ASIC1a affects hypothalamic signaling and regulates the daily rhythm of body temperature in mice.

The body temperature of mice is higher at night than during the day. We show here that global deletion of acid-sensing ion channel 1a (ASIC1a) results in lower body temperature during a part of the night. ASICs are pH sensors that modulate neuronal activity. The deletion of ASIC1a decreased the voluntary activity at night of mice that had access to a running wheel but did not affect their spontaneous activity. Daily rhythms of thyrotropin-releasing hormone mRNA in the hypothalamus and of thyroid-stimulating hormone ß mRNA in the pituitary, and of prolactin mRNA in the hypothalamus and pituitary were suppressed in ASIC1a-/- mice. The serum thyroid hormone levels were however not significantly changed by ASIC1a deletion. Our findings indicate that ASIC1a regulates activity and signaling in the hypothalamus and pituitary. This likely leads to the observed changes in body temperature by affecting the metabolism or energy expenditure.

Severe Resistance to Thyroid Hormone Beta in a Patient with Athyreosis.

We report a patient with congenital hypothyroidism due to athyreosis complicated by a heterozygous thyroid hormone receptor beta (THRß) gene mutation (R320L), resulting in a severe resistance to thyroid hormone beta phenotype. The proband inherited the mutant allele from his father, presenting a very mild phenotype. While the precise reason for this discrepancy remains unknown, we postulate the possibility of de novo mutation and mosaicism in the father. Correlating thyrotropin (TSH) with free thyroxine (fT4) allowed us to predict the amount of fT4 required to normalize the proband's TSH, which supported the treatment with high dose of levothyroxine.

XB130 Deficiency Causes Congenital Hypothyroidism in Mice due to Disorganized Apical Membrane Structure and Function of Thyrocytes.

Background: Congenital hypothyroidism is often caused by genetic mutations that impair thyroid hormone (TH) production, resulting in growth and development defects. XB130 (actin filament associated protein 1 like 2) is an adaptor/scaffold protein that plays important roles in cell proliferation, migration, intracellular signal transduction, and tumorigenesis. It is highly expressed in thyrocytes, however, its function in the thyroid remains largely unexplored. Methods:Xb130-/- mice and their littermates were studied. Postnatal growth and growth hormone levels were measured, and responses to low or high-iodine diet, and levothyroxine treatment were examined. TH and thyrotropin in the serum and TH in the thyroid glands were quantified. Structure and function of thyrocytes in embryos and postnatal life were studied with histology, immunohistochemistry, immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction. Results:Xb130-/- mice exhibited transient growth retardation postnatally, due to congenital hypothyroidism with reduced TH synthesis and secretion, which could be rescued by exogenous thyroxine supplementation. The thyroid glands of Xb130-/- mice displayed diminished thyroglobulin iodination and release at both embryonic and early postnatal stages. XB130 was found mainly on the apical membrane of thyroid follicles. Thyroid glands of embryonic and postnatal Xb130-/- mice exhibited disorganized apical membrane structure, delayed folliculogenesis, and abnormal formation of thyroid follicle lumina. Conclusion: XB130 critically regulates folliculogenesis by maintaining apical membrane structure and function of thyrocytes, and its deficiency leads to congenital hypothyroidism.

Homozygous loss-of-function mutations in SLC26A7 cause goitrous congenital hypothyroidism.

Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear.

TRH Action Is Impaired in Pituitaries of Male IGSF1-Deficient Mice.

Loss-of-function mutations in the X-linked immunoglobulin superfamily, member 1 (IGSF1) gene cause central hypothyroidism. IGSF1 is a transmembrane glycoprotein of unknown function expressed in thyrotropin (TSH)-producing thyrotrope cells of the anterior pituitary gland. The protein is cotranslationally cleaved, with only its C-terminal domain (CTD) being trafficked to the plasma membrane. Most intragenic IGSF1 mutations in humans map to the CTD. In this study, we used CRISPR-Cas9 to introduce a loss-of-function mutation into the IGSF1-CTD in mice. The modified allele encodes a truncated protein that fails to traffic to the plasma membrane. Under standard laboratory conditions, Igsf1-deficient males exhibit normal serum TSH levels as well as normal numbers of TSH-expressing thyrotropes. However, pituitary expression of the TSH subunit genes and TSH protein content are reduced, as is expression of the receptor for thyrotropin-releasing hormone (TRH). When challenged with exogenous TRH, Igsf1-deficient males release TSH, but to a significantly lesser extent than do their wild-type littermates. The mice show similarly attenuated TSH secretion when rendered profoundly hypothyroid with a low iodine diet supplemented with propylthiouracil. Collectively, these results indicate that impairments in pituitary TRH receptor expression and/or downstream signaling underlie central hypothyroidism in IGSF1 deficiency syndrome.

Genetic causes of congenital hypothyroidism due to dyshormonogenesis.

PURPOSE OF REVIEW: Overview of congenital hypothyroidism caused by thyroid hormone synthesis defects, the current understanding of their pathophysiology, and clinical implications of molecular diagnoses. RECENT FINDINGS: Genetic defects in all known thyroid-specific factors required for thyroid hormone synthesis have been described. These include defects in iodide trapping (NIS), in the facilitated iodide efflux across the apical membrane (PDS), the organification of iodide within the follicular lumen (thyroid peroxidase, DUOX2, DUOXA2), the substrate for thyroid hormone synthesis (thyroglobulin) and the ability to recover and retain intrathyroidal iodine (iodotyrosine deiodinase). Clinical and biochemical evaluation aids in selecting the most appropriate candidate gene(s). A definite molecular diagnosis of thyroid dyshormonogenesis allows genetic counseling and has prognostic value in differentiating transient from permanent congenital hypothyroidism and predicting the response of patients to iodine supplementation as adjunct or alternative treatment to L-T4 replacement. SUMMARY: Congenital hypothyroidism due to thyroid dyshormonogenesis is a heterogenic disorder that may be caused by mutations in any of the known steps in the thyroid hormone biosynthesis pathway. An exact molecular diagnosis allows genetic counseling and the identification of asymptomatic mutation carriers at risk of recurrent hypothyroidism, and provides a rationale for adjunct iodide supplementation.

The syndrome of inherited partial SBP2 deficiency in humans.

Selenium (Se) is an essential trace element required for the biosynthesis of selenoproteins. Selenocysteine insertion sequence (SECIS) binding protein 2 (SBP2) represents a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins. In 2005, we reported the first mutations in the SBP2 gene in two families in which the probands presented with transient growth retardation associated with abnormal thyroid function tests. Intracellular metabolism of thyroid hormone (TH) and availability of the active hormone, triiodothyronine, is regulated by three selenoprotein iodothyronine deiodinases (Ds). While acquired changes in D activities are common, inherited defects in humans were not known. Affected children were either homozygous or compound heterozygous for SBP2 mutations. Other selenoproteins, glutathione peroxidase, and selenoprotein P were also reduced in affected subjects. Since our initial report, another family manifesting a similar phenotype was found to harbor a novel SBP2 mutation. In vivo studies of these subjects have explored the effects of Se and TH supplementation. In vitro experiments have provided new insights into the effect of SBP2 mutations. In this review we discuss the clinical presentation of SBP2 mutations, their effect on protein function, consequence for selenoproteins, and the clinical course of subjects with SBP2 defects.

Clinical and molecular characterization of a novel selenocysteine insertion sequence-binding protein 2 (SBP2) gene mutation (R128X).

CONTEXT: Although acquired abnormalities of thyroid hormone metabolism are common, inherited defects in humans involving the synthesis of selenoproteins, including iodothyronine deiodinases, have been described in only one recent publication. OBJECTIVE: We report the study of a novel selenocysteine insertion sequence-binding protein 2 (SBP2) gene mutation (R128X) and its clinical and molecular characterization. SUBJECTS AND METHODS: A family of African origin was studied. The proband presented with growth retardation, low serum selenium level, and thyroid test abnormalities consisting of high serum total and free T(4) concentrations associated with low T(3), high rT(3), and normal TSH. The entire coding region of the SBP2 gene was sequenced and minigenes constructed to explain the nature of the defect. RESULTS: The proband was homozygous for a nonsense gene mutation that produces an early stop codon (R128X). Both parents and a sister were heterozygous but showed no growth or thyroid test abnormalities. Despite the severity of the defect, the patient had a relatively mild phenotype, similar to that associated with partial SBP2 deficiency. In vitro analysis showed that the mutant minigene synthesized SBP2 from at least three downstream ATGs capable of generating molecules containing the essential functional domains. Treatment with l-T(3) accelerated the growth velocity and advanced the bone age. CONCLUSIONS: We identified a novel SBP2 gene mutation producing an early arrest in the synthesis of a full-length molecule. The demonstration that SBP2 isoforms containing all functional domains could be synthesized from three downstream ATGs explains the relatively mild phenotype caused by this defect.

Selenium supplementation fails to correct the selenoprotein synthesis defect in subjects with SBP2 gene mutations.

BACKGROUND: Selenium (Se) is an essential trace element needed for the biosynthesis of selenoproteins. Selenocysteine incorporation sequence binding protein 2 (SBP2) represents a key trans-acting factor for the co-translational insertion of selenocysteine into selenoproteins. We recently described children with mutations in the SBP2 gene who displayed abnormal thyroid function tests and reduced selenoprotein concentrations. We have tried to improve selenoprotein biosynthesis and thyroid hormone metabolism in SBP2 deficient subjects by supplementing an organic and an inorganic Se form. METHODS: Three affected and two unaffected siblings received daily doses of 100, 200, or 400 microg selenomethionine-rich yeast and 400 microg sodium selenite for one month each. Serum was drawn at baseline and after supplementations. Thyroid function tests, extracellular glutathione peroxidase activity, Se, and selenoprotein P concentrations were determined. RESULTS: Selenomethionine-rich yeast increased serum Se concentrations in all subjects irrespective of genotype. Sodium selenite was effective in increasing the selenoprotein P concentration in normal and to a lesser degree in affected subjects. Both forms failed to increase the glutathione peroxidase activity or to correct the thyroid function abnormalities in the SBP2 deficient individuals indicating that impaired deiodinase expression was not positively affected. No adverse side effects were observed. CONCLUSIONS: Total serum Se concentrations in SBP2 deficient subjects respond to selenomethionine supplementation but this effect is not indicative for improved selenoprotein synthesis. Se is obviously not a limiting factor in the SBP2 deficient individuals when regular daily Se intake is provided. These findings might help to identify and diagnose more individuals with selenoprotein biosynthesis defects who might present at young age irrespective of their Se supply with characteristic thyroid function test abnormalities, growth retardation, and reduced Se and selenoprotein concentrations.

Type 3 deiodinase deficiency results in functional abnormalities at multiple levels of the thyroid axis.

The type 3 deiodinase (D3) is a selenoenzyme that inactivates thyroid hormones and is highly expressed during development and in the adult central nervous system. We have recently observed that mice lacking D3 activity (D3KO mice) develop perinatal thyrotoxicosis followed in adulthood by a pattern of hormonal levels that is suggestive of central hypothyroidism. In this report we describe the results of additional studies designed to investigate the regulation of the thyroid axis in this unique animal model. Our results demonstrate that the thyroid and pituitary glands of D3KO mice do not respond appropriately to TSH and TRH stimulation, respectively. Furthermore, after induction of severe hypothyroidism by antithyroid treatment, the rise in serum TSH in D3KO mice is only 15% of that observed in wild-type mice. In addition, D3KO animals rendered severely hypothyroid fail to show the expected increase in prepro-TRH mRNA in the paraventricular nucleus of the hypothalamus. Finally, treatment with T(3) results in a serum T(3) level in D3KO mice that is much higher than that in wild-type mice. This is accompanied by significant weight loss and lethality in mutant animals. In conclusion, the absence of D3 activity results in impaired clearance of T(3) and significant defects in the mechanisms regulating the thyroid axis at all levels: hypothalamus, pituitary, and thyroid.

Tissue-specific thyroid hormone deprivation and excess in monocarboxylate transporter (mct) 8-deficient mice.

Mutations of the X-linked thyroid hormone (TH) transporter (monocarboxylate transporter, MCT8) produce in humans unusual abnormalities of thyroid function characterized by high serum T3 and low T4 and rT3. The mechanism of these changes remains obscure and raises questions regarding the regulation of intracellular availability and metabolism of TH. To study the pathophysiology of MCT8 deficiency, we generated Mct8 knockout mice. Male mice deficient in Mct8 (Mct8(-/y)) replicate the thyroid abnormalities observed in affected men. TH deprivation and replacement with L-T3 showed that suppression of TSH required higher serum levels T3 in Mct8(-/y) than wild-type (WT) littermates, indicating hypothalamus and/or thyrotroph resistance to T3. Furthermore, T4 is required to maintain the high serum T3 level because the latter was not different between the two genotypes during administration of T3. Mct8(-/y) mice have 2.3-fold higher T3 content in liver associated with 6.1- and 3.1-fold increase in deiodinase 1 mRNA and enzymatic activity, respectively. The relative T3 excess in liver of Mct8(-/y) mice produced a decrease in serum cholesterol (79 +/- 18 vs. 137 +/- 38 mg/dl in WT) and an increase in alkaline phosphatase (107 +/- 23 vs. 58 +/- 3 U/liter in WT) levels. In contrast, T3 content in cerebrum was 1.8-fold lower in Mct8(-/y) mice, associated with a 1.6- and 10.6-fold increase in D2 mRNA and enzymatic activity, respectively, as previously observed in TH-deprived WT mice. We conclude that cell-specific differences in intracellular TH content due to differences in contribution of the various TH transporters are responsible for the unusual clinical presentation of this defect, in contrast to TH deficiency.

Dominant role of thyrotropin-releasing hormone in the hypothalamic-pituitary-thyroid axis.

Hypothalamic thyrotropin-releasing hormone (TRH) stimulates thyroid-stimulating hormone (TSH) secretion from the anterior pituitary. TSH then initiates thyroid hormone (TH) synthesis and release from the thyroid gland. Although opposing TRH and TH inputs regulate the hypothalamic-pituitary-thyroid axis, TH negative feedback is thought to be the primary regulator. This hypothesis, however, has yet to be proven in vivo. To elucidate the relative importance of TRH and TH in regulating the hypothalamic-pituitary-thyroid axis, we have generated mice that lack either TRH, the beta isoforms of TH receptors (TRbeta KO), or both (double KO). TRbeta knock-out (KO) mice have significantly higher TH and TSH levels compared with wild-type mice, in contrast to double KO mice, which have reduced TH and TSH levels. Unexpectedly, hypothyroid double KO mice also failed to mount a significant rise in serum TSH levels, and pituitary TSH immunostaining was markedly reduced compared with all other hypothyroid mouse genotypes. This impaired TSH response, however, was not due to a reduced number of pituitary thyrotrophs because thyrotroph cell number, as assessed by counting TSH immunopositive cells, was restored after chronic TRH treatment. Thus, TRH is absolutely required for both TSH and TH synthesis but is not necessary for thyrotroph cell development.

Genomic organization of mouse ZAKI-4 gene that encodes ZAKI-4 alpha and beta isoforms, endogenous calcineurin inhibitors, and changes in the expression of these isoforms by thyroid hormone in adult mouse brain and heart.

OBJECTIVE: ZAKI-4 was identified as a thyroid hormone-responsive gene in cultured human fibroblasts. A single ZAKI-4 gene encodes two isoforms, ZAKI-4 alpha and beta, both inhibiting calcineurin activity. ZAKI-4 alpha and beta differ at their N termini, and show distinct distribution profiles in human tissues. The aim of this study was to elucidate the organization of the mouse ZAKI-4 gene and to determine the effect of thyroid hormone on the expression of ZAKI-4 isoforms in vivo. DESIGN: We cloned mouse homologues of human ZAKI-4 alpha and beta cDNA. Fluorescence in situ hybridization and bioinformatics analysis were employed to determine the gene organization. The effect of thyroid hormone on the expression of ZAKI-4 isoforms in mouse brain and heart was also studied. METHODS: Total RNA extracted from mouse cerebellum was used to clone ZAKI-4 alpha and beta cDNAs by RT-PCR followed by rapid amplification of cDNA ends. Mice were rendered hypothyroid by feeding a low iodine diet supplemented with propylthiouracil for 2 weeks. In one group (hyperthyroid) L-T(3) was injected i.p. for the last 4 days whereas another group (hypothyroid) received vehicle only. Non-treated mice were controls. RESULTS AND CONCLUSION: Mouse ZAKI-4 alpha and beta cDNAs were highly homologous to the human isoforms. The gene was mapped on chromosome 17qC, syntenic to human chromosome 6 where the human ZAKI-4 gene is located. As observed in human, ZAKI-4 alpha mRNA was expressed only in brain whereas beta mRNA was distributed in other tissues as well, such as heart and skeletal muscle. ZAKI-4 alpha mRNA was lower in the cerebral cortex of hypothyroid mice. Injection of L-T(3) caused an increase in ZAKI-4 beta mRNA in heart; however, expression of neither ZAKI-4 alpha nor beta mRNA was influenced by thyroid status in other tissues. These results indicate that expression of ZAKI-4 alpha and beta isoforms is regulated by thyroid hormone in vivo, and the regulation is isoform- and tissue-specific.

Hypothyroidism in thyroid transcription factor 1 haploinsufficiency is caused by reduced expression of the thyroid-stimulating hormone receptor.

Humans expressing one allele of the thyroid transcription factor 1 (TTF1) gene have neurological symptoms and increased serum TSH with variable degrees of hypothyroidism. Ttf1+/- mice have also poor coordination and increased serum TSH concentration (205 +/- 22 vs. 92 +/- 12 mU/liter; P < 0.001) and slightly lower T4 (46 +/- 3 vs. 63 +/- 6 nmol/liter; P < 0.02) as compared with Ttf1+/+ mice. To determine whether the hypothyroidism is of central or primary origin, we examined the bioactivity of TSH, thyroidal response to exogenous TSH and the expression of genes regulated by TTF1. TSH bioactivity was normal, but T4 response to a low but not high dose of TSH was significantly reduced in the Ttf1+/- mice (5.5 +/- 2.2 vs. 15.3 +/- 4.1 nmol/liter; P < 0.03), indicating a reduced thyroidal response. Thyroid mRNAs were measured by real-time PCR (Ttf1+/+ littermates = 100%). Ttf1+/- mice had half the levels of TTF1 mRNA (54 +/- 9; P < 0.01) and protein, confirming their haploinsufficiency. Significantly lower levels of mRNAs were observed for two of the three genes with TTF1 cis elements: TSH receptor (TSHr, 57 +/- 4%; P < 0.002), thyroglobulin (63 +/- 7%; P < 0.005), but not thyroid peroxidase (81 +/- 12%; P > 0.05). No significant difference between the two genotypes was found for Pax8, sodium iodide symporter, and iodothyronine deiodinase 1. These results show that Ttf1 haploinsufficiency causes a reduction in the expression of TSHr and thyroglobulin, genes with TTF1 binding sites in their promoter regions. The low TSHr is only partially compensated by an increase in TSH secretion because T4 remains mildly reduced. However, administration of a larger amount of TSH obliterates the response differences by saturating a reduced amount of receptor.

Type 1 iodothyronine deiodinase in the house musk shrew (Suncus murinus, Insectivora: Soricidae): cloning and characterization of complementary DNA, unique tissue distribution and regulation by T(3).

The house musk shrew Suncus murinus (Insectivora: Soricidae) has been reported as having low thyroxine to 3,3'5-triiodothyronine (T(3)) converting activity in liver and kidney homogenates and was assumed to be type 1 iodothyronine deiodinase (D1)-deficient. To study whether this is due to structural abnormality of shrew D1, we cloned the cDNA and characterized the enzyme. The deduced amino acid sequence of shrew D1 was found to be highly homologous to other known D1s and the enzyme itself to have similar catalytic activity. However, unlike in other species, the D1 activity was detected only in liver. Moreover, the D1 activity in liver of the shrew was less than half of that in rat liver and its expression was not up-regulated by T(3). In contrast, a very high activity of D2 was demonstrated in brain and brown adipose tissue. The present study also revealed that the serum level of T(3) in the shrew was in the same range as these in other mammals. These results suggest that D2 contributes to the production and maintenance of T(3) levels in the house musk shrew.

Steroid receptor coactivator-1 deficiency causes variable alterations in the modulation of T(3)-regulated transcription of genes in vivo.

Thyroid hormone exerts its biological effect by binding to a TR. Both liganded and unliganded TRs regulate the transcription of T(3)-responsive genes. Cofactors with activating or repressing function modulate the transcriptional regulation by TRs. We showed that steroid receptor coactivator 1 (SRC-1)-deficient mice (SRC-1(-/-)) exhibit partial resistance to thyroid hormone at the level of the pituitary thyrotrophs. To determine whether SRC-1 deficiency affects globally T(3)-dependent transcriptional regulation, we studied the effects of thyroid hormone deprivation and replacement on the expression of several genes in different tissues of SRC-1(-/-) and wild-type mice (SRC-1(+/+)). Thyroid hormone deficiency was induced by a low iodine diet (LoI) supplemented with propylthiouracil (PTU) for 2 wk. L-T(3) was injected ip for the last 4 d in one group (PTU+T(3) group), and another group (PTU group) received only vehicle. Levels of mRNAs for T(3)-responsive genes were determined by Northern blotting: GH and TSH beta in pituitary; type 1 iodothyronine 5'-deiodinase, spot 14 (S14), and malic enzyme in liver; and sarcoplasmic reticulum calcium adenosine triphosphatase 2 and myosin heavy chain alpha and beta in heart. Serum parameters, TSH, total cholesterol, creatine kinase, and alkaline phosphatase (AP), were also measured. Hypothyroidism produced a comparable increase in TSH beta mRNA in both genotypes, but its suppression by L-T(3) was attenuated in SRC-1(-/-) mice. In contrast, hypothyroidism failed to reduce S14 mRNA levels in SRC-1(-/-) mice. As a consequence, the response to L-T(3) was not observed in these mice. SRC-1 deficiency had no effect on the expression of the rest of the T(3)-responsive genes examined. Of the four serum parameters, the T(3)-mediated decrease in TSH and changes in AP were attenuated in SRC-1(-/-) mice. We conclude that SRC-1 deficiency altered the expression of only some of the T(3)-responsive genes. SRC-1 appears to be involved not only in transcriptional activation by liganded TRs, but also in the suppression by liganded or unliganded TRs. Some of the effects of SRC-1 may be TR isoform specific.

Characterization and primary structures of bovine and porcine thyroxine-binding globulin.

Thyroxine-binding globulin (TBG) is the major serum transport protein for iodothyronines in most of the large, omni- or herbivorous mammals. Characterization of human TBG (hTBG), including its 20 known natural variants, allowed the identification of the ligand-binding site and a correlation of diminished synthesis or loss of function with mutations in the TBG gene. Further refinement of the structure-function correlation, especially the high binding affinity and heat stability, requires characterization of other mammalian TBGs, of which only rat and sheep TBG were available. We now present some of the chemical and physical properties of bovine TBG (bTBG) and porcine TBG (pTBG) and their primary structures deduced from their cDNA sequences. The serum concentrations of bTBG and pTBG estimated by Scatchard analysis of T(4)-binding were similar to hTBG. The T(4)-binding affinity of human, bovine and porcine TBGs were all similar, at 1.2x10(10) M(-1). However, heat stability of the animal TBGs was reduced, with a half life of denaturation of 7 min (bTBG) and 5 min (pTBG) at 55 degreeC, compared with 21 min for hTBG. Nucleotide alignment revealed identity with hTBG of 85.5% (bTBG) and 83.7% (pTBG) and amino acid identity of 82.8% (bTBG) and 82.6% (pTBG). As expected, the relevant parts of the ligand-binding domain (amino acids 215-291, and 363-395) were highly conserved at more than 95% similarity. Comparison of the five known mammalian TBGs allows focusing of future mutagenesis experiments to further characterize the properties of the molecule.