolic and tyrosyl rings of thyroxine (T4), respectively. Chemically verifying whether I atoms with different chemical properties undergo deiodination through a common mechanism is challenging. Herein, we report the modeling of ID-1 using aliphatic diselenide (Se-Se) and selenenylsulfide (Se-S) compounds. Mechanistic investigations of deiodination using the ID-1-like reagents suggested that the 5'-I and 5-I deiodinations proceed via the same mechanism through an unstable intermediate containing a Se⋅⋅⋅I halogen bond between a selenolate anion, reductively produced from Se-Se (or Se-S) in the compound, and an I atom in T4. Moreover, imidazolium and thiol groups, which may act as general acid catalysts, promoted the heterolytic cleavage of the C-I bond in the Se⋅⋅⋅I intermediate, which is the rate-determining step, by donating a proton to the C atom.

L-3,3',5-triiodothyronine and pregnenolone sulfate inhibit Torpedo nicotinic acetylcholine receptors.

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand-gated ion channel (pLGIC), homologous to the inhibitory γ-aminobutyric acid (GABA) type A receptor targeted by pharmaceuticals and endogenous sedatives. Activation of the GABAA receptor by the neurosteroid allopregnanolone can be inhibited competitively by thyroid hormone (L-3,3',5-triiodothyronine, or T3), but modulation of nAChR by T3 or neurosteroids has not been investigated. Here we show that allopregnanolone inhibits the nAChR from Torpedo californica at micromolar concentrations, as do T3 and the anionic neurosteroid pregnenolone sulfate (PS). We test for the role of protein and ligand charge in mediated receptor inhibition by varying pH in a narrow range around physiological pH. We find that both T3 and PS become less potent with increasing pH, with remarkably similar trends in IC50 when T3 is neutral at pH < 7.3. After deprotonation of T3 (but no additional deprotonation of PS) at pH 7.3, T3 loses potency more slowly with increasing pH than PS. We interpret this result as indicating the negative charge is not required for inhibition but does increase activity. Finally, we show that both T3 and PS affect nAChR channel desensitization, which may implicate a binding site homologous to one that was recently indicated for accelerated desensitization of the GABAA receptor by PS.

Alternative ligands for thyroid hormone receptors.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that activate or repress gene transcription, resulting in the regulation of numerous physiological programs. While 3,3',5-L-triiodothyronine is the TR cognate ligand, these receptors can also be activated by various alternative ligands, including endogenous and synthetic molecules capable of inducing diverse active receptor conformations that influence thyroid hormone-dependent signaling pathways. This review mainly discusses current knowledge on 3,5-diiodo-L-thyronine and 3,5,3'-triiodothyroacetic acid, two endogenous molecules that bind to TRs and regulate gene expression; and the molecular interactions between TRs and ligands, like synthetic thyromimetics developed to target specific TR isoforms for tissue-specific regulation of thyroid-related disorders, or endocrine disruptors that have allowed the design of new analogues and revealed essential amino acids for thyroid hormone binding.

Strategic design of peptide-decorated aligned nanofibers impregnated with triiodothyronine for neural regeneration.

Nerve injuries are often debilitating as its regeneration occurs in a slow and laborious manner. Remediation of nerve injury is a colossal task as functional restoration in larger gaps seldom occurs due to the complex nerve regeneration mechanism. A nanofiber-based graft material has been fabricated to provide topographical and biochemical cues to encourage neural differentiation. Laminin plays a crucial role in supporting peripheral nerve regeneration and hence aligned polyvinyl cinnamate nanofibers surface-conjugated with laminin-derived cell-adhesion peptides have been fabricated to improve selective neural adhesion and regeneration. Further, triiodothyronine has been encapsulated within the nanofibers enabling its sustained release so as to bolster regeneration and reinstate the lost functionality to the damaged nerve. The fabricated nanofibers were characterized for its physicochemical, morphological, and topographical properties. Nanofibers were biocompatible, improved cell adhesion rate, and illustrated favourable interaction with cells. Gene expression (showed 9.5 and 4.1 fold increase in ß-tubulin and MAP 2 expression, respectively) and protein expression (immunofluorescence, flow cytometry, and western blot) studies confirmed the positive influence of the scaffold over cell differentiation. The studies were extrapolated to adult zebrafish model with a surgical incision in posterior lateral line. The biocomposite treated group showed earlier functional restoration of the nerve compared with control groups detected by touch-evoked response. Thus, the combination of aligned nanofibers providing topographical cue, along with the peptides and triiodothyronine serving as biochemical cues, has a robust potential to restore functionality to the injured nerve, thereby opening avenues for fabrication of regenerative nerve grafts.

Environmental photochemical fate and UVC degradation of sodium levothyroxine in aqueous medium.

The synthetic hormone sodium levothyroxine (LTX) is one of the most prescribed drugs in the world and the most effective in hypothyroidism treatment. The presence of LTX in the environment has become a matter of major concern due to the widespread use of this hormone and by the fact that it is only partially removed in conventional water and sewage treatment plants. However, information regarding the photochemical fate of this hormone in environmental or engineered systems is scarce in the literature. In this work, the sunlight-driven direct and indirect LTX degradation was investigated by determining the photolysis quantum yield, ΦLTX = 3.80 (± 0.02) × 10-5, as well as the second-order kinetic constants of the reactions with hydroxyl radicals, kLTX,•OH = 1.50 (± 0.01) × 1010 L mol-1 s-1 and singlet oxygen, kLTX,1O2 = 1.47 (± 0.66) × 108 L mol-1 s-1. Mathematical simulations indicate that LTX photodegradation is favored in shallow, nitrite-rich, and dissolved organic matter (DOM)-poor environments, with LTX half-life times varying from less than 10 days to about 80 days. LTX removals of 85 and 95% were achieved by UVC photolysis and UVC/H2O2 after 120 min, respectively. Three transformation products, triiodothyronine, diiodothyronine, and diiodotyrosine, were identified during LTX degradation by the UVC-based processes studied. The results herein regarding photo-induced kinetics coupled with environmental fate simulations may help evaluate LTX persistence and also the design of water and wastewater treatment processes.

Biotin interference in TSH, FT4, and FT3 assays based on the LOCI technology: Identifying interference by dilution.

BACKGROUND: Although biotin interferences in TSH, FT3, FT4, and other biotinylated antibody-based assays manufactured by Roche Diagnostics have been well studied, there are relatively few reports on biotin interference in biotin-based assays manufactured by other companies. We investigated biotin interferences in TSH, FT4, and FT3 assays based on the LOCI (luminescent oxygen channeling assay) technology using the Dimension Vista 1500 analyzer (Siemens). METHODS: We prepared four serum pools using leftover specimens. Three serum pools were prepared initially for the original study but the 4th pool was prepared three months later. The aliquots of serum pool one and two were supplemented with various amounts of biotin (50 -1200 ng/mL) followed by determination of TSH, FT4, and FT3 concentrations. The aliquots of third pool were also supplemented with biotin to investigate whether 1:3 dilution could identify biotin interference. Aliquots of serum pool four were supplemented with biotin in order to study reproducibility of our original data. RESULTS: We observed significantly elevated FT3 levels at biotin concentration of 100 ng/mL. In contrast, FT4 levels were falsely elevated but TSH levels were falsely decreased at a biotin level of 500 ng/mL. We also observed nonlinearity in dilution experiment. CONCLUSIONS: We conclude that FT3 assay is most susceptible to biotin interference (threshold: 100 ng/mL) while the FT4 and TSH assays are less affected (threshold: 500 ng/mL). In addition, we also observed nonlinearity upon 1:3 dilution, which may indicate biotin interference (or interference from other compounds).

Printing T3 and T4 oral drug combinations as a novel strategy for hypothyroidism.

Hypothyroidism is a chronic and debilitating disease that is estimated to affect 3% of the general population. Clinical experience has highlighted the synergistic value of combining triiodothyronine (T3) and thyroxine (T4) for persistent or recurrent symptoms. However, thus far a platform that enables the simultaneous and independent dosing of more than one drug for oral administration has not been developed. Thermal inkjet (TIJ) 2D printing is a potential solution to enable the dual deposition of T3 and T4 onto orodispersible films (ODFs) for therapy personalisation. In this study, a two-cartridge TIJ printer was modified such that it could print separate solutions of T3 and T4. Dose adjustments were achieved by printing solutions adjacent to each other, enabling therapeutic T3 (15-50 µg) and T4 dosages (60-180 µg) to be successfully printed. Excellent linearity was observed between the theoretical and measured dose for both T3 and T4 (R2 = 0.982 and 0.985, respectively) by changing the length of the print objective (Y-value). Rapid disintegration of the ODFs was achieved (<45 s). As such, this study for the first time demonstrates the ability to produce personalised dose combinations by TIJ printing T3 and T4 onto the same substrate for oral administration.

AlloFinder: a strategy for allosteric modulator discovery and allosterome analyses.

Allostery tweaks innumerable biological processes and plays a fundamental role in human disease and drug discovery. Exploration of allostery has thus been regarded as a crucial requirement for research on biological mechanisms and the development of novel therapeutics. Here, based on our previously developed allosteric data and methods, we present an interactive platform called AlloFinder that identifies potential endogenous or exogenous allosteric modulators and their involvement in human allosterome. AlloFinder automatically amalgamates allosteric site identification, allosteric screening and allosteric scoring evaluation of modulator-protein complexes to identify allosteric modulators, followed by allosterome mapping analyses of predicted allosteric sites and modulators in human proteome. This web server exhibits prominent performance in the reemergence of allosteric metabolites and exogenous allosteric modulators in known allosteric proteins. Specifically, AlloFinder enables identification of allosteric metabolites for metabolic enzymes and screening of potential allosteric compounds for disease-related targets. Significantly, the feasibility of AlloFinder to discover allosteric modulators was tested in a real case of signal transduction and activation of transcription 3 (STAT3) and validated by mutagenesis and functional experiments. Collectively, AlloFinder is expected to contribute to exploration of the mechanisms of allosteric regulation between metabolites and metabolic enzymes, and to accelerate allosteric drug discovery. The AlloFinder web server is freely available to all users at http://mdl.shsmu.edu.cn/ALF/.

An AOP-based alternative testing strategy to predict the impact of thyroid hormone disruption on swim bladder inflation in zebrafish.

The adverse outcome pathway (AOP) framework can be used to help support the development of alternative testing strategies aimed at predicting adverse outcomes caused by triggering specific toxicity pathways. In this paper, we present a case-study demonstrating the selection of alternative in chemico assays targeting the molecular initiating events of established AOPs, and evaluate use of the resulting data to predict higher level biological endpoints. Based on two AOPs linking inhibition of the deiodinase (DIO) enzymes to impaired posterior swim bladder inflation in fish, we used in chemico enzyme inhibition assays to measure the molecular initiating events for an array of 51 chemicals. Zebrafish embryos were then exposed to 14 compounds with different measured inhibition potentials. Effects on posterior swim bladder inflation, predicted based on the information captured by the AOPs, were evaluated. By linking the two datasets and setting thresholds, we were able to demonstrate that the in chemico dataset can be used to predict biological effects on posterior chamber inflation, with only two outliers out of the 14 tested compounds. Our results show how information organized using the AOP framework can be employed to develop or select alternative assays, and successfully forecast downstream key events along the AOP. In general, such in chemico assays could serve as a first-tier high-throughput system to screen and prioritize chemicals for subsequent acute and chronic fish testing, potentially reducing the need for long-term and costly toxicity tests requiring large numbers of animals.

Simple synthesis of novel copper metal-organic framework nanoparticles: biosensing and biological applications.

Novel copper metal organic framework nanoparticles Cu-MOF-NPs (C1) were prepared via two simple alternative methods and confirmed by analytical characterization using mass, IR, Raman, XRD spectrum, HR-TEM and TGA-DSC. Mass spectroscopy revealed the molecular ion peak at 647 m/z for the monomeric unit structure n[Cu(AIP)2(PIY)(H2O)2]·4H2O, the presence of which was further supported by mass fragmentation. The Raman spectrum revealed two separate peaks corresponding to D and G bands of carbon in the structure of C1. Moreover, TGA-DSC showed the presence of CuO. XRD data were typically consistent with Raman and TGA-DSC data. In addition, HR-TEM revealed that the morphology of the C1 nanoparticles is uniform with well-distributed elliptical/spherical particles with a size range from 7 to 19 nm. The spectrophotometric and biological activity studies based on Cu-MOF-NPs were analyzed. The results indicated that Cu-MOF-NPs (C1) were successfully used as biosensors for the assessment of the triiodothyronine hormone (T3). The calibration plot was achieved over the concentration range of 40.0-100.0 ng dl-1 T3 with limits of detection (LOD) and quantitation (LOQ) of 1.46 and 4.85 ng dl-1, respectively, and a correlation coefficient (r) of 0.973. Moreover, the Cu-MOF-NPs (C1) show more enhanced biological activity against various pathogens (five strains of bacteria: Gram positive and Gram negative) when compared to an antibacterial agent and the effectiveness of Cu-MOF-NPs increases with increasing particle dose. The interactions of MOF-NPs (C1) with the biological targets were studied.

Relationship between the dimerization of thyroglobulin and its ability to form triiodothyronine.

Thyroglobulin (TG) is the most abundant thyroid gland protein, a dimeric iodoglycoprotein (660 kDa). TG serves as the protein precursor in the synthesis of thyroid hormones tetraiodothyronine (T4) and triiodothyronine (T3). The primary site for T3 synthesis in TG involves an iodotyrosine acceptor at the antepenultimate Tyr residue (at the extreme carboxyl terminus of the protein). The carboxyl-terminal region of TG comprises a cholinesterase-like (ChEL) domain followed by a short unique tail sequence. Despite many studies, the monoiodotyrosine donor residue needed for the coupling reaction to create T3 at this evolutionarily conserved site remains unidentified. In this report, we have utilized a novel, convenient immunoblotting assay to detect T3 formation after protein iodination in vitro, enabling the study of T3 formation in recombinant TG secreted from thyrocytes or heterologous cells. With this assay, we confirm the antepenultimate residue of TG as a major T3-forming site, but also demonstrate that the side chain of this residue intimately interacts with the same residue in the apposed monomer of the TG dimer. T3 formation in TG, or the isolated carboxyl-terminal region, is inhibited by mutation of this antepenultimate residue, but we describe the first substitution mutation that actually increases T3 hormonogenesis by engineering a novel cysteine, 10 residues upstream of the antepenultimate residue, allowing for covalent association of the unique tail sequences, and that helps to bring residues Tyr2744 from apposed monomers into closer proximity.

The Affinity of Brominated Phenolic Compounds for Human and Zebrafish Thyroid Receptor ß: Influence of Chemical Structure.

Brominated phenolic compounds (BPCs) are found in the environment, and in human and wildlife tissues, and some are considered to have endocrine disrupting activities. The goal of this study was to determine how structural differences of 3 BPC classes impact binding affinities for the thyroid receptor beta (TRß) in humans and zebrafish. BPC classes included halogenated bisphenol A derivatives, halogenated oxidative transformation products of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and brominated phenols. Affinities were assessed using recombinant TRß protein in competitive binding assays with 125I-triiodothyronine (125I-T3) as the radioligand. Zebrafish and human TRß displayed similar binding affinities for T3 (Ki = 0.40 and 0.49 nM) and thyroxine (T4, Ki = 6.7 and 6.8 nM). TRß affinity increased with increasing halogen mass and atomic radius for both species, with the iodinated compounds having the highest affinity within their compound classes. Increasing halogen mass and radius increases the molecular weight, volume, and hydrophobicity of a compound, which are all highly correlated with increasing affinity. TRß affinity also increased with the degree of halogenation for both species. Human TRß displayed higher binding affinities for the halogenate bisphenol A compounds, whereas zebrafish TRß displayed higher affinities for 2,4,6-trichlorophenol and 2,4,6-trifluorophenol. Observed species differences may be related to amino acid differences within the ligand binding domains. Overall, structural variations impact TRß affinities in a similar manner, supporting the use of zebrafish as a model for TRß disruption. Further studies are necessary to investigate how the identified structural modifications impact downstream receptor activities and potential in vivo effects.

A novel lactoferrin-modified stealth liposome for hepatoma-delivery of triiodothyronine.

Triiodothyronine (T3), a thyroid hormone synthesized and secreted by the thyroid gland, plays an essential role in morphogenesis and differentiation through interaction with its nuclear receptors (TRs). However, there are increasing evidences for its role in hepatocellular carcinoma (HCC) suppression. The aim of this work was to develop an effective hepatocellular carcinoma targeting drug delivery system to improve T3 delivery to hepatic cancer cells as well as to reduce toxic side effects. Three different liposomal systems, such as unmodified, Stealth (PEGylated) and Lactoferrin (Lf)-modified-Stealth liposomes were successfully prepared by the film hydration method, and fully characterized. Liposome cell interactions and cellular uptake were evaluated in three different HCC target cells (FaO, HepG2 and SKHep) by confocal microscopy. Finally, in vitro cytotoxicity studies were carried out by using MTT assay to evaluate toxicity of the liposome delivery system and to test the effect of T3 when incorporated into liposomes. Internalization studies, performed using Lf-modified-liposomes labeled with the lipophilic marker Rho-PE and loaded with the hydrophilic probe CF, clearly demonstrated the effective internalization of both hydrophilic and lipophilic markers. Lf-liposomes might markedly enhance the specific cell binding and cellular uptake in hepatoma cells due to the mediating of Lf that could bind with high affinity to multiple receptors on cell surface, such as ASGP-R. Results obtained from this study highlight that the Lf- modified-liposomal delivery system may ensure a specific and sustained T3 delivery, thus, allowing reduced therapeutic doses and deleterious side effects of T3.

The inhibition of UDP-glucuronosyltransferases (UGTs) by tetraiodothyronine (T4) and triiodothyronine (T3).

1. UDP-glucuronosyltransferases (UGTs) are important drug-metabolizing enzymes (DMEs) catalyzing the glucuronidation elimination of various xenobiotics and endogenous substances. Endogenous substances are important regulators for the activity of various UGT isoforms. Triiodothyronine (T3) and thyroxine (T4) are important thyroid hormones essential for normal cellular differentiation and growth. The present study aims to elucidate the inhibition behavior of T3 and T4 on the activity of UGT isoforms. 2. In vitro recombinant UGTs-catalyzed glucuronidation of 4-methylumbelliferone (4-MU) was used to screen the inhibition potential of T3 and T4 on the activity of various UGT isoforms. Initial screening results showed that T4 exerted stronger inhibition potential than T3 on the activity of various UGT isoforms at 100 µM. Inhibition kinetics was determined for the inhibition of T4 on the representative UGT isoforms, including UGT1A1, -1A3, -1A7, -1A8, -1A10 and -2B7. The results showed that T4 competitively inhibited the activity of UGT1A1, -1A3, -1A7, 1A10 and -2B7, and noncompetitively inhibited the activity of UGT1A8. The inhibition kinetic parameters were calculated to be 1.5, 2.4, 11, 9.6, 4.8 and 3.0 µM for UGT1A1, -1A3, -1A7, -1A8, -1A10 and -2B7, respectively. In silico docking method was employed to demonstrate why T4 exerted stronger inhibition than T3 towards UGT1A1. Stronger hydrogen bonds and hydrophobic interaction between T4 and activity cavity of UGT1A1 than T3 contributed to stronger inhibition of T4 towards UGT1A1. 3. In conclusion, more clinical monitoring should be given for the patients with the elevation of T4 level due to stronger inhibition of UGT isoforms-catalyzed metabolism of drugs or endogenous substances by T4.

Kinetic Analysis of Drug Release from Compounded Slow-release Capsules of Liothyronine Sodium (T3).

The purpose of this study was to formulate extemporaneously compounded Liothyronine Sodium (T3) slow-release capsules and to evaluate their in vitro drug release performance. Twenty-one formulations containing T3 (7.5 µg) with various compositions of two different grades of Methocel E4M and K100M premium (30% to 90%), and/or SimpleCap/Lactose (10% to 70%) were examined. Quality assessment of the capsules was conducted by standard quality control criteria of the United States Pharmacopeia (i.e., weight variation, content uniformity) to ensure their compliance. The dissolution release profile of the formulations was evaluated using United States Pharmacopeia Apparatus type II (paddle method) at a speed of 50 rpm and temperature of 37°C in phosphate buffered saline media ( pH = 7.2 to 7.4). Aliquots from the media were taken periodically up to 24 hours and analyzed using a validated enzyme-linked immunosorbent assay method. The cumulative percentage of drug release for each formulation was fitted to eleven major release kinetic equations to determine the best-fit model of drug release, as well as the mechanism of release. Assay sensitivity was as low as 1 ng/mL and the optimal calibration range was found to be between 0 ng/mL and 7.5 ng/mL, which corresponded well with the average physiological plasma concentrations of T3. Liothyronine sodium with either SimpleCap (100%) or Methocel E4M (100%) exhibited slowrelease kinetic patterns of Peppas and Zero Order, respectively. The formulation with SimpleCap (100%) had a higher percentage of drug release (as compared to 100% Methocel E4M) within the first four hours; this formulation released 80% of the drug within 12 hours when the release was plateaued thereafter. The formulation with 30% Methocel E4M and 70% SimpleCap released 100% of the drug within the initial 12 hours and exhibited a Zero Order slow-release kinetic pattern. In general, the release kinetic rate of the formulations containing Methocel K100M appeared to be slower than Methocel E4M. This alteration may be due to a higher molecular weight and apparent viscosity of Methocel K100M. While most of the formulations were fitted to a slow-release kinetic pattern, several others including Methocel E4M 100%, 30% Methocel E4M+ 70% Simple Cap, 40% Methocel K100M+ 60% SimpleCap, 50% Methocel K100M+ 50% SimpleCap, 30% Methocel E4M+ 70% Lactose, 90% Methocel E4M+ 10% Lactose, 40% Methocel K100M+ 60% Lactose, and 50% Methocel K100M+ 50% Lactose followed an ideal slow-release kinetic pattern of Zero Order or Higuchi. The results of this study successfully demonstrated the optiomal composition of slow-release compounded capsules of T3. Future studies are warranted to evaluate the in vivo performance of the optimal formulations and to establish an in vitro-in vivo correlation.

Tailored release of triiodothyronine and retinoic acid from a spatio-temporally fabricated nanofiber composite instigating neuronal differentiation.

Regeneration of the central and peripheral nervous system is challenging since the functional restoration of injured nerves is an incredible task. The fabrication of an ideal nerve guide that fulfills the requirement to regenerate nerve tissue is a herculean challenge requiring a combination of both biochemical and topographical cues. The present study explores the combinatorial effect of aligned nanofibers and the regulated delivery of triiodothyronine and retinoic acid on nerve regeneration. A sequential release mechanism is adopted in fabricating the nanofiber scaffold, with triiodothyronine incorporated into the nanofiber shell ensuring its prior release, followed by retinoic acid (entrapped within zein nanoparticles) from the core. The composite nanofibers thus fabricated possess excellent mechanical, physical and thermal properties and good topographical morphology and were highly biocompatible. The nanofibers were scrutinized for their efficacy in stimulating differentiation to a neuronal phenotype. The elongation factor (E-factor) of the neural cells had doubled in the bioactive incorporated composite compared to other scaffolds, as observed on phalloidin staining of their cytoskeleton, which endorsed enhanced neural differentiation on the fabricated nanofiber scaffold. There was a significant increase in the expression of neural-lineage specific markers on investigation of mRNA by real time PCR, showing a 10 fold increase in the gene expression of ß-III-tubulin, a 5.5 fold increase for microtubule associated protein 2 gene and 3.5 fold for neurofilament M gene in the cells cultured over bioactive incorporated aligned nanofiber composites. Similarly protein expression was analyzed by immunofluorescence and flow cytometry studies, which showed an increase in the expression of ß-III-tubulin in the composite nanofiber. This corroborates that neuronal differentiation is enhanced by the aligned nanotopography and spatio-temporal delivery of triiodothyronine and retinoic acid, opening avenues for nerve regenerative graft fabrication.

The distribution and mechanism of iodotyrosine deiodinase defied expectations.

Iodotyrosine deiodinase (IYD) is unusual for its reliance on flavin to promote reductive dehalogenation under aerobic conditions. As implied by the name, this enzyme was first discovered to catalyze iodide elimination from iodotyrosine for recycling iodide during synthesis of tetra- and triiodothyronine collectively known as thyroid hormone. However, IYD likely supports many more functions and has been shown to debrominate and dechlorinate bromo- and chlorotyrosines. A specificity for halotyrosines versus halophenols is well preserved from humans to bacteria. In all examples to date, the substrate zwitterion establishes polar contacts with both the protein and the isoalloxazine ring of flavin. Mechanistic data suggest dehalogenation is catalyzed by sequential one electron transfer steps from reduced flavin to substrate despite the initial expectations for a single two electron transfer mechanism. A purported flavin semiquinone intermediate is stabilized by hydrogen bonding between its N5 position and the side chain of a Thr. Mutation of this residue to Ala suppresses dehalogenation and enhances a nitroreductase activity that is reminiscent of other enzymes within the same structural superfamily.

Structure-activity relationship of a novel series of inhibitors for cancer type transporter L-type amino acid transporter 1 (LAT1).

L-type amino acid transporter 1 (LAT1) is known as a cancer-type amino acid transporter. In cancer cells, LAT1 is responsible for the cellular uptake of many essential amino acids including leucine that activates mechanistic/mammalian target of rapamycin (mTOR), regulating cancer cell growth. In this study, we designed a novel series of LAT1 inhibitors, SKN101-105, based on the structure of triiodothyronine (T3), a known LAT1 blocker. The compounds consist of core structure of 2-amino-3-[3,5-dichloro-4-(naphthalene-1-methoxy)-phenyl]-propanoic acid and different modifications on the naphthalene. Among them, the compounds including SKN103 with a modified phenyl group at C-7 position of naphthalene inhibited LAT1-mediated leucine transport, whereas SKN102 with a phenyl group at C-6 position did not, indicating the importance of the position of substituents on the naphthalene for the interaction with LAT1. SKN103 was suggested to be a non-transportable blocker rather than a substrate of LAT1 and inhibited LAT1 in a competitive manner with the Ki value of 2.1 µM. SKN103 suppressed mTOR activity and the growth of cancer cells. Moreover, SKN103 in combination with cisplatin additively enhanced the growth inhibition in cancer cells. This study provides an additional insight into the structure-activity relationship of LAT1 ligands, which could lead to designing desirable LAT1 inhibitors.