Factors affecting composition of fatty acids in wild-growing forest mushrooms.

The importance of mushrooms as a food source is continually increasing. To investigate how environmental factors affect the nutritional value of mushrooms, we harvested them in eastern Poland, south-central Germany, and northwestern Belgium in plots with similar environmental conditions but varying in tree species composition and richness. We used gas chromatography-mass spectrometry (GC-MS) to analyze the fatty acid (FA) content of the mushrooms. Fungal species identity explained the largest part (40%) of the total variation in FA concentration and composition. Environmental factors accounted for 1-12% of variation. The concentration of FA, especially saturated fatty acids, decreased with increasing understory cover and increasing nitrogen concentration in the topsoil. The effect of tree species richness or tree species identity was negligible. Our results suggest that the nutritional value of mushrooms depends mainly on the species identity of fungi, but that their FA content is slightly higher in forests with less undergrowth and in nitrogen-poor soils.

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction.

The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.

Decision Support Tool for Water Reclamation Beyond Technical Considerations-Egyptian, Moroccan, and Tunisian Case Studies.

While the Middle East and North African (MENA) region is facing challenges to sustain water security, water reclamation has received increasing consideration as a favorable mitigating solution. Despite the availability of adequate technologies, economic, political, legal, social, and environmental constraints often hamper stakeholders, and especially decision makers, from exploiting the existing potential into solution implementation. In the present paper, a comprehensive assessment for water reclamation and reuse was developed. This assessment consisted of 4 objectives: 1) apply a decision-support tool (DST) for water reclamation potential for municipal wastewater, 2) apply a DST for simulating and estimating the lifecycle costs of project-related technologies for water reclamation (municipal and industrial wastewater, as well as drainage canal water), 3) assess the national-level conditions for water reuse with a multicriteria decision analysis (MCA), and 4) establish exemplary strategies, barriers, and measures for water reuse. The present MCA considered 6 thematic subjects: policy and institution, economy, society, water management, legislation, and environment. The assessment was applied to food and nonfood crop irrigation in Egyptian, Moroccan, and Tunisian case studies. For all defined case studies, adapted treatment trains that could treat wastewater to the desired quality at reasonable costs were identified and are presented in the present paper. Results showed that technological options are available for water reuse, but the concept is not widely implemented in Egypt, Morocco, and Tunisia. The present paper identifies key barriers and drivers for the implementation of water reclamation for irrigation. In particular, the considered countries showed different characteristics regarding efficient water management, water pricing, subsidies and wastewater tariffs, implementation of monitoring and reporting systems, or legal aspects regarding the use of reclaimed water for food crop irrigation. Further exploration of case studies on high potential water reuse and financially affordable wastewater reclamation, particularly case studies that explore the impacts of policies and practices across countries, would be useful for helping the MENA region improve their water security situation. Integr Environ Assess Manag 2020;16:885-897. © 2020 SETAC.

Sclerosing mucoepidermoid carcinoma with eosinophilia of the thyroid: Case report of a rare lesion with novel genetic mutation.

Sclerosing mucoepidermoid carcinoma with eosinophilia (SMECE) is a rare primary cancer of the thyroid. This tumor is analogous to other primary tumors of the salivary glands, breast, pancreas, and esophagus. We present a case of this rare tumor with characteristic clinical features, ultrasound images, cytopathology, histopathology, and a heretofore undocumented somatic gene mutation. Additionally, we provide a succinct review of the controversial literature for this uncommon lesion.

Characterization of a drug-targetable allosteric site regulating vascular endothelial growth factor signaling.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases (VEGFRs). The extracellular domain of VEGFRs consists of seven Ig-homology domains, of which D2-3 form the ligand-binding site, while the membrane proximal domains D4-7 are involved in homotypic interactions in ligand-bound receptor dimers. Based on low-resolution structures, we identified allosteric sites in D4-5 and D7 of vascular endothelial growth factor receptor 2 (VEGFR-2) accomplishing regulatory functions. Allosteric inhibition of VEGFR-2 signaling represents an attractive option for the treatment of neovascular diseases. We showed earlier that DARPin® binders to domains D4 or D7 are potent VEGFR-2 inhibitors. Here we investigated in detail the allosteric inhibition mechanism of the domain D4 binding inhibitor D4b. The 2.38 Å crystal structure of D4b in complex with VEGFR-2 D4-5, the first high-resolution structure of this VEGFR-2 segment, indicates steric hindrance by D4b as the mechanism of inhibition of receptor activation. At the cellular level, D4b triggered quantitative internalization of VEGFR-2 in the absence of ligand and thus clearance of VEGFR-2 from the surface of endothelial cells. The allosteric VEGFR-2 inhibition was sufficiently strong to efficiently inhibit the growth of human endothelial cells at suboptimal dose in a mouse xenograft model in vivo, underlining the therapeutic potential of the approach.

Giant cell tumor of temporomandibular joint presenting as a parotid tumor: Challenges in the accurate subclassification of giant cell tumors in an unusual location.

Fine needle aspiration is frequently used as the initial diagnostic procedure in the work-up of head and neck lesions, including soft tissue masses and salivary gland neoplasms. Giant cell tumors (GCTs), both osseous and extraosseous, are benign tumors that occur, albeit rarely, in the head and neck region. Extraosseous GCTs may be further classified based on their tissue of origin and specific anatomic location. Regardless of location, giant cell tumors are morphologically similar and share cytologic and histologic diagnostic criteria. Evaluation of imaging is therefore essential to the correct classification of these tumors. Accurate diagnosis is crucial since the clinical behavior and treatment is significantly different among the subtypes of GCTs. The case presented herein illustrates the diagnostic dilemma between two uncommon entities in an unusual site: GCT of parotid gland and tenosynovial GCT.

A framework for evaluating the accessibility of raw materials from end-of-life products and the Earth's crust.

An increasing number of geochemically scarce metallic raw materials are entering into our lives via new technologies. A reversal of this trend is not foreseeable, leading to concerns regarding the security of their supply. However, the evaluation of raw material supply is currently hampered by inconsistent use of fundamental terminologies and incomplete assessment criteria. In this paper, we aim to establish a consistent framework for evaluating raw material supply from both anthropogenic and geological sources. A method for concept extraction was applied to evaluate systematically the use of fundamental terms in the evaluation of raw material supply. The results have shown that 'availability' is commonly used in raw material supply evaluations, whilst other researchers suggest that raw material supply should be evaluated based on 'accessibility'. It was revealed that 'accessibility' actually comprises two aspects: 'availability' and 'approachability'. Raw material 'approachability' has not previously been explicitly addressed at a system level. A novel, consistent framework for evaluating raw material supply was therefore developed. To demonstrate the application of the established framework, we evaluated the raw material supply of four rare earth element case studies. Three case studies are End-of-Life products (the anthroposphere) from Switzerland: (i) phosphors in fluorescent lamps, (i) permanent magnets in the drive motors of electric cars and (iii) fibre optic cable. The fourth case study source is the Earth's crust (the geosphere): Mount Weld deposit in Australia. The framework comprises a comprehensive evaluation of six components relating to raw material mining and processing: their geological knowledge, eligibility, technology, economic, societal and environmental impacts. Our results show that metals are not considered to be fully accessible in any of the case studies due to a lack of necessary technologies and potential societal and environmental impacts. The framework presented here can serve as a starting point for the development of an evaluation framework for raw material accessibility at an early project development stage.

THADA Regulates the Organismal Balance between Energy Storage and Heat Production.

Human susceptibility to obesity is mainly genetic, yet the underlying evolutionary drivers causing variation from person to person are not clear. One theory rationalizes that populations that have adapted to warmer climates have reduced their metabolic rates, thereby increasing their propensity to store energy. We uncover here the function of a gene that supports this theory. THADA is one of the genes most strongly selected during evolution as humans settled in different climates. We report here that THADA knockout flies are obese, hyperphagic, have reduced energy production, and are sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and acts on it as an uncoupler. Reducing SERCA activity in THADA mutant flies rescues their obesity, pinpointing SERCA as a key effector of THADA function. In sum, this identifies THADA as a regulator of the balance between energy consumption and energy storage, which was selected during human evolution.

Patients with non-substance-related disorders report a similar profile of childhood trauma experiences compared to heroin-dependent patients.

BACKGROUND AND OBJECTIVES: Exposure to traumatic events is common among patients with substance use disorders (SUD). In patients with non-substance-related disorders, especially with gambling disorders (GD) and internet addiction (IA), traumatic childhood experiences have not been investigated extensively. The objective of this study was to compare trauma histories in patients with GD and IA to patients with heroin dependence. METHODS: Cross-sectional surveys including the childhood trauma questionnaire (CTQ) and clinical data among 107 participants; 59 patients with non-substance-related disorders (GD [n = 39]; IA [n = 20]) were compared to 28 patients prescribed injectable heroin for opioid dependence in heroin-assisted treatment (HAT) and to a healthy control group (HC) (n = 20). RESULTS: The findings revealed a high prevalence of trauma exposure in all three clinical groups, with 74.4% of patients with GD, 80.0% of patients with IA, and 93.0% of patients in HAT compared to 40% in HC. All three groups (GD, IA, HAT) reported significantly higher levels of "emotional neglect" compared to HC. CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: The results provide clinically relevant information suggesting that the burden of childhood traumatic experiences may be as common in patients with GD and IA as in patients with heroin dependence. These findings could pose an important starting-point for treatment. (Am J Addict 2017;26:215-220).

Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A.

Vascular endothelial growth factors (VEGFs) regulate blood and lymph vessel development upon activation of three receptor tyrosine kinases: VEGFR-1, -2, and -3. Partial structures of VEGFR/VEGF complexes based on single-particle electron microscopy, small-angle X-ray scattering, and X-ray crystallography revealed the location of VEGF binding and domain arrangement of individual receptor subdomains. Here, we describe the structure of the full-length VEGFR-1 extracellular domain in complex with VEGF-A at 4 Å resolution. We combined X-ray crystallography, single-particle electron microscopy, and molecular modeling for structure determination and validation. The structure reveals the molecular details of ligand-induced receptor dimerization, in particular of homotypic receptor interactions in immunoglobulin homology domains 4, 5, and 7. Functional analyses of ligand binding and receptor activation confirm the relevance of these homotypic contacts and identify them as potential therapeutic sites to allosterically inhibit VEGFR-1 activity.

Biotic homogenization can decrease landscape-scale forest multifunctionality.

Many experiments have shown that local biodiversity loss impairs the ability of ecosystems to maintain multiple ecosystem functions at high levels (multifunctionality). In contrast, the role of biodiversity in driving ecosystem multifunctionality at landscape scales remains unresolved. We used a comprehensive pan-European dataset, including 16 ecosystem functions measured in 209 forest plots across six European countries, and performed simulations to investigate how local plot-scale richness of tree species (α-diversity) and their turnover between plots (ß-diversity) are related to landscape-scale multifunctionality. After accounting for variation in environmental conditions, we found that relationships between α-diversity and landscape-scale multifunctionality varied from positive to negative depending on the multifunctionality metric used. In contrast, when significant, relationships between ß-diversity and landscape-scale multifunctionality were always positive, because a high spatial turnover in species composition was closely related to a high spatial turnover in functions that were supported at high levels. Our findings have major implications for forest management and indicate that biotic homogenization can have previously unrecognized and negative consequences for large-scale ecosystem multifunctionality.

A geological reconnaissance of electrical and electronic waste as a source for rare earth metals.

The mining of material resources requires knowledge about geogenic and anthropogenic deposits, in particular on the location of the deposits with the comparatively highest concentration of raw materials. In this study, we develop a framework that allows the establishment of analogies between geological and anthropogenic processes. These analogies were applied to three selected products containing rare earth elements (REE) in order to identify the most concentrated deposits in the anthropogenic cycle. The three identified anthropogenic deposits were characterised according to criteria such as "host rock", "REE mineralisation" and "age of mineralisation", i.e. regarding their "geological" setting. The results of this characterisation demonstrated that anthropogenic deposits have both a higher concentration of REE and a longer mine life than the evaluated geogenic deposit (Mount Weld, Australia). The results were further evaluated by comparison with the geological knowledge category of the United Nations Framework Classification for Fossil Energy and Mineral Reserves and Resources 2009 (UNFC-2009) to determine the confidence level in the deposit quantities. The application of our approach to the three selected cases shows a potential for recovery of REE in anthropogenic deposits; however, further exploration of both potential and limitations is required.

Two-hour algorithm for triage toward rule-out and rule-in of acute myocardial infarction using high-sensitivity cardiac troponin T.

BACKGROUND: High-sensitivity cardiac troponin (hs-cTn) may allow an earlier diagnosis of acute myocardial infarction (AMI). METHODS: We prospectively enrolled 1148 (derivation cohort) and 517 (external validation cohort) unselected patients presenting with suspected AMI to the emergency department. Final diagnosis was adjudicated by 2 independent cardiologists. Hs-cTnT was measured at presentation and after 2 hours. A diagnostic algorithm incorporating hs-cTnT values at presentation and absolute changes within the first 2 hours was derived. RESULTS: AMI was the final diagnosis in 16% of patients in the derivation and 9.1% in the validation cohort. The 2-hour algorithm developed in the derivation cohort classified 60% of patients as "rule-out," 16% as "rule-in," and 24% in the "observational-zone." Resulting sensitivity and negative predictive value (NPV) were 99.5% and 99.9%, respectively, for rule-out, and specificity and positive predictive value (PPV) were 96% and 78%, respectively, for rule-in. Applying the 2-hour triage algorithm in the external validation cohort, 78% of patients could be classified as "rule-out," 8% as "rule-in," and 14% in the "observational-zone." Resulting sensitivity and NPV were 96% and 99.5%, respectively, for rule-out, and specificity and PPV were 99% and 85%, respectively, for rule-in. Cumulative 30-day survival rates were 100%, 98.9%, and 95.2% (P < .001), and 100%, 100%, and 95% (P < .001) in patients classified as "rule-out," "observational-zone," and "rule-in" in the 2 cohorts, respectively. CONCLUSIONS: A simple algorithm incorporating hs-cTnT baseline values and absolute changes over 2 hours allowed a triage toward safe rule-out, or accurate rule-in, of AMI in the vast majority of patients, with only 20% requiring more prolonged monitoring and serial blood sampling.

A randomized, controlled trial of combined cognitive-behavioral therapy plus prize-based contingency management for cocaine dependence.

BACKGROUND: Cocaine has become one of the drugs of most concern in Switzerland, being associated with a wide range of medical, psychiatric and social problems. Available treatment options for cocaine dependence are rare. The study sought to compare combined prize-based contingency management (prizeCM) plus cognitive-behavioral therapy (CBT) to CBT alone in cocaine-dependent patients. METHODS: Sixty cocaine-dependent patients participated in a randomized, controlled trial with two treatment conditions. The participants were randomly assigned to the experimental group (EG; n = 29), who received CBT combined with prizeCM, or to the control group (CG; n = 31), who received CBT only during 24 weeks. The primary outcome measures were retention, at least 3 consecutive weeks of cocaine abstinence, the maximum number of consecutive weeks of abstinence and proportions of cocaine-free urine samples during the entire 24-week and at 6-month follow-up. RESULTS: Sixty-three percent of the participants completed the study protocol. Participants in both groups significantly reduced cocaine use over time. Overall, no difference in cocaine-free urine screens was found across the two treatment groups, except at weeks 8, 9, 10, 17 and 21 in favor of the EG. CONCLUSIONS: The addition of prizeCM to CBT seems to enhance treatment effects, especially in the early treatment period, supporting results from previous studies. Both the combined intervention and CBT alone, led to significant reductions in cocaine use during treatment and these effects were sustained at 6-month follow-up. These findings underline the importance in implementing CM and CBT interventions as treatment options for cocaine dependence in the European context.

A stem-loop structure directs oskar mRNA to microtubule minus ends.

mRNA transport coupled with translational control underlies the intracellular localization of many proteins in eukaryotic cells. This is exemplified in Drosophila, where oskar mRNA transport and translation at the posterior pole of the oocyte direct posterior patterning of the embryo. oskar localization is a multistep process. Within the oocyte, a spliced oskar localization element (SOLE) targets oskar mRNA for plus end-directed transport by kinesin-1 to the posterior pole. However, the signals mediating the initial minus end-directed, dynein-dependent transport of the mRNA from nurse cells into the oocyte have remained unknown. Here, we show that a 67-nt stem-loop in the oskar 3' UTR promotes oskar mRNA delivery to the developing oocyte and that it shares functional features with the fs(1)K10 oocyte localization signal. Thus, two independent cis-acting signals, the oocyte entry signal (OES) and the SOLE, mediate sequential dynein- and kinesin-dependent phases of oskar mRNA transport during oogenesis. The OES also promotes apical localization of injected RNAs in blastoderm stage embryos, another dynein-mediated process. Similarly, when ectopically expressed in polarized cells of the follicular epithelium or salivary glands, reporter RNAs bearing the oskar OES are apically enriched, demonstrating that this element promotes mRNA localization independently of cell type. Our work sheds new light on how oskar mRNA is trafficked during oogenesis and the RNA features that mediate minus end-directed transport.

Structural and mechanistic insights into VEGF receptor 3 ligand binding and activation.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.

Bone turnover and bone mineral density are independently related to selenium status in healthy euthyroid postmenopausal women.

CONTEXT: Selenium status may have direct effects on bone and indirect effects through changes in thyroid hormone sensitivity. OBJECTIVE: We hypothesized that variation in selenium status in healthy euthyroid postmenopausal women is associated with differences in bone turnover, bone mineral density (BMD) and fracture susceptibility. DESIGN: The Osteoporosis and Ultrasound Study (OPUS) is a 6-yr prospective study of fracture-related factors. SETTING: The study was comprised of a population-based cohort from five European cities. PARTICIPANTS: A total of 2374 postmenopausal women participated. Subjects with thyroid disease and nonthyroidal illness and those receiving drugs affecting thyroid status or bone metabolism were excluded, leaving a study population of 1144. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: We measured selenium (micrograms per liter); selenoprotein P (milligrams per liter); free T(4) (picomoles per liter); free T(3) (picomoles per liter); TSH (milliunits per liter); bone turnover markers; BMD; and vertebral, hip, and nonvertebral fractures. RESULTS: Higher selenium levels were associated with higher hip BMD at study entry (ß = 0.072, P = 0.004) and lower levels of bone formation (osteocalcin: ß = -0.101, P < 0.001; procollagen type 1 N-terminal propeptide: ß = -0.074, P = 0.013) and resorption markers (C-telopeptide of type 1 collagen: ß = -0.058, P = 0.050; N-telopeptide of type 1 collagen: ß = -0.095, P = 0.002). Higher selenoprotein P was associated with higher hip (ß = 0.113, P < 0.001) and lumbar spine BMD (ß = 0.088, P = 0.003) at study entry, higher hip BMD after the 6-yr follow-up (ß = 0.106, P = 0.001) and lower osteocalcin (ß = -0.077, P = 0.009), C-telopeptide of type 1 collagen (ß = -0.075, P = 0.012), and N-telopeptide of type 1 collagen (ß = -0.110, P < 0.001). CONCLUSION: Selenium status is inversely related to bone turnover and positively correlated with BMD in healthy euthyroid postmenopausal women independent of thyroid status.

The hinge region of the TSH receptor stabilizes ligand binding and determines different signaling profiles of human and bovine TSH.

The hinge region (HinR) is a variable structure of glycoprotein hormone receptors. Its amino acid composition and length is different for glycoprotein hormone receptors and connects the ligand binding domain with the serpentine domain. The role of the HinR of the receptors for TSH, follitropin (FSH), and LH/choriogonadotropin (LHCG) in receptor and signaling specificity is unknown. To investigate the role of the HinR for ligand binding, signal generation, and for the transmission of the signal towards the serpentine domain, we replaced the HinR of the TSH receptor (TSHR) by those of LHCG receptor and FSH receptor and introduced constitutively activating mutations and one mutation deficient for bovine (b)TSH binding in these chimeras. Functional characterization of the TSHR variants was carried out after transient transfection of COS-7 cells by determination of the cell surface expression, ligand binding, and recombinant human (rh)TSH or bTSH activation of second messengers. We show that the HinR of the TSHR stabilizes hormone binding regarding ligand affinity and retention time of the bound ligand as determined by dissociation experiments. Introduction of a constitutively activating extracellular loop mutation in these constructs lead to partially restored binding patterns. These findings indicate that the HinR-extracellular loop interface is besides signaling also important for bTSH binding. Furthermore, data for G protein signaling reveal that the activity of bTSH, but not of rhTSH, depends on the TSHR HinR, which was indicated by a significant right shift in the dose-response curves for G(s) and G(q) activation for TSHR chimeras harboring the LHCG receptor and FSH receptor HinR, respectively. Moreover, we identified different G protein signaling profiles for bTSH and rhTSH, which cannot be explained by the characterized HinR. For future studies regarding structure and function of the TSHR, it will be necessary to characterize TSHR variants with both or more ligands.

Identification of novel TSH interaction sites by systematic binding analysis of the TSHR hinge region.

In which ways the binding of the thyroid stimulating hormone to the extracellular domain of its receptor leads to activation of the thyroid-stimulating hormone receptor (TSHR) is currently only incompletely understood. It is known that TSH binding to the TSHR depends on the interaction with the leucine-rich repeat and sulfation at Y385 of the hinge region. Recently it was also shown that electrostatic interactions between positive charges of bovine (b) TSH and the residues E297, E303, and D382 of the hinge region contribute to hormone-TSHR binding. After the identification of these first TSH binding sites in the hinge region, it was apparent that multiple positions in this region remained to be characterized for their roles in hormone binding. The goal of this study was therefore to clarify whether further contact points of TSH exist in the structurally undefined hinge region. Therefore, we systematically analyzed 41 uncharacterized residues of the TSHR hinge region as single mutants regarding differences between cell surface expression and bTSH binding. Indeed, we identified further amino acids of the hinge region with influence on bTSH binding. Some of these contribute to a new binding domain from human TSHR position F381 to D386. These hinge mutants with influence on bTSH binding were also analyzed for binding of the superagonistic human TSH analog TR1401 demonstrating that these positions also have an impact on TR1401 binding. Moreover, side chain variations revealed that different amino acid properties like the negative charge, aromatic as well as hydrophilic characteristics, contribute to maintain the hormone-TSHR hinge interaction.