An integrated transcriptomic and computational analysis for biomarker identification in gastric cancer.

This report describes an integrated study on identification of potential markers for gastric cancer in patients' cancer tissues and sera based on: (i) genome-scale transcriptomic analyses of 80 paired gastric cancer/reference tissues and (ii) computational prediction of blood-secretory proteins supported by experimental validation. Our findings show that: (i) 715 and 150 genes exhibit significantly differential expressions in all cancers and early-stage cancers versus reference tissues, respectively; and a substantial percentage of the alteration is found to be influenced by age and/or by gender; (ii) 21 co-expressed gene clusters have been identified, some of which are specific to certain subtypes or stages of the cancer; (iii) the top-ranked gene signatures give better than 94% classification accuracy between cancer and the reference tissues, some of which are gender-specific; and (iv) 136 of the differentially expressed genes were predicted to have their proteins secreted into blood, 81 of which were detected experimentally in the sera of 13 validation samples and 29 found to have differential abundances in the sera of cancer patients versus controls. Overall, the novel information obtained in this study has led to identification of promising diagnostic markers for gastric cancer and can benefit further analyses of the key (early) abnormalities during its development.

Conserved amino acids participate in the structure networks deputed to intramolecular communication in the lutropin receptor.

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor (GPCR) particularly susceptible to spontaneous pathogenic gain-of-function mutations. Protein structure network (PSN) analysis on wild-type LHR and two constitutively active mutants, combined with in vitro mutational analysis, served to identify key amino acids that are part of the regulatory network responsible for propagating communication between the extracellular and intracellular poles of the receptor. Highly conserved amino acids in the rhodopsin family GPCRs participate in the protein structural stability as network hubs in both the inactive and active states. Moreover, they behave as the most recurrent nodes in the communication paths between the extracellular and intracellular sides in both functional states with emphasis on the active one. In this respect, non-conservative loss-of-function mutations of these amino acids is expected to impair the most relevant way of communication between activating mutation sites or hormone-binding domain and G protein recognition regions.

Functional differences of invariant and highly conserved residues in the extracellular domain of the glycoprotein hormone receptors.

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in ß-strands 2 (Lys(74)), 4 (Tyr(124), Asn(129), and Thr(130)), and 5 (Asp(150) and Asp(153)) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in ß-strands 2 (Ser(78)) and 8 (Asp(224) and Ser(226)) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.

Regulation of gene expression in ovarian cancer cells by luteinizing hormone receptor expression and activation.

BACKGROUND: Since a substantial percentage of ovarian cancers express gonadotropin receptors and are responsive to the relatively high concentrations of pituitary gonadotropins during the postmenopausal years, it has been suggested that receptor activation may contribute to the etiology and/or progression of the neoplasm. The goal of the present study was to develop a cell model to determine the impact of luteinizing hormone (LH) receptor (LHR) expression and LH-mediated LHR activation on gene expression and thus obtain insights into the mechanism of gonadotropin action on ovarian surface epithelial (OSE) carcinoma cells. METHODS: The human ovarian cancer cell line, SKOV-3, was stably transfected to express functional LHR and incubated with LH for various periods of time (0-20 hours). Transcriptomic profiling was performed on these cells to identify LHR expression/activation-dependent changes in gene expression levels and pathways by microarray and qRT-PCR analyses. RESULTS: Through comparative analysis on the LHR-transfected SKOV-3 cells exposed to LH, we observed the differential expression of 1,783 genes in response to LH treatment, among which five significant families were enriched, including those of growth factors, translation regulators, transporters, G-protein coupled receptors, and ligand-dependent nuclear receptors. The most highly induced early and intermediate responses were found to occupy a network impacting transcriptional regulation, cell growth, apoptosis, and multiple signaling transductions, giving indications of LH-induced apoptosis and cell growth inhibition through the significant changes in, for example, tumor necrosis factor, Jun and many others, supportive of the observed cell growth reduction in in vitro assays. However, other observations, e.g. the substantial up-regulation of the genes encoding the endothelin-1 subtype A receptor, stromal cell-derived factor 1, and insulin-like growth factor II, all of which are potential therapeutic targets, may reflect a positive mediation of ovarian cancer growth. CONCLUSION: Overall, the present study elucidates the extensive transcriptomic changes of ovarian cancer cells in response to LH receptor activation, which provides a comprehensive and objective assessment for determining new cancer therapies and potential serum markers, of which over 100 are suggested.

Luteinizing hormone-induced up-regulation of ErbB-2 is insufficient stimulant of growth and invasion in ovarian cancer cells.

The effects of luteinizing hormone (LH), a gonadotropic hormone implicated in the development of ovarian cancer, are mediated by specific binding to its G protein-coupled receptor, the LH receptor (LHR). Activated LHR initiates second messenger responses, including cyclic AMP (cAMP) and inositol phosphate. Because cAMP increases expression of ErbB-2, a receptor tyrosine kinase whose overexpression in cancers correlates with poor survival, we hypothesized that LH may regulate ErbB-2 expression. Cell surface LHR expression in stable transformants of the ErbB-2-overexpressing ovarian cancer cell line SKOV3 was confirmed by PCR and whole-cell ligand binding studies. Second messenger accumulation in the LHR-expressing cells confirmed signaling through Gs and Gq. Western blots of total protein revealed that LHR introduction up-regulated ErbB-2 protein expression 2-fold and this was further up-regulated in a time- and dose-dependent manner in response to LH. Forskolin and 8Br-cAMP also up-regulated ErbB-2 in both LHR-expressing and mock-transfected cells, indicating that regulation of ErbB-2 is a cAMP-mediated event. Kinase inhibitor studies indicated the involvement of protein kinase A-mediated, protein kinase C-mediated, epidermal growth factor receptor-mediated, and ErbB-2-mediated mechanisms. The LH-induced up-regulation of ErbB-2 was insufficient to overcome the negative effects of LH on proliferation, invasion, and migration. A molecular signature for this nonaggressive phenotype was determined by Taqman array to include increased and decreased expression of genes encoding adhesion proteins and metalloproteinases, respectively. These data establish a role for LH and LHR in the regulation of ErbB-2 expression and suggest that, in some systems, ErbB-2 up-regulation alone is insufficient in producing a more aggressive phenotype.

Computational prediction of human proteins that can be secreted into the bloodstream.

We present a novel computational method for predicting which proteins from highly and abnormally expressed genes in diseased human tissues, such as cancers, can be secreted into the bloodstream, suggesting possible marker proteins for follow-up serum proteomic studies. A main challenging issue in tackling this problem is that our understanding about the downstream localization after proteins are secreted outside the cells is very limited and not sufficient to provide useful hints about secretion to the bloodstream. To bypass this difficulty, we have taken a data mining approach by first collecting, through extensive literature searches, human proteins that are known to be secreted into the bloodstream due to various pathological conditions as detected by previous proteomic studies, and then asking the question: 'what do these secreted proteins have in common in terms of their physical and chemical properties, amino acid sequence and structural features that can be used to predict them?' We have identified a list of features, such as signal peptides, transmembrane domains, glycosylation sites, disordered regions, secondary structural content, hydrophobicity and polarity measures that show relevance to protein secretion. Using these features, we have trained a support vector machine-based classifier to predict protein secretion to the bloodstream. On a large test set containing 98 secretory proteins and 6601 non-secretory proteins of human, our classifier achieved approximately 90% prediction sensitivity and approximately 98% prediction specificity. Several additional datasets are used to further assess the performance of our classifier. On a set of 122 proteins that were found to be of abnormally high abundance in human blood due to various cancers, our program predicted 62 as blood-secreted proteins. By applying our program to abnormally highly expressed genes in gastric cancer and lung cancer tissues detected through microarray gene expression studies, we predicted 13 and 31 as blood secreted, respectively, suggesting that they could serve as potential biomarkers for these two cancers, respectively. Our study demonstrated that our method can provide highly useful information to link genomic and proteomic studies for disease biomarker discovery. Our software can be accessed at http://csbl1.bmb.uga.edu/cgi-bin/Secretion/secretion.cgi.

Contributions of intracellular loops 2 and 3 of the lutropin receptor in Gs coupling.

A number of amino acids essential for Gs coupling, i.e. hot spots, were identified after in vitro Ala-scanning mutagenesis of the cytosolic extensions of helices 3, 5, and 6 and of intracellular loops 2 and 3 (IL2 and IL3) of the human LH receptor (LHR). Consistent with the results of in vitro experiments involving ligand binding and ligand-mediated signaling in transiently transfected human embryonic kidney 293 cells, computational modeling of the isolated receptor and of the receptor-G protein complexes suggests an important role of the cytosolic extension of helix 3 and the N-terminal portion of the IL2 in Gs(alpha) interaction, whereas the contribution of IL3 is marginal. Mapping the hot spots into the computational models of LHR and the LHR-Gs complexes allowed for a distinction between receptor sites required for intramolecular structural changes (i.e. I460, T461, H466, and I549) and receptor sites more likely involved in G protein recognition (i.e. R464, T467, I468, Y470, Y550, and D564). The latter sites include the highly conserved arginine of the (E/D)R(Y/W) motif, which is therefore likely to be a receptor recognition point for Gs rather than a switch of receptor activation. The results of in vitro and in silico experiments carried out in this study represent the first comprehensive delineation of functionality of the individual residues in the intracellular domains of LHR and establish potential switches of receptor activation as well as a map of the primary receptor recognition sites for Gs. A novel way to consider constitutively active mutants was inferred from this study, i.e. receptor states with improved complementarity for the G protein compared to the wild-type receptor.

Shear wave elastography and Afirma™ gene expression classifier in thyroid nodules with indeterminate cytology: a comparison study.

PURPOSE: To compare shear wave elastography (SWE) and Afirma™ gene expression classifier (GEC) for diagnosis of malignancy in thyroid nodules (TNs) with Bethesda Classification (BC) III or IV indeterminate cytology. METHODS: This preliminary single-center prospective study was approved by the Institutional Review Board. We evaluated 151 consented patients with 151 indeterminate TNs (123 BC III, 28 BC IV) on fine-needle aspiration biopsy (FNAB). B-mode ultrasound, vascularity, and SWE were performed prior to FNAB. TN stiffness was measured as shear wave velocity (SWV) in meters per second (m/s). The stiffest area of the TN was selected for SWV measurement. GEC testing was performed with a second FNAB. Surgery was recommended for GEC-suspicious TNs, or GEC-benign TNs with two or more worrisome B-mode US features. RESULTS: Surgical pathology confirmed 31 malignant TNs. Among the GEC-suspicious group, 28 of 59 TNs were malignant. The SWV value of ≥3.59 m/s was the best cut-off for malignancy risk based on the receiver operating curve (ROC). Twenty-six malignant TNs had SWV ≥ 3.59 m/s. The sensitivity and specificity for SWV ≥ 3.59 m/s were 83.9 and 79.2%, respectively. Positive predictive value (PPV) was 51.0% and negative predictive value (NPV) was 95.0%. For the GEC-suspicious group, sensitivity, specificity, PPV, and NPV were 90.3, 74.2, 47.5, and 96.7%, respectively. In multivariate analysis, SWV and GEC-suspicious were significant predictors of malignancy, but B-mode features and vascularity were not. CONCLUSION: This preliminary study indicates that SWE and GEC are independent predictors of malignancy in TNs with BC III or IV.

Determination of hyperglycosylated human chorionic gonadotropin produced by malignant gestational trophoblastic neoplasias and male germ cell tumors using a lectin-based immunoassay and surface plasmon resonance.

The ability to reliably detect aberrant glycosylation of human chorionic gonadotropin (hCG) may have profound implications for the diagnosis and monitoring of malignant gestational trophoblastic neoplasia, germ cell tumors, other malignancies, and pregnancy complications. To become a clinically useful assay, however, this discrimination of glycoforms should be possible on minimally treated biological specimens. Towards this end, we have developed a lectin-based sandwich-type immunoassay to compare the glycosylation patterns of hCG among urine specimens from patients presenting with a normal pregnancy, invasive mole, choriocarcinoma, and male germ cell tumors using carbohydrate-free antibody fragments as capture reagents and a panel of eight lectins, five recognizing neutral sugars and three recognizing sialic acid. There was no significant difference in the binding of any of the lectins to hCG in the urine of women over the gestational range of 6-38 weeks. Three lectins, however, exhibited differential binding to urinary hCG derived from these normal pregnant controls and that from patients with malignant forms of gestational trophoblastic disease and male germ cell tumors. Galanthus nivalis agglutinin and Maackia amurensis lectin, which bind terminal mannose and alpha(2-3)sialic acid, respectively, preferentially bound pregnancy-derived hCG, whereas the lectin, wheat germ agglutinin, which binds sialic acid and beta(1-4)N-acetylglucosamine, exhibited decreased binding to pregnancy-derived hCG compared to that from patients with male germ cell tumors and malignant gestational trophoblastic neoplasia. The differential binding observed with these three promising lectins is most encouraging and warrants further examination. The experimental paradigm also holds promise for the development of comparable assays for other glycosylated tumor markers.

Steroidogenesis and early response gene expression in MA-10 Leydig tumor cells following heterologous receptor down-regulation and cellular desensitization.

The Leydig tumor cell line, MA-10, expresses the luteinizing hormone receptor, a G protein-coupled receptor that, when activated with luteinizing hormone or chorionic gonadotropin (CG), stimulates cAMP production and subsequent steroidogenesis, notably progesterone. These cells also respond to epidermal growth factor (EGF) and phorbol esters with increased steroid biosynthesis. In order to probe the intracellular pathways along with heterologous receptor down-regulation and cellular desensitization, cells were preincubated with EGF or phorbol esters and then challenged with CG, EGF, dibutryl-cyclic AMP, and a phorbol ester. Relative receptor numbers, steroid biosynthesis, and expression of the early response genes, JUNB and c-FOS, were measured. It was found that in all cases but one receptor down-regulation and decreased progesterone production were closely coupled under the conditions used; the exception involved preincubation of the cells with EGF followed by addition of CG where the CG-mediated stimulation of steroidogenesis was considerably lower than the level of receptor down-regulation. In a number of instances JUNB and c-FOS expression paralleled the decreases in receptor number and progesterone production, while in some cases these early response genes were affected little if at all by the changes in receptor number. This finding may indicate that even low levels of activated signaling kinases, e.g. protein kinase A, protein kinase C, or receptor tyrosine kinase, may suffice to yield good expression of JUNB and c-FOS, or it may suggest alternative pathways for regulating expression of these two early response genes.

Shear Wave Elastography and Cervical Lymph Nodes: Predicting Malignancy.

This prospective study evaluates the accuracy of virtual touch imaging quantification (VTIQ), a non-invasive shear wave elastography method for measuring cervical lymph nodes (LN) stiffness in differentiating benign from malignant LN. The study evaluated 270 LN in 236 patients with both conventional B-mode ultrasound and VTIQ shear wave elastography before fine-needle aspiration biopsy (FNAB). LN stiffness was measured as shear wave velocity (SWV) in m/s. Surgical resection was advised for FNAB results that were not clearly benign. Surgical pathology confirmed 54 malignant LN. The receiver operating curve (ROC) identified a single cut-off value of 2.93 m/s as the maximum SWV for predicting a malignant cervical LN. The sensitivity and specificity were 92.59% and 75.46%, respectively. Positive predictive value (PPV) was 48.54% and negative predictive value (NPV) was 97.60%. LN stiffness measured by VTIQ-generated shear wave elastography is an independent predictor of malignancy.

Shear wave elastography and parathyroid adenoma: A new tool for diagnosing parathyroid adenomas.

OBJECTIVES: This study prospectively determines the shear wave elastography characteristics of parathyroid adenomas using virtual touch imaging quantification, a non-invasive ultrasound based shear wave elastography method. METHODS: This prospective study examined 57 consecutive patients with biochemically proven primary hyperparathyroidism and solitary parathyroid adenoma identified by ultrasound and confirmed by at least one of the following: surgical resection, positive Technetium-99m Sestamibi Scintigraphy (MIBI) scan, or fine needle aspiration biopsy with positive PTH washout (performed only in MIBI negative patients). Vascularity and shear wave elastography were performed for all patients. Parathyroid adenoma stiffness was measured as shear wave velocity in meters per second. RESULTS: The median (range) pre-surgical value for PTH and calcium were 58pg/mL (19, 427) and 10.8mg/dL (9.5, 12.1), respectively. 37 patients had positive MIBI scan. 20 patients had negative MIBI scan but diagnosis was confirmed with positive PTH washout. 42 patients underwent parathyroidectomy, and an adenoma was confirmed in all. The median (range) shear wave velocity for all parathyroid adenomas enrolled in this study was 2.02m/s (1.53, 2.50). The median (range) shear wave velocity for thyroid tissue was 2.77m/s (1.89, 3.70). The shear wave velocity of the adenomas was independent of adenoma size, serum parathyroid hormone concentration, or plasma parathyroid hormone concentration. CONCLUSIONS: Tissue elasticity of parathyroid adenoma is significantly lower than thyroid tissue. B-mode features and distinct vascularity pattern are helpful tools in diagnosing parathyroid adenoma with ultrasound. Shear wave elastography may provide valuable information in diagnosing parathyroid adenoma.

Gonadal defects and hormonal alterations in transgenic mice expressing a single chain human chorionic gonadotropin-lutropin receptor complex.

To study the effects of premature and chronic ligand-mediated luteinizing hormone receptor (LHR) activation on reproductive development, we have generated transgenic mice expressing a genetically engineered, constitutively active yoked hormone-receptor complex (YHR), in which a fusion protein of human chorionic gonadotropin (hCG) is covalently linked to the N-terminus of rat LHR. YHR-expressing mice (YHR(+)) were analyzed at pre- and post-pubertal ages. Relative to wild type (WT) controls, male mice exhibited prepubertal increases in testosterone levels and seminal vesicle weights, and decreases in serum FSH, serum LH, testes weight, and the size of the seminiferous tubules. In adult male YHR(+) mice, testosterone and LH levels are not significantly different from WT controls. However, FSH levels and testes weights remain decreased. Female YHR(+) mice undergo precocious puberty with early vaginal opening, accelerated uterine development, enhanced follicular development, including the presence of corpora lutea, and an increase in serum progesterone. At 12 weeks of age, the ovary exhibits a relative increase in the amount of interstitial tissue, comprised of cells that are hypertrophic and luteinized, as well as follicles that are degenerating. Additionally, hemorrhagic cysts develop in approximately 25% of the transgenic mice. These degenerative changes are consistent with an aging ovary suggesting that CG-induced LHR activation in female mice leads to precocious sexual development and ovarian lesions. Taken together, these data indicate that the single chain YHR is functional in vivo and demonstrate that YHR(+) mice provide a novel system to further understand the reproductive consequences of aberrant LHR activation.

Structure-function relationships of the luteinizing hormone receptor.

Of the 800-900 genes in the human genome that appear to encode G-protein-coupled receptors (GPCRs), two are known to encode receptors that bind the three heterodimeric human gonadotropins, luteinizing hormone (LH), chorionic gonadotropin (CG), and follicle-stimulating hormone (FSH). LH and CG bind to a common receptor, LHR, and FSH binds to a paralogous receptor. These GPCRs contain a relatively large ectodomain (ECD), responsible for high-affinity ligand binding, and a transmembrane portion, as in the other GPCRs. The ECD contains nine leucine-rich repeats capped by N-terminal and C-terminal cysteine-rich regions. The overall goal of this research is to elucidate the molecular mechanisms by which CG and LH bind to and activate LHR and the latter, in turn, activates Gs alpha. A combination of molecular modeling and site-directed mutagenesis, coupled with binding and signaling studies in transiently transfected HEK 293 cells expressing wild-type and mutant forms of LHR, has been used to develop and test models for the LHR ECD, the CG-LHR ECD complex, and the structural changes in the transmembrane helices and intracellular loops, particularly loop 2, that accompany receptor activation. In addition, a single-chain CG-LHR complex was designed in which a fusion protein of the two subunits of human CG was linked to full-length LHR. This ligand-receptor complex was shown to be constitutively active in cellular models and in transgenic mice, the latter of which exhibit precocious puberty. From a combination of molecular modeling, site-directed mutagenesis, genetic/protein engineering, and receptor characterization in cellular and animal models, considerable insight is being developed on the mechanisms of normal and aberrant activation of LHR.

Yoked complexes of human choriogonadotropin and the lutropin receptor: evidence that monomeric individual subunits are inactive.

Human choriogonadotropin (hCG) contains an alpha-subunit, common to other members of the glycoprotein hormone family, and a unique beta-subunit that determines hormone specificity. It is generally thought that heterodimer formation is obligatory for full hormonal activity, although other studies have indicated that individual subunits and homodimeric hCGbeta were capable of low affinity binding to the LH receptor (LHR) and subsequent activation. Previously, we constructed two yoked hormone (hCG)-LHR complexes, where the two hormone subunits and the heptahelical receptor were engineered to form single polypeptide chains, i.e. N-beta-alpha-LHR-C and N-alpha-beta-LHR-C. Expression of both complexes led to constitutive stimulation of cAMP production. In the present study, we investigated whether the human alpha-subunit and hCGbeta can act as functional agonists when covalently attached to or coexpressed with the LH receptor. Our initial results showed that hCGbeta, but not alpha, was able to activate LHR with an increase in intracellular cAMP in human embryonic kidney 293 cells but not in Chinese hamster ovary or COS-7 cells. Further examination of this apparent cell-specific agonist activity of hCGbeta revealed that low levels of endogenous alpha-subunit were expressed in human embryonic kidney 293 cells, thus enabling sufficient amounts of active heterodimer to form with the transfected hCGbeta to activate LHR. The studies in Chinese hamster ovary and COS-7 cells clearly demonstrate that, even under experimental conditions where hormone-receptor interactions are maximized, individual subunits of hCG can not act as functional agonists, at least in their monomeric form.

Consequences of single-chain translation on the structures of two chorionic gonadotropin yoked analogs in alpha-beta and beta-alpha configurations.

Human chorionic gonadotropin (hCG) is a placental-derived heterodimeric glycoprotein hormone, which, through the binding and activation of the LH receptor, rescues the corpus luteum and maintains pregnancy. The three-dimensional structure of hCG is known; however, the relevance of its fold to bioactivity is unclear. Although both subunits (alpha and beta) are required for activity, recent data with single-chain analogs have suggested a diminished role for the cystine knot and an intact heterodimeric interface in binding and receptor activation in vitro. Herein, we report the purification and structural characterization of two yoked (Y) hCG analogs, YhCG1 (beta-alpha) and YhCG3 (alpha-beta). The fusion proteins yielded higher IC50s and EC50s than those of hCG; the maximal hCG-mediated cAMP production, however, was the same. Circular dichroic spectroscopy revealed that the three proteins exhibit distinct far UV circular dichroic spectra, with YhCG1 containing somewhat more secondary structure than YhCG3 and hCG. Limited proteolysis with proteinase K indicated that heterodimeric hCG was much more resistant to cleavage than the single-chain analogs. YhCG1 was more susceptible to proteolysis than YhCG3, and the fragmentation patterns were different in the two proteins. Taken together, the data presented herein provide direct structural evidence for altered three-dimensional conformations in the two single-chain hCG analogs. Thus, the cognate G protein-coupled receptor can recognize and functionally respond to multiple ligand conformations.

Structural analysis of yoked chorionic gonadotropin-luteinizing hormone receptor ectodomain complexes by circular dichroic spectroscopy.

Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1-341 (encoded by exons 1-10 and a portion of 11), and two C-terminal ECD deletion fragments, 1-294 (encoded by exons 1-10) and 1-180 (encoded by exons 1-7). The fusion proteins, including yoked CG (N-beta-alpha-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating beta-strand/alpha-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8-10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD.

Thyroid Nodules and Shear Wave Elastography: A New Tool in Thyroid Cancer Detection.

This study determines the performance of virtual touch imaging quantification (VTIQ), a non-invasive shear wave elastography method for measuring thyroid nodule (TN) stiffness, in distinguishing benign from malignant TNs. This prospective study evaluates 707 TNs in 676 patients with fine-needle aspiration biopsy (FNAB). Before FNAB, both conventional B-mode ultrasound and shear wave elastography were performed. Surgical resection was recommended for FNAB results that were not clearly benign. Surgical pathology confirmed 82 malignant TNs. The receiver operating curve identified a single cut-off of 3.54 m/s as the maximum shear wave velocity (SWV) for predicting thyroid cancer (TC). The sensitivity and specificity were 79.27% and 71.52%, respectively. Positive predictive value (PPV) was 26.75% and negative predictive value (NPV) was 96.34%. Compared with B-mode US features for predicting malignancy, SWV ≥3.54 m/s has a higher sensitivity, specificity, PPV and NPV. TN stiffness measured by VTIQ-generated shear wave elastography is an independent predictor of TC.

Specificity of cognate ligand-receptor interactions: fusion proteins of human chorionic gonadotropin and the heptahelical receptors for human luteinizing hormone, thyroid-stimulating hormone, and follicle-stimulating hormone.

The family of glycoprotein hormones and their homologous heptahelical receptors represent an excellent system for comparative structure-function studies. We have engineered single chain molecules of human chorionic gonadotropin (hCG) fused to its cognate receptor, LH receptor (LHR), and to the noncognate receptors, TSH receptor (TSHR) and FSH receptor (FSHR; N-beta-alpha-receptor-C), to create the yoked (Y) complexes YCG/LHR, YCG/TSHR, and YCG/FSHR. The expression and bioactivity of these fusion proteins were examined in transiently transfected HEK 293 cells. Western blot analysis and antibody binding assays demonstrated that each of the proteins was expressed. In the case of YCG/LHR, minimal binding of exogenous hormone was observed due to the continued occupation of receptor by the fused ligand. The presence of hCG in the YCG/TSHR and YCG/FSHR, however, did not prevent binding of exogenous cognate ligand, presumably due to the lower affinity of hCG. The basal cAMP levels in cells expressing the YCG/LHR complex was approximately 20-fold higher than that in cells expressing LHR. Increases in basal cAMP production were also observed with YCG/TSHR and YCG/FSHR, e.g. 13- and 4-fold increases, respectively. Whereas the affinity and specificity of hCG for LHR are extraordinarily high, the hormone is capable of binding to and activating both TSHR and FSHR under these conditions that mimic high ligand concentrations. These findings were confirmed by adding high concentrations of hCG to cells expressing TSHR and FSHR. Although the functional interaction of hCG and TSHR has been recognized in gestational hyperthyroidism, there are no reports linking hCG to FSHR activation. This study, however, suggests that such a functional interaction is capable of occurring under conditions of high circulating levels of hCG, e.g. the first trimester of pregnancy and in patients with hCG-secreting tumors.