The Monothiol Glutaredoxin Grx4 Influences Iron Homeostasis and Virulence in Ustilago maydis.

The corn smut fungus, Ustilago maydis, is an excellent model for studying biotrophic plant-pathogen interactions, including nutritional adaptation to the host environment. Iron acquisition during host colonization is a key aspect of microbial pathogenesis yet less is known about this process for fungal pathogens of plants. Monothiol glutaredoxins are central regulators of key cellular functions in fungi, including iron homeostasis, cell wall integrity, and redox status via interactions with transcription factors, iron-sulfur clusters, and glutathione. In this study, the roles of the monothiol glutaredoxin Grx4 in the biology of U. maydis were investigated by constructing strains expressing a conditional allele of grx4 under the control of the arabinose-inducible, glucose-repressible promoter Pcrg1. The use of conditional expression was necessary because Grx4 appeared to be essential for U. maydis. Transcriptome and genetic analyses with strains depleted in Grx4 revealed that the protein participates in the regulation of iron acquisition functions and is necessary for the ability of U. maydis to cause disease on maize seedlings. Taken together, this study supports the growing appreciation of monothiol glutaredoxins as key regulators of virulence-related phenotypes in pathogenic fungi.

Organic acids and glucose prime late-stage fungal biotrophy in maize.

Many plant-associated fungi are obligate biotrophs that depend on living hosts to proliferate. However, little is known about the molecular basis of the biotrophic lifestyle, despite the impact of fungi on the environment and food security. In this work, we show that combinations of organic acids and glucose trigger phenotypes that are associated with the late stage of biotrophy for the maize pathogen Ustilago maydis. These phenotypes include the expression of a set of effectors normally observed only during biotrophic development, as well as the formation of melanin associated with sporulation in plant tumors. U. maydis and other hemibiotrophic fungi also respond to a combination of carbon sources with enhanced proliferation. Thus, the response to combinations of nutrients from the host may be a conserved feature of fungal biotrophy.

The phosphate language of fungi.

Phosphate is an essential macronutrient for fungal proliferation as well as a key mediator of antagonistic, beneficial, and pathogenic interactions between fungi and other organisms. In this review, we summarize recent insights into the integration of phosphate metabolism with mechanisms of fungal adaptation that support growth and survival. In particular, we highlight aspects of phosphate sensing important for responses to stress and regulation of cell-surface changes with an impact on fungal pathogenesis, host immune responses, and disease outcomes. Additionally, new studies provide insights into the influence of phosphate availability on cooperative or antagonistic interactions between fungi and other microbes, the associations of mycorrhizal and endophytic fungi with plants, and connections with plant immunity. Overall, phosphate homeostasis is emerging as an integral part of fungal metabolism and communication to support diverse lifestyles.

Chloroplasts and Plant Immunity: Where Are the Fungal Effectors?

Chloroplasts play a central role in plant immunity through the synthesis of secondary metabolites and defense compounds, as well as phytohormones, such as jasmonic acid and salicylic acid. Additionally, chloroplast metabolism results in the production of reactive oxygen species and nitric oxide as defense molecules. The impact of viral and bacterial infections on plastids and chloroplasts has been well documented. In particular, bacterial pathogens are known to introduce effectors specifically into chloroplasts, and many viral proteins interact with chloroplast proteins to influence viral replication and movement, and plant defense. By contrast, clear examples are just now emerging for chloroplast-targeted effectors from fungal and oomycete pathogens. In this review, we first present a brief overview of chloroplast contributions to plant defense and then discuss examples of connections between fungal interactions with plants and chloroplast function. We then briefly consider well-characterized bacterial effectors that target chloroplasts as a prelude to discussing the evidence for fungal effectors that impact chloroplast activities.

Clinical relevance of metal artefact reduction in computed tomography (iMAR) in the pelvic and head and neck region: Multi-institutional contouring study of gross tumour volumes and organs at risk on clinical cases.

INTRODUCTION: Artefacts caused by dental implants and hip replacements may impede target volume definition and dose calculation accuracy. The iterative metal artefact reduction (iMAR) algorithm can provide a solution for this problem. The present study compares delineation of gross tumour volumes (GTVs) and organs at risk (OARs) in the pelvic and the head and neck (H & N) regions using computed tomography (CT) with and without iMAR, and thus the practical applicability of iMAR for routine clinical use. METHODS: The native planning CT and CT-iMAR data of two typical clinical cases with image-distorting artefacts were used for multi-institutional contouring and analysis using the Dice similarity coefficient (DSC). GTV/OAR contours were compared with an intraobserver approach and compared to predefined reference structures. RESULTS: Mean volume for GTVprostate in the intraobserver approach decreased from 87 ± 44 cm3 (native CT) to 75 ± 22 cm3 (CT-iMAR) (P = 0.168). Compared to the reference, DSC values for GTVP rostate increased from 0.68 ± 0.15 to 0.78 ± 0.07 (CT vs. iMAR) (P < 0.05). In the H & N region, the reference for GTVT ongue (34 cm3 ) was underestimated on both data sets. No significant improvement in DSC values (0.83 ± 0.06 (native CT) versus 0.86 ± 0.06 (CT-iMAR)) was observed. CONCLUSION: The use of iMAR improves the anatomical delineation at the transition of prostate and bladder in cases of bilateral hip replacement. In the H & N region, anatomical residual structures and experience were apparently sufficient for precise contouring.

Transcripts and tumors: regulatory and metabolic programming during biotrophic phytopathogenesis.

Biotrophic fungal pathogens of plants must sense and adapt to the host environment to complete their life cycles. Recent transcriptome studies of the infection of maize by the biotrophic pathogen Ustilago maydis are providing molecular insights into an ordered program of changes in gene expression and the deployment of effectors as well as key features of nutrient acquisition. In particular, the transcriptome data provide a deeper appreciation of the complexity of the transcription factor network that controls the biotrophic program of invasion, proliferation, and sporulation. Additionally, transcriptome analysis during tumor formation, a key late stage in the life cycle, revealed features of the remodeling of host and pathogen metabolism that may support the formation of tremendous numbers of spores. Transcriptome studies are also appearing for other smut species during interactions with their hosts, thereby providing opportunities for comparative approaches to understand biotrophic adaptation.

Acetate provokes mitochondrial stress and cell death in Ustilago maydis.

The fungal pathogen Ustilago maydis causes disease on maize by mating to establish an infectious filamentous cell type that invades the host and induces tumours. We previously found that ß-oxidation mutants were defective in virulence and did not grow on acetate. Here, we demonstrate that acetate inhibits filamentation during mating and in response to oleic acid. We therefore examined the influence of different carbon sources by comparing the transcriptomes of cells grown on acetate, oleic acid or glucose, with expression changes for the fungus during tumour formation in planta. Guided by the transcriptional profiling, we found that acetate negatively influenced resistance to stress, promoted the formation of reactive oxygen species, triggered cell death in stationary phase and impaired virulence on maize. We also found that acetate induced mitochondrial stress by interfering with mitochondrial functions. Notably, the disruption of oxygen perception or inhibition of the electron transport chain also influenced filamentation and mating. Finally, we made use of the connections between acetate and ß-oxidation to test metabolic inhibitors for an influence on growth and virulence. These experiments identified diclofenac as a potential inhibitor of virulence. Overall, these findings support the possibility of targeting mitochondrial metabolic functions to control fungal pathogens.

Impurities in Oligonucleotide Drug Substances and Drug Products.

This white paper, which is the 10th in a series intended to address issues associated with the development of therapeutic oligonucleotides, examines the subject of product-related impurities. The authors consider chemistry and safety aspects and advance arguments in favor of platform approaches to impurity identification and qualification. Reporting, identification, and qualification thresholds suitable for product-related impurities of therapeutic oligonucleotides are proposed.

Planning benchmark study for SBRT of early stage NSCLC : Results of the DEGRO Working Group Stereotactic Radiotherapy.

PURPOSE: The aim was to evaluate stereotactic body radiation therapy (SBRT) treatment planning variability for early stage nonsmall cell lung cancer (NSCLC) with respect to the published guidelines of the Stereotactic Radiotherapy Working Group of the German Society for Radiation Oncology (DEGRO). MATERIALS AND METHODS: Planning computed tomography (CT) scan and the structure sets (planning target volume, PTV; organs at risk, OARs) of 3 patients with early stage NSCLC were sent to 22 radiotherapy departments with SBRT experience: each department was asked to prepare a treatment plan according to the DEGRO guidelines. The prescription dose was 3 fractions of 15 Gy to the 65% isodose. RESULTS: In all, 87 plans were generated: 36 used intensity-modulated arc therapy (IMAT), 21 used three-dimensional conformal radiation therapy (3DCRT), 6 used static field intensity-modulated radiation therapy (SF-IMRT), 9 used helical radiotherapy and 15 used robotic radiosurgery. PTV dose coverage and simultaneously kept OARs doses were within the clinical limits published in the DEGRO guidelines. However, mean PTV dose (mean 58.0 Gy, range 52.8-66.4 Gy) and dose conformity indices (mean 0.75, range 0.60-1.00) varied between institutions and techniques (p ≤ 0.02). OARs doses varied substantially between institutions, but appeared to be technique independent (p = 0.21). CONCLUSION: All studied treatment techniques are well suited for SBRT of early stage NSCLC according to the DEGRO guidelines. Homogenization of SBRT practice in Germany is possible through the guidelines; however, detailed treatment plan characteristics varied between techniques and institutions and further homogenization is warranted in future studies and recommendations. Optimized treatment planning should always follow the ALARA (as low as reasonably achievable) principle.

Phosphorus-rich structures and capsular architecture in Cryptococcus neoformans.

AIM: In this study, we aimed to analyze the relationship of phosphorus-rich structures with surface architecture in Cryptococcus neoformans. METHODS: Phosphorus-rich structures in C. neoformans were analyzed by combining fluorescence microscopy, biochemical extraction, scanning electron microscopy, electron probe x-ray microanalysis and 3D reconstruction of high pressure frozen and freeze substituted cells by focused ion beam-scanning electron microscopy (FIB-SEM). RESULTS & CONCLUSION: Intracellular and surface phosphorus-enriched structures were identified. These molecules were required for capsule assembly, as demonstrated in experiments using polysaccharide incorporation by capsule-deficient cells and mutants with defects in polyphosphate synthesis. The demonstration of intracellular and cell wall-associated polyphosphates in C. neoformans may lead to future studies involving their participation in both physiologic and pathogenic events.

A P4-ATPase subunit of the Cdc50 family plays a role in iron acquisition and virulence in Cryptococcus neoformans.

The pathogenic fungus Cryptococcus neoformans delivers virulence factors such as capsule polysaccharide to the cell surface to cause disease in vertebrate hosts. In this study, we screened for mutants sensitive to the secretion inhibitor brefeldin A to identify secretory pathway components that contribute to virulence. We identified an ortholog of the cell division control protein 50 (Cdc50) family of the noncatalytic subunit of type IV P-type ATPases (flippases) that establish phospholipid asymmetry in membranes and function in vesicle-mediated trafficking. We found that a cdc50 mutant in C. neoformans was defective for survival in macrophages, attenuated for virulence in mice and impaired in iron acquisition. The mutant also showed increased sensitivity to drugs associated with phospholipid metabolism (cinnamycin and miltefosine), the antifungal drug fluconazole and curcumin, an iron chelator that accumulates in the endoplasmic reticulum. Cdc50 is expected to function with catalytic subunits of flippases, and we previously documented the involvement of the flippase aminophospholipid translocases (Apt1) in virulence factor delivery. A comparison of phenotypes with mutants defective in genes encoding candidate flippases (designated APT1, APT2, APT3, and APT4) revealed similarities primarily between cdc50 and apt1 suggesting a potential functional interaction. Overall, these results highlight the importance of membrane composition and homeostasis for the ability of C. neoformans to cause disease.

The putative phospholipase Lip2 counteracts oxidative damage and influences the virulence of Ustilago maydis.

Ustilago maydis is an obligate biotrophic fungal pathogen which causes common smut disease of corn. To proliferate in host tissue, U. maydis must gain access to nutrients and overcome plant defence responses, such as the production of reactive oxygen species. The elucidation of the mechanisms by which U. maydis meets these challenges is critical for the development of strategies to combat smut disease. In this study, we focused on the contributions of phospholipases (PLs) to the pathogenesis of corn smut disease. We identified 11 genes encoding putative PLs and characterized the transcript levels for these genes in the fungus grown in culture and during infection of corn tissue. To assess the contributions of specific PLs, we focused on two genes, lip1 and lip2, which encode putative phospholipase A2 (PLA2 ) enzymes with similarity to platelet-activating factor acetylhydrolases. PLA2 enzymes are known to counteract oxidative damage to lipids in other organisms. Consistent with a role in the mitigation of oxidative damage, lip2 mutants were sensitive to oxidative stress provoked by hydrogen peroxide and by increased production of reactive oxygen species caused by inhibitors of mitochondrial functions. Importantly, mutants defective in lip2, but not lip1, were attenuated for virulence in corn seedlings. Finally, a comparative analysis of fatty acid and cardiolipin profiles in the wild-type strain and a lip2 mutant revealed differences consistent with a protective role for Lip2 in maintaining lipid homeostasis and mitochondrial health during proliferation in the hostile host environment.

Chloroplast-associated metabolic functions influence the susceptibility of maize to Ustilago maydis.

Biotrophic fungal pathogens must evade or suppress plant defence responses to establish a compatible interaction in living host tissue. In addition, metabolic changes during disease reflect both the impact of nutrient acquisition by the fungus to support proliferation and the integration of metabolism with the plant defence response. In this study, we used transcriptome analyses to predict that the chloroplast and associated functions are important for symptom formation by the biotrophic fungus Ustilago maydis on maize. We tested our prediction by examining the impact on disease of a genetic defect (whirly1) in chloroplast function. In addition, we examined whether disease was influenced by inhibition of glutamine synthetase by glufosinate (impacting amino acid biosynthesis) or inhibition of 3-phosphoshikimate 1-carboxyvinyltransferase by glyphosate (influencing secondary metabolism). All of these perturbations increased the severity of disease, thus suggesting a contribution to resistance. Overall, these findings provide a framework for understanding the components of host metabolism that benefit the plant versus the pathogen during a biotrophic interaction. They also reinforce the emerging importance of the chloroplast as a mediator of plant defence.

Maize susceptibility to Ustilago maydis is influenced by genetic and chemical perturbation of carbohydrate allocation.

The ability of biotrophic fungi to metabolically adapt to the host environment is a critical factor in fungal diseases of crop plants. In this study, we analysed the transcriptome of maize tumours induced by Ustilago maydis to identify key features underlying metabolic shifts during disease. Among other metabolic changes, this analysis highlighted modifications during infection in the transcriptional regulation of carbohydrate allocation and starch metabolism. We confirmed the relevance of these changes by establishing that symptom development was altered in an id1 (indeterminate1) mutant that showed increased accumulation of sucrose as well as being defective in the vegetative to reproductive transition. We further established the relevance of specific metabolic functions related to carbohydrate allocation by assaying disease in su1 (sugary1) mutant plants with altered starch metabolism and in plants treated with glucose, sucrose and silver nitrate during infection. We propose that specific regulatory and metabolic changes influence the balance between susceptibility and resistance by altering carbon allocation to promote fungal growth or to influence plant defence. Taken together, these studies reveal key aspects of metabolism that are critical for biotrophic adaptation during the maize-U. maydis interaction.

Treatment of breast cancer with simultaneous integrated boost in hybrid plan technique : Influence of flattening filter-free beams.

OBJECTIVE: The present study compares in silico treatment plans using hybrid plan technique during hypofractionated radiation of mammary carcinoma with simultaneous integrated boost (SIB). The influence of 6 MV photon radiation in flattening filter free (FFF) mode against the clinical standard flattening filter (FF) mode is to be examined. PATIENTS AND METHODS: RT planning took place with FF and FFF radiation plans for 10 left-sided breast cancer patients. Hybrid plans were realised with two tangential IMRT fields and one VMAT field. The dose prescription was in line with the guidelines in the ARO-2010-01 study. The dosimetric verification took place with a manufacturer-independent measurement system. RESULTS: Required dose prescriptions for the planning target volumes (PTV) were achieved for both groups. The average dose values of the ipsi- and contralateral lung and the heart did not differ significantly. The overall average incidental dose to the left anterior descending artery (LAD) of 8.24 ± 3.9 Gy in the FFF group and 9.05 ± 3.7 Gy in the FF group (p < 0.05) were found. The dosimetric verifications corresponded to the clinical requirements. FFF-based RT plans reduced the average treatment time by 17 s/fraction. CONCLUSION: In comparison to the FF-based hybrid plan technique the FFF mode allows further reduction of the average LAD dose for comparable target volume coverage without adverse low-dose exposure of contralateral structures. The combination of hybrid plan technique and 6 MV photon radiation in the FFF mode is suitable for use with hypofractionated dose schemes. The increased dose rate allows a substantial reduction of treatment time and thus beneficial application of the deep inspiration breath hold technique.

Heart dose reduction in breast cancer treatment with simultaneous integrated boost: Comparison of treatment planning and dosimetry for a novel hybrid technique and 3D-CRT.

OBJECTIVE: The present study compares in silico treatment plans of clinically established three-dimensional conformal radiotherapy (3D-CRT) with a hybrid technique consisting of intensity-modulated radiotherapy (IMRT) and volumetric modulated arc radiotherapy (VMAT) during normally fractionated radiation of mammary carcinomas with simultaneous integrated boost on the basis of dose-volume histogram (DVH) parameters. PATIENTS AND METHODS: Radiation treatment planning was performed with a hybrid and a 3D-CRT treatment plan for 20 patients. Hybrid plans were implemented with two tangential IMRT fields and a VMAT field in the angular range of the tangents. Verification of the plan was performed with a manufacturer-independent measurement system consisting of a detector array and rotation unit. RESULTS: The mean values of the heart dose for the entire patient collective were 3.6 ± 2.5 Gy for 3D-CRT and 2.9 ± 2.1 Gy for the hybrid technique (p < 0.01). For the left side (n = 10), the mean values for the left anterior descending artery were 21.8 ± 7.4 Gy for 3D-CRT and 17.6 ± 7.4 Gy for the hybrid technique (p < 0.01). The mean values of the ipsilateral lung were 11.9 ± 1.6 Gy for 3D-CRT and 10.5 ± 1.3 Gy for the hybrid technique (p < 0.01). Calculated dose distributions in the hybrid arm were in good accordance with measured dose (on average 95.6 ± 0.5 % for γ < 1 and 3 %/3 mm). The difference of the mean treatment time per fraction was 7 s in favor of 3D-CRT. CONCLUSION: Compared with the established 3D-CRT technique, the hybrid technique allows for a decrease in dose, particularly of the mean heart and lung dose with comparable target volume acquisition and without disadvantageous low-dose load of contralateral structures. Uncomplicated implementation of the hybrid technique was demonstrated in this context. The hybrid technique combines the advantages of tangential IMRT with the superior sparing of organs at risk by VMAT.

Defects in phosphate acquisition and storage influence virulence of Cryptococcus neoformans.

Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous transcriptional profiling indicated that the fungal pathogen Cryptococcus neoformans, which causes meningoencephalitis in immunocompromised individuals, encounters phosphate limitation during proliferation in phagocytic cells. We therefore tested the hypothesis that phosphate acquisition and polyphosphate metabolism are important for cryptococcal virulence. Deletion of the high-affinity uptake system interfered with growth on low-phosphate medium, perturbed the formation of virulence factors (capsule and melanin), reduced survival in macrophages, and attenuated virulence in a mouse model of cryptococcosis. Additionally, analysis of nutrient sensing functions for C. neoformans revealed regulatory connections between phosphate acquisition and storage and the iron regulator Cir1, cyclic AMP (cAMP)-dependent protein kinase A (PKA), and the calcium-calmodulin-activated protein phosphatase calcineurin. Deletion of the VTC4 gene encoding a polyphosphate polymerase blocked the ability of C. neoformans to produce polyphosphate. The vtc4 mutant behaved like the wild-type strain in interactions with macrophages and in the mouse infection model. However, the fungal load in the lungs was significantly increased in mice infected with vtc4 deletion mutants. In addition, the mutant was impaired in the ability to trigger blood coagulation in vitro, a trait associated with polyphosphate. Overall, this study reveals that phosphate uptake in C. neoformans is critical for virulence and that its regulation is integrated with key signaling pathways for nutrient sensing.

Incidental dose to coronary arteries is higher in prone than in supine whole breast irradiation. A dosimetric comparison in adjuvant radiotherapy of early stage breast cancer.

PURPOSE: Sparing of normal lung is best achieved in prone whole breast irradiation (WBI). However, exposure of the heart and coronary arteries might increase due to anterior movement of the heart in prone WBI. PATIENTS AND METHODS: Treatment plans of 46 patients with large breasts irradiated for mammary cancer after breast-conserving surgery were retrospectively analyzed. The average treated breast volume of right-sided breasts (n = 33) was 1,804 ccm and 1,500 ccm for left-sided breasts (n = 13). The majority had invasive cancer (96 %) of which 61 % were pT1 and 39 % pT2 tumors. All patients received radiation therapy to the breast only. For three-dimensional (3D) treatment planning, all patients underwent a noncontrast-enhanced CT in the supine position with a wingboard and a second CT in the prone position using a prone breastboard. Nontarget volumes of the lung, heart, and coronary arteries were contoured. A total dose of 50.4 Gy was prescribed to the breast only. Differences were calculated for each patient and compared using the Wilcoxon signed-rank test. RESULTS: Treatment of left-sided breasts resulted in similar average mean heart doses in prone versus supine WBI (4.16 vs. 4.01 Gy; p = 0.70). The left anterior descending artery (LAD) had significantly higher dose exposure in left versus right WBI independent of position. Prone WBI always resulted in significantly higher exposures of the right circumflex artery (RCA) and LAD as compared to supine WBI. In left WBI, the mean LADprone was 33.5 Gy vs. LADsupine of 25.6 Gy (p = 0.0051). The V20prone of the LAD was 73.6 % vs. V20supine 50.4 % (p = 0.0006). CONCLUSION: The heart dose is not different between supine and prone WBI. However, in left WBI the incidental dose to the LAD with clinically relevant doses can be significantly higher in prone WBI. This is discussed controversially in the literature as it might depend on contouring and treatment techniques. We recommend contouring of LAD if patients are treated in prone WBI and evaluation of alternative treatment techniques for optimal sparing of coronary arteries.

The impact of flattening-filter-free beam technology on 3D conformal RT.

BACKGROUND: The removal of the flattening filter (FF) leads to non-uniform fluence distribution with a considerable increase in dose rate. It is possible to adapt FFF beams (flattening-filter-free) in 3D conformal radiation therapy (3D CRT) by using field in field techniques (FiF). The aim of this retrospective study is to clarify whether the quality of 3D CRT plans is influenced by the use of FFF beams. METHOD: This study includes a total of 52 CT studies of RT locations that occur frequently in clinical practice. Dose volume targets were provided for the PTV of breast (n=13), neurocranium (n=11), lung (n=7), bone metastasis (n=10) and prostate (n=11) in line with ICRU report 50/62. 3D CRT planning was carried out using FiF methods. Two clinically utilized photon energies are used for a Siemens ARTISTE linear accelerator in FFF mode at 7MVFFF and 11MVFFF as well as in FF mode at 6MVFF and 10MVFF. The plan quality in relation to the PTV coverage, OAR (organs at risk) and low dose burden as well as the 2D dosimetric verification is compared with FF plans. RESULTS: No significant differences were found between FFF and FF plans in the mean dose for the PTV of breast, lung, spine metastasis and prostate. The low dose parameters V5Gy and V10Gy display significant differences for FFF and FF plans in some subgroups. The DVH analysis of the OAR revealed some significant differences. Significantly more fields (1.9-4.5) were necessary in the use of FFF beams for each location (p<0.0001) in order to achieve PTV coverage. All the tested groups displayed significant increases (1.3-2.2 times) in the average number of necessary MU with the use of FFF beams (p<0.001). CONCLUSIONS: This study has shown that the exclusive use of a linear accelerator in FFF mode is feasible in 3D CRT. It was possible to realize RT plans in comparable quality in typical cases of clinical radiotherapy. The 2D dosimetric validation of the modulated fields verified the dose calculation and thus the correct reproduction of the characteristic FFF parameters in the planning system that was used.