The Arabidopsis Leucine-Rich Repeat Receptor Kinase BIR3 Negatively Regulates BAK1 Receptor Complex Formation and Stabilizes BAK1.

BAK1 is a coreceptor and positive regulator of multiple ligand binding leucine-rich repeat receptor kinases (LRR-RKs) and is involved in brassinosteroid (BR)-dependent growth and development, innate immunity, and cell death control. The BAK1-interacting LRR-RKs BIR2 and BIR3 were previously identified by proteomics analyses of in vivo BAK1 complexes. Here, we show that BAK1-related pathways such as innate immunity and cell death control are affected by BIR3 in Arabidopsis thaliana BIR3 also has a strong negative impact on BR signaling. BIR3 directly interacts with the BR receptor BRI1 and other ligand binding receptors and negatively regulates BR signaling by competitive inhibition of BRI1. BIR3 is released from BAK1 and BRI1 after ligand exposure and directly affects the formation of BAK1 complexes with BRI1 or FLAGELLIN SENSING2. Double mutants of bak1 and bir3 show spontaneous cell death and constitutive activation of defense responses. BAK1 and its closest homolog BKK1 interact with and are stabilized by BIR3, suggesting that bak1 bir3 double mutants mimic the spontaneous cell death phenotype observed in bak1 bkk1 mutants via destabilization of BIR3 target proteins. Our results provide evidence for a negative regulatory mechanism for BAK1 receptor complexes in which BIR3 interacts with BAK1 and inhibits ligand binding receptors to prevent BAK1 receptor complex formation.

Variations in radioiodine ablation: decision-making after total thyroidectomy.

BACKGROUND: The role of radioiodine treatment following total thyroidectomy for differentiated thyroid cancer is changing. The last major revision of the American Thyroid Association (ATA) Management Guidelines for Patients with Thyroid Nodules and Differentiated Thyroid Cancer in 2015 changed treatment recommendations dramatically in comparison with the European Association of Nuclear Medicine (EANM) 2008 guidelines. We hypothesised that there is marked variability between the different treatment regimens used today. METHODS: We analysed decision-making in all Swiss hospitals offering radioiodine treatment to map current practice within the community and identify consensus and discrepancies. RESULTS AND CONCLUSION: We demonstrated that for low-risk DTC patients after thyroidectomy, some institutions offered only follow-up, while RIT with significant activities is recommended in others. For intermediate- and high-risk patients, radioiodine treatment is generally recommended. Dosing and treatment preparation (recombinant human thyroid stimulation hormone (rhTSH) vs. thyroid hormone withdrawal (THW)) vary significantly among centres.

REACH alternative testing strategy for skin sensitization in practice: Fact or fiction?

From October 2016 the REACH Regulation requires an alternative testing strategy for skin sensitization. The current paper describes our experience when putting into practice the REACH alternative testing strategy with a modification for 50 industrial chemicals in total, including mono-constituents, multi-constituents and UVCBs. For mono- and multi-constituents, a tiered approach was followed starting with an in silico (Derek Nexus) assessment, DPRA and KeratinoSens™ assay, followed by a weight of evidence conclusion based on the generated data, or further testing using the U-SENS™ assay. For UVCBs testing started with the KeratinoSens™ assay followed by the U-SENS™ assay if additional relevant information could be gained for an overall conclusion. From the 50 substances tested, for 46% a conclusion on skin sensitization potential and potency could be drawn based on the non-animal testing strategy; however, 54% of the substances still needed to be studied in vivo due to discordant results from non-animal testing or the need for reliable potency data. Important issues with the currently available, validated non-animal methods are the lack or comparability of skin metabolism and lack of potency indication, which is present in the in vivo assays. Skin sensitization testing for UVCBs and multi-constituents is still in a grey area, as neither the in chemico, in vitro assays, and in vivo LLNA have been validated for UVCBs and multi-constituents.

Axenfeld-Rieger syndrome.

Axenfeld-Rieger syndrome (ARS) is a clinically and genetically heterogeneous group of developmental disorders affecting primarily the anterior segment of the eye, often leading to secondary glaucoma. Patients with ARS may also present with systemic changes, including dental defects, mild craniofacial dysmorphism, and umbilical anomalies. ARS is inherited in an autosomal-dominant fashion; the underlying defect in 40% of patients is mutations in PITX2 or FOXC1. Here, an overview of the clinical spectrum of ARS is provided. As well, the known underlying genetic defects, clinical diagnostic possibilities, genetic counseling and treatments of ARS are discussed in detail.

Pharmacokinetics of coadministration of levothyroxine sodium and alendronate sodium new effervescent formulation.

No clinically important pharmacokinetic interference of alendronate occurred between a new effervescent formulation of alendronate and levothyroxine when coadministered. The combination does not materially affect levothyroxine absorption. INTRODUCTION: Concurrent treatment of osteoporosis with alendronate (Aln) and hypothyroidism with levothyroxine (LT4) may be problematic because both drugs are to be taken separately after fasting overnight. The primary objective was to assess pharmacokinetic interactions between a new effervescent formulation of Aln (Aln-NEF) and LT4. METHODS: A randomized, open-label, 3-way crossover study was conducted in 30 healthy adults (15 women). Subjects were dosed 3 times, separated by 35 days, after overnight fasts, with Aln-NEF alone (70 mg), LT4 alone (600 µg), or Aln-NEF and LT4 concurrently. Samples were analyzed for plasma Aln and serum LT4. Pharmacokinetic drug-drug interaction was assessed using 90% confidence intervals (CIs) for the test/reference ratio of the geometric means for area under the concentration-time curve from time zero to last measureable time point (AUC0-t ) and maximum concentration (C max). Results were compared to the default no-effect boundaries of 80 to 125% for the ratio Aln-NEF and LT4 concurrently/Aln-NEF alone and the ratio Aln-NEF and LT4 concurrently/LT4 alone. RESULTS: Geometric mean ratios (Aln-NEF with LT4/Aln-NEF alone) were 0.927 (90% CI 0.795-1.081) for AUC0-8 and 0.912 (90% CI 0.773-1.077) for C max, demonstrating LT4 does not appreciably affect the pharmacokinetics of Aln. Geometric mean ratios (LT4 with Aln-NEF/LT4 alone) were 1.049 (90% CI 0.983-1.119) for AUC0-48 and 1.075 (90% CI 1.006-1.148) for C max, demonstrating LT4 is bioequivalent between the 2 treatments. Coadministration of Aln-NEF and LT4 was well tolerated. CONCLUSIONS: There was no clinically important pharmacokinetic interference between the Aln-NEF formulation and LT4. Aln-NEF does not materially affect LT4 absorption.

Somatostatin-based radiopeptide therapy with [177Lu-DOTA]-TOC versus [90Y-DOTA]-TOC in neuroendocrine tumours.

PURPOSE: Somatostatin-based radiopeptide treatment is generally performed using the ß-emitting radionuclides (90)Y or (177)Lu. The present study aimed at comparing benefits and harms of both therapeutic approaches. METHODS: In a comparative cohort study, patients with advanced neuroendocrine tumours underwent repeated cycles of [(90)Y-DOTA]-TOC or [(177)Lu-DOTA]-TOC until progression of disease or permanent adverse events. Multivariable Cox regression and competing risks regression were employed to examine predictors of survival and adverse events for both treatment groups. RESULTS: Overall, 910 patients underwent 1,804 cycles of [(90)Y-DOTA]-TOC and 141 patients underwent 259 cycles of [(177)Lu-DOTA]-TOC. The median survival after [(177)Lu-DOTA]-TOC and after [(90)Y-DOTA]-TOC was comparable (45.5 months versus 35.9 months, hazard ratio 0.91, 95% confidence interval 0.63-1.30, p = 0.49). Subgroup analyses revealed a significantly longer survival for [(177)Lu-DOTA]-TOC over [(90)Y-DOTA]-TOC in patients with low tumour uptake, solitary lesions and extra-hepatic lesions. The rate of severe transient haematotoxicities was lower after [(177)Lu-DOTA]-TOC treatment (1.4 vs 10.1%, p = 0.001), while the rate of severe permanent renal toxicities was similar in both treatment groups (9.2 vs 7.8%, p = 0.32). CONCLUSION: The present results revealed no difference in median overall survival after [(177)Lu-DOTA]-TOC and [(90)Y-DOTA]-TOC. Furthermore, [(177)Lu-DOTA]-TOC was less haematotoxic than [(90)Y-DOTA]-TOC.

Robust array-based coregulator binding assay predicting ERα-agonist potency and generating binding profiles reflecting ligand structure.

Testing chemicals for their endocrine-disrupting potential, including interference with estrogen receptor (ER) signaling, is an important aspect of chemical safety testing. Because of the practical drawbacks of animal testing, the development of in vitro alternatives for the uterotrophic assay and other in vivo (anti)estrogenicity tests has high priority. It was previously demonstrated that an in vitro assay that profiles ligand-induced binding of ERα to a microarray of coregulator-derived peptides might be a valuable candidate for a panel of in vitro assays aiming at an ultimate replacement of the uterotrophic assay. In the present study, the reproducibility and robustness of this coregulator binding assay was determined by measuring the binding profiles of 14 model compounds that are recommended by the Office of Prevention, Pesticides and Toxic Substances for testing laboratory proficiency in estrogen receptor transactivation assays. With a median coefficient of variation of 5.0% and excellent correlation (R(2) = 0.993) between duplicate measurements, the reproducibility of the ERα-coregulator binding assay was better than the reproducibility of other commonly used in vitro ER functional assays. In addition, the coregulator binding assay is correctly predicting the estrogenicity for 13 out of 14 compounds tested. When the potency of the ER-agonists to induce ERα-coregulator binding was compared to their ER binding affinity, their ranking was similar, and the correlation between the EC50 values was excellent (R(2) = 0.96), as was the correlation with their potency in a transactivation assay (R(2) = 0.94). Moreover, when the ERα-coregulator binding profiles were hierarchically clustered using Euclidian cluster distance, the structurally related compounds were found to cluster together, whereas the steroid test compounds having an aromatic A-ring were separated from those with a cyclohexene A-ring. We concluded that this assay is capable of distinguishing ERα agonists and antagonists and that it even reflects the structural similarity of ERα agonists, indicating a potential to achieve identification and classification of ERα endocrine disruptors with high fidelity.

A method for constructing radiation hybrid maps of whole genomes.

In radiation hybrid mapping, chromosomes in human-rodent hybrid cells are fragmented by X-rays and fragments rescued by fusion of the donor cell to a recipient rodent cell. The co-retention frequencies of markers in 100-200 hybrids are used to map individual chromosomes, but mapping the whole genome in this way is impractical. We have reverted to the original protocols of Goss and Harris and have produced a panel of 44 hybrids using irradiated human fibroblasts as donors. This panel has been used to make a map of human chromosome 14 containing 40 ordered markers. The map integrates previously published maps and localizes nine new markers. We suggest that the construction of a high resolution map of the whole human genome is feasible with a single panel of 100-200 hybrids.

Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells.

In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these cells of a functionally active p53 protein, a functionally competent DNA-repair system, active enzymes for phase-I and -II metabolism, and an active Nrf2 electrophile responsive system. These properties may result in an assay with a high predictivity for in vivo genotoxicity. The assays with CHO-k1 and HepG2 cells were both evaluated by testing a set of compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM), among which are in vivo genotoxins and non-genotoxins. The CHO-k1 cell line showed a high sensitivity (percentage of genotoxic compounds that gave a positive result: 80%; 16/20) and specificity (percentage of non-genotoxic compounds that came out negative: 88%; 37/42). Although the sensitivity of the HepG2 cell line was lower (60%; 12/20), the specificity was high (88%; 37/42). These results were confirmed by testing an additional series of 16 genotoxic compounds. For both the CHO-k1 and the HepG2 cell line it was possible to size-classify micronuclei, enabling distinguishing aneugens from clastogens. It is concluded that two high-throughput micronucleus assays were developed that can detect genotoxic potential and allow differentiation between clastogens and aneugens. The performance scores of the CHO-k1 and HepG2 cell lines for in vivo genotoxicity were high. Application of these assays in the early discovery phase of drug development may prove to be a useful strategy to assess genotoxic potential at an early stage.

A deletion map of the human immunoglobulin heavy chain variable region.

Analysis of VH gene segments deleted in the process of immunoglobulin heavy chain (IGH) variable region assembly in three series of monoclonal B cell lines has been used to determine the human VH region organization. A deletion map of the relative positions of 21 different VH gene segments has been determined. The characterization of B cell lines from three unrelated adults of two racial groups yielded the same relative VH gene segment order, suggesting that the overall order of VH genes in the normal population is constant. This VH gene segment order was consistent with what we had previously generated from physical mapping techniques. DH segments from the second DH cluster, distinct from the major DH locus 3' of the VH region, were not observed to be used in 32 different rearrangements. Approximately 77% of the VH-(D)JH rearrangements involved VH gene segments within 500 kb of the JH region, indicating that human B cell lines preferentially rearrange JH-proximal VH gene segments. The switch, observed in mice, from the fetal use of JH-proximal VH gene segments to an adult VH use dependent upon VH family size may therefore not occur in humans. This detailed map of the VH gene segments is a necessary prerequisite for understanding VH usage in development and disease.

The development of RAD51C, Cystatin A, p53 and Nrf2 luciferase-reporter assays in metabolically competent HepG2 cells for the assessment of mechanism-based genotoxicity and of oxidative stress in the early research phase of drug development.

Four different mechanism-based high-throughput luciferase-reporter assays were developed in human HepG2 cells, which contain phase I and II metabolic activity and a functionally active p53 protein. The promoter regions of RAD51C and Cystatin A, as well as the responsive element of the p53 protein, were selected for the generation of the genotoxicity reporter assays. Moreover, a luciferase-based reporter assay was generated that measures the activation of the Nrf2 oxidative stress pathway. Validation with respect to the ECVAM compound list [D. Kirkland, P. Kasper, L. Muller, R. Corvi, G. Speit, Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop, Mutat. Res. 653 (2008) 99-108] resulted in an overall sensitivity of the HepG2 genotoxicity reporter assays for genotoxicity of 85% (17/20). The specificity and predictivity were high with 81% (34/42) and 82% (51/62), respectively. Various compounds had a positive score although metabolic activation was needed. The HepG2 reporter data were also compared with the available data on bacterial mutagenicity (Ames test), in vitro clastogenicity and in vivo clastogenicity for an additional set of 192 compounds. The predictivity for mutagenicity results was 74% (sensitivity, 61%, 30/49; specificity, 80%, 77/96) and for in vitro clastogenicity 59% (sensitivity, 45%, 35/78; specificity 83%, 38/46). The correlation between results from the HepG2 genotoxicity reporter assays and in vivo clastogenicity was much higher with 77% (sensitivity, 74%, 28/38; specificity 81%, 26/32). Results from the Nrf2 reporter assay showed that a large number of genotoxic compounds activated the Nrf2 oxidative stress pathway. In conclusion, four high-throughput mechanism-based reporter assays in the HepG2 cell line were developed, which can be applied for screening in the early research phase of drug development. The use of these assays in combination with the previously validated Vitotox and RadarScreen assays will certainly reduce the attrition rate due to genotoxicity in the developmental phase of drug development.

The kinetic direct peptide reactivity assay (kDPRA): Intra- and inter-laboratory reproducibility in a seven-laboratory ring trial

While the skin sensitization hazard of substances can be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide are evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times. The reaction is stopped by addition of monobromobimane, which forms a fluorescent complex with the free cysteine of the model peptide. The relative remaining non-depleted amount of peptide is determined. Kinetic rate constants are derived from the depletion vs concentration and time matrix and used to distinguish between UN GHS sub-category 1A sensitizers and test substances in sub-category 1B/not classified test substances. In this study, we present a ring trial of the kDPRA with 24 blind-coded test substances in seven laboratories. The intra- and inter-laboratory reproducibility were 96% and 88%, respectively (both for differentiating GHS Cat 1A sensitizers from GHS Cat 1B/not classified). Following an independent peer review, the kDPRA was considered to be acceptable for the identification of GHS Cat 1A skin sensitizers. Besides GHS Cat 1A identification, the kDPRA can be used as part of a defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment. For this aim, next to reproducibility of classification, the quantitative reproducibility and variability of the rate constants were quantified in this study.

High-throughput screening for analysis of in vitro toxicity.

The influence of combinatorial chemistry and high-throughput screening (HTS) technologies in the pharmaceutical industry during the last 10 years has been enormous. However, the attrition rate of drugs in the clinic due to toxicity during this period still remained 40-50%. The need for reduced toxicity failure led to the development of early toxicity screening assays. This chapter describes the state of the art for assays in the area of genotoxicity, cytotoxicity, carcinogenicity, induction of specific enzymes from phase I and II metabolism, competition assays for enzymes of phase I and II metabolism, embryotoxicity as well as endocrine disruption and reprotoxicity. With respect to genotoxicity, the full Ames, Ames II, Vitotox, GreenScreen GC, RadarScreen, and non-genotoxic carcinogenicity assays are discussed. For cytotoxicity, cellular proliferation, calcein uptake, oxygen consumption, mitochondrial activity, radical formation, glutathione depletion as well as apoptosis are described. For high-content screening (HCS), the possibilities for analysis of cytotoxicity, micronuclei, centrosome formation and phospholipidosis are examined. For embryotoxicity, endocrine disruption and reprotoxicity alternative assays are reviewed for fast track analysis by means of nuclear receptors and membrane receptors. Moreover, solutions for analyzing enzyme induction by activation of nuclear receptors, like AhR, CAR, PXR, PPAR, FXR, LXR, TR and RAR are given.

Different strategies to overcome the effect of carbimazole on high- and low-dose radioiodine therapy: results from continuous dose-effect models.

BACKGROUND: Until now, it remains elusive which strategy - antithyroid drug withdrawal or increased radioiodine target doses - should be preferred to avoid the detrimental effect of antithyroid drugs in high- and low-dose radioiodine therapy, respectively. METHODS: We explored the effects of carbimazole on the 1-year post-radioiodine success and hypothyroidism rates by continuous dose-effect models, whereas success was defined as elimination of hyperthyroidism. Euthyroidism rates with and without carbimazole were calculated by numerical integration of the area between success and hypothyroidism curves. Target dose amplification factors for equal chance of success with and without carbimazole were calculated using logistic regression. RESULTS: Two hundred and twenty-eight patients were included in this study. Radioiodine target doses between 33 and 839 Gy were applied. Overall, the euthyroidism rates were 16.5% and 64.8%, while the hypothyroidism rates were 37.6% and 14.8% in Graves' disease and toxic nodular goitre, respectively. The success rate with simultaneous carbimazole (median dose 15 mg day(-1); range 2.5-60 mg day(-1)) was reduced over the entire target dose range in Graves' disease and toxic nodular goitre. The areas between curves for euthyroidism without and with simultaneous carbimazole were 127 and 43 Gy in Graves' disease and 178 and 128 Gy in toxic nodular goitre. The estimated radioiodine target dose amplification factor was 5.5 for Graves' disease and 3.0 for toxic nodular goitre. CONCLUSIONS: Simultaneous carbimazole reduces the euthyroidism rate, the aim of low-dose radioiodine therapy, over the entire target dose range in both Graves' disease and toxic nodular goitre. Therefore, antithyroid drug discontinuation should be preferred in low-dose radioiodine therapy. Conversely, escalation of the target dose should be preferred in high-dose radioiodine therapy.

Evaluation of the Vitotox and RadarScreen assays for the rapid assessment of genotoxicity in the early research phase of drug development.

The Vitotox and RadarScreen assays were evaluated as early screens for mutagenicity and clastogenicity, respectively. The Vitotox assay is a bacterial reporter assay in Salmonella typhimurium based on the SOS-response, and it contains a luciferase gene under control of the recN promoter. The RadarScreen assay is a RAD54 promoter-linked beta-galactosidase reporter assay in yeast. The expression of this beta-galactosidase can easily be quantified by use of the substrate d-luciferin-o-beta-galactopyranoside, which is converted into galactose and luciferin that can be measured luminometrically. Recently, an ECVAM workgroup defined a list of 20 genotoxic and 42 non-genotoxic compounds [D. Kirkland, P. Kasper, L. Muller, R. Corvi, G. Speit, Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop, Mutat. Res. 653 (2008) 99-108.] that can be used for the validation and/or optimization of in vitro genotoxicity assays. In the present study, this compound set was used for the validation of the assays. Moreover, an additional set of 192 compounds was used to broaden this validation study. The compounds of this additional set can be classified as non-genotoxins and genotoxins and consists of both in-house and reference compounds. In case of the ECVAM compound list, the results from the Vitotox and RadarScreen assays were compared to the genotoxic/non-genotoxic classification of the compounds in this list. In case of the additionally tested compounds, the results of the Vitotox and RadarScreen assays were compared, respectively, with bacterial mutagenicity (Ames) results or in vitro clastogenicity data obtained in-house or from the literature. The validation with respect to the ECVAM compound list resulted in a sensitivity for both the Vitotox and RadarScreen assay of 70% (14/20). If both assays were combined the sensitivity increased to 85% (17/20). Both tests also gave a low number of false positive results. The specificity of the Vitotox and RadarScreen assays was 93% (39/42) and 83% (35/42), respectively. This resulted in a predictivity of the Vitotox and RadarScreen assay of 85% (53/62) and 79% (49/62), respectively. In case both tests were combined the specificity and the predictivity of the Vitotox and RadarScreen assay turned out to be 81% (34/42) and 82% (51/62), respectively. The results from the additional list of 192 compounds confirmed the results found with the ECVAM compound list. The results from the Vitotox assay showed a high correlation with Ames test of 91% (132/145). Subsequently, the RadarScreen assay had a correlation with in vitro clastogenicity of 76% (93/123). The specificity of the Vitotox assay was 94% (90/96) for Ames test results and that of the RadarScreen assay was 74% (34/46) for clastogenicity. Moreover, the sensitivities of the Vitotox and RadarScreen assays were 86% (42/49) and 77% (59/77), respectively. Implementation of the Vitotox and RadarScreen assays in the early research phase of drug development can lead to fast de-selection for genotoxicity. It is expected that this application will reduce the number of compounds that have a positive score in the regulatory Ames and clastogenicity tests. Moreover, problems with a complete compound class can be foreseen at an early time point in the research phase, which gives more time for issue resolution than late detection of these problems with the regulatory tests.

Pharmacologic profiling of human and rat cytochrome P450 1A1 and 1A2 induction and competition.

Strong activation of the AhR can lead to various toxic effects such as (non-genotoxic) carcinogenicity. Moreover, drug-drug interactions by non- or competitive inhibition of CYP1A1 and 1A2 may cause adverse side effects. Normally the majority of toxicity studies are performed in rats, while for the prediction of human toxicity human AhR activation and CYP1A competition should be studied. The present study focused on the deselection of strong AhR activators and/or CYP1A inducers and (non-)competitive inhibitors in the early phase of drug development, as well as on species differences between humans and rats. Induction studies were performed in the human HepG2 and rat H4IIE cell lines. A set of 119 compounds, including known AhR ligands were tested. CYP1A induction was observed for 24 compounds. In H4IIE cells, more compounds showed induction and most EC50 values were below those of HepG2 cells. Species specific CYP1A induction in H4IIE and HepG2 cells was obtained for eight and three compounds, respectively. The same compounds except four in-house NCEs were used to study differences between CYP1A1 and 1A2 competition in human and rat supersomes. Of the 115 compounds 46 showed CYP1A1 competition. Competition was human and rat specific for 12 and 10 compounds, respectively. CYP1A2 competition was observed for 37 compounds of which 14 and 3 compounds showed human and rat specific inhibition, respectively. In conclusion, for several compounds species differences between CYP1A induction and competition in human and rat were found. Therefore, parallel screening in both species might be a very useful strategy.

[Somatostatin receptor PET/CT (SSTR-PET/CT)]. / Somatostatinrezeptor-PET/CT.

The present guideline is focused on quality assurance of somatostatin receptor PET/CT (SSTR-PET/CT) in oncology patients. The document has been developed by a multidisciplinary board of specialists providing consensus of definitions, prerequisites, methodology, operating procedures, assessment, and standardized reporting. In particular, imaging procedures for the two most commonly used radioligands of human SSTR, i. e. 68Ga-DOTATOC and 68Ga-DOTATATE are presented. Overall, SSTR-PET/CT requires close interdisciplinary communication and cooperation of referring and executing medical disciplines, taking into account existing guidelines and recommendations of the European and German medical societies, including the European Association of Nuclear Medicine (EANM), German Society for Endocrinology (DGE), German Society for Nuclear Medicine (DGN) and German Society for Radiology (DRG).

Exploring natural genetic variation in tomato sucrose synthases on the basis of increased kinetic properties.

Sucrose synthase (SuSy) is one key enzyme directly hydrolyzing sucrose to supply substrates for plant metabolism, and is considered to be a biomarker for plant sink strength. Improvement in plant sink strength could lead to enhanced plant growth and yield. Cultivated tomatoes are known to have a narrow genetic diversity, which hampers further breeding for novel and improved traits in new cultivars. In this study, we observed limited genetic variation in SuSy1, SuSy3 and SuSy4 in 53 accessions of cultivated tomato and landraces, but identified a wealth of genetic diversity in 32 accessions of related wild species. The variation in the deduced amino acid sequences was grouped into 23, 22, and 17 distinct haplotypes for SuSy1/3/4, respectively. Strikingly, all known substrate binding sites were highly conserved, as well as most of the phosphorylation sites except in SuSy1. Two SuSy1 and three SuSy3 protein variants were heterologously expressed to study the effect of the amino acid changes on enzyme kinetic properties, i.e. maximal sucrose hydrolyzing capacity (Vmax), affinity for sucrose (Km), and catalytic efficiency (Vmax/Km) at 25°C and 16°C. SuSy1-haplotype#3 containing phosphorylation site Ser-16 did not have an improvement in the kinetic properties compared to the reference SuSy1-haplotype#1 containing Arg-16. Meanwhile SuSy3-haplotype#9 from a wild accession, containing four amino acid changes S53A, S106I, E727D and K741E, showed an increase in Vmax/Km at 16°C compared to the reference SuSy3-haplotype#1. This study demonstrates that SuSy kinetic properties can be enhanced by exploiting natural variation, and the potential of this enzyme to improve sucrose metabolism and eventually sink strength in planta.

Radiation hybrids: irradiation and fusion gene transfer.

Irradiation and fusion gene transfer can be used to construct detailed genetic maps of complex genomes. This technique is complementary to mapping methods based on both physical distance and genetic recombination.