The Fdb3 transcription factor of the Fusarium Detoxification of Benzoxazolinone gene cluster is required for MBOA but not BOA degradation in Fusarium pseudograminearum.

A number of cereals produce the benzoxazolinone class of phytoalexins. Fusarium species pathogenic towards these hosts can typically degrade these compounds via an aminophenol intermediate, and the ability to do so is encoded by a group of genes found in the Fusarium Detoxification of Benzoxazolinone (FDB) cluster. A zinc finger transcription factor encoded by one of the FDB cluster genes (FDB3) has been proposed to regulate the expression of other genes in the cluster and hence is potentially involved in benzoxazolinone degradation. Herein we show that Fdb3 is essential for the ability of Fusarium pseudograminearum to efficiently detoxify the predominant wheat benzoxazolinone, 6-methoxy-benzoxazolin-2-one (MBOA), but not benzoxazoline-2-one (BOA). Furthermore, additional genes thought to be part of the FDB gene cluster, based upon transcriptional response to benzoxazolinones, are regulated by Fdb3. However, deletion mutants for these latter genes remain capable of benzoxazolinone degradation, suggesting that they are not essential for this process.

A γ-lactamase from cereal infecting Fusarium spp. catalyses the first step in the degradation of the benzoxazolinone class of phytoalexins.

The benzoxazolinone class of phytoalexins are released by wheat, maize, rye and other agriculturally important species in the Poaceae family upon pathogen attack. Benzoxazolinones show antimicrobial effects on plant pathogens, but certain fungi have evolved mechanisms to actively detoxify these compounds which may contribute to the virulence of the pathogens. In many Fusarium spp. a cluster of genes is thought to be involved in the detoxification of benzoxazolinones. However, only one enzyme encoded in the cluster has been unequivocally assigned a role in this process. The first step in the detoxification of benzoxazolinones in Fusarium spp. involves the hydrolysis of a cyclic ester bond. This reaction is encoded by the FDB1 locus in F. verticillioides but the underlying gene is yet to be cloned. We previously proposed that FDB1 encodes a γ-lactamase, and here direct evidence for this is presented. Expression analyses in the important wheat pathogen F. pseudograminearum demonstrated that amongst the three predicted γ-lactamase genes only the one designated as FDB1, part of the proposed benzoxazolinone cluster in F. pseudograminearum, was strongly responsive to exogenous benzoxazolinone application. Analysis of independent F. pseudograminearum and F. graminearum FDB1 gene deletion mutants, as well as biochemical assays, demonstrated that the γ-lactamase enzyme, encoded by FDB1, catalyses the first step in detoxification of benzoxazolinones. Overall, our results support the notion that Fusarium pathogens that cause crown rot and head blight on wheat have adopted strategies to overcome host-derived chemical defences.

Degradation of the benzoxazolinone class of phytoalexins is important for virulence of Fusarium pseudograminearum towards wheat.

Wheat, maize, rye and certain other agriculturally important species in the Poaceae family produce the benzoxazolinone class of phytoalexins on pest and pathogen attack. Benzoxazolinones can inhibit the growth of pathogens. However, certain fungi can actively detoxify these compounds. Despite this, a clear link between the ability to detoxify benzoxazolinones and pathogen virulence has not been shown. Here, through comparative genome analysis of several Fusarium species, we have identified a conserved genomic region around the FDB2 gene encoding an N-malonyltransferase enzyme known to be involved in benzoxazolinone degradation in the maize pathogen Fusarium verticillioides. Expression analyses demonstrated that a cluster of nine genes was responsive to exogenous benzoxazolinone in the important wheat pathogen Fusarium pseudograminearum. The analysis of independent F. pseudograminearum FDB2 knockouts and complementation of the knockout with FDB2 homologues from F. graminearum and F. verticillioides confirmed that the N-malonyltransferase enzyme encoded by this gene is central to the detoxification of benzoxazolinones, and that Fdb2 contributes quantitatively to virulence towards wheat in head blight inoculation assays. This contrasts with previous observations in F. verticillioides, where no effect of FDB2 mutations on pathogen virulence towards maize was observed. Overall, our results demonstrate that the detoxification of benzoxazolinones is a strategy adopted by wheat-infecting F. pseudograminearum to overcome host-derived chemical defences.

2009 EANM parathyroid guidelines.

The present guidelines were issued by the Parathyroid Task Group of the European Association of Nuclear Medicine. The main focus was imaging of primary hyperparathyroidism. Dual-tracer and single-tracer parathyroid scintigraphy protocols were discussed as well as the various modalities of image acquisition. Primary hyperparathyroidism is an endocrine disorder with high prevalence, typically caused by a solitary parathyroid adenoma, less frequently (about 15%) by multiple parathyroid gland disease (MGD) and rarely (1%) by parathyroid carcinoma. Patients with MGD may have a double adenoma or hyperplasia of three or all four parathyroid glands. Conventional surgery has consisted in routine bilateral neck exploration. The current trend is toward minimally invasive surgery. In this new era, the success of targeted parathyroid surgery depends not only on an experienced surgeon, but also on a sensitive and accurate imaging technique. Recognizing MGD is the major challenge for pre-operative imaging, in order to not direct a patient towards inappropriate minimal surgery. Scintigraphy should also report on thyroid nodules that may cause confusion with a parathyroid adenoma or require concurrent surgical resection. The two main reasons for failed surgery are ectopic glands and undetected MGD. Imaging is mandatory before re-operation, and scintigraphy results should be confirmed with a second imaging technique (usually US for a neck focus, CT or MRI for a mediastinal focus). Hybrid SPECT/CT instruments should be most helpful in this setting. SPECT/CT has a major role for obtaining anatomical details on ectopic foci. However, its use as a routine procedure before target surgery is still investigational. Preliminary data suggest that SPECT/CT has lower sensitivity in the neck area compared to pinhole imaging. Additional radiation to the patient should also be considered. The guidelines also discuss aspects related to radio-guided surgery of hyperparathyroidism and imaging of chronic kidney disease patients with secondary hyperparathyroidism.

Parathyroid imaging: how good is it and how should it be done?

Hypersecretion of parathormone in primary hyperparathyroidism is common, occurring in approximately 1 in 500 women and 1 in 2,000 men per year in their fifth to seventh decades of life. This has been suggested from the literature to be primarily the result of a parathyroid adenoma (80-85% of cases), hyperplasia involving more than 1 gland, usually with all 4 glands being involved (10-15% of cases), or the result, albeit rarely, of parathyroid carcinoma (0.5-1% of cases). Surgical removal of the hypersecreting gland is the primary treatment; this procedure is best performed by a skilled surgeon who would normally find the abnormality in 95% of cases. Imaging, however, should be used to identify the site of abnormality, potentially reducing inpatient stay and improving the patient experience. Functional imaging of parathyroid tissue using thallium was introduced in the 1980s but has largely been superceded by the use of (99m)Tc-labeled isonitriles. The optimum techniques have used (99m)Tc-sestamibi with subtraction imaging or washout imaging. A recent systematic review reported the percentage sensitivity (95% confidence intervals) for sestamibi in the identification of solitary adenomas as 88.44 (87.48-89.40), multigland hyperplasia 44.46 (41.13-47.8), double adenomas 29.95 (-2.19 to 62.09), and carcinoma 33 (33). This review does not separate the washout and subtraction techniques. The subtraction technique using (99m)Tc-sestamibi and (123)I is the optimal technique enabling the site to be related to the thyroid tissue when the parathyroid gland is in the neck in a normal position. If there is an equivocal scan then confirmation with high resolution ultrasound should be used. With ectopic glands, the combined use of single-photon emission computed tomography may then provide anatomical information to enable localization of the functional abnormality. In patients who have had surgical exploration by an experienced parathyroid surgeon in a unit with an experienced nuclear medicine team and negative sestamibi imaging, it is reasonable to image the patient with (11)C methionine. It is debatable whether patients with a high likelihood of secondary hyperparathyroidism should be imaged. The only possible justification for this is to exclude an ectopic site. There is no substitute for an experienced surgeon and an experienced imaging unit to provide a parathyroid service.

Optimization of parathyroid imaging by simultaneous dual energy planar and single photon emission tomography.

BACKGROUND: Single photon emission tomography (SPET) gives valuable 3-dimensional information, but prolongs the time of imaging and increases the possibility of patient movement. We therefore investigated a method for the optimization of SPET. METHOD: Using an in-house fabricated thyroid/parathyroid neck phantom simultaneous dual energy (DE) 1223I/99mTc imaging to localize parathyroid glands was assessed both in planar and SPET modes. RESULTS: Experiments demonstrated improved spatial resolution and contrast for planar pinhole imaging compared to parallel collimation. For DE-SPET compared to planar pinhole imaging more glands in the phantom were visualized by the improved contrast. CONCLUSION: We conclude that DE-SPET for parathyroid imaging is feasible to aid the accurate localization of parathyroid adenomas. For planar imaging pinhole collimation is superior to parallel hole collimation.