Immunomodulatory Effects of Histone Variant H2A.J in Ionizing Radiation Dermatitis.

PURPOSE: Histone variant H2A.J is associated with premature senescence after ionizing radiation (IR) and modulates senescence-associated secretory phenotype (SASP). Using constitutive H2A.J knock-out mice, the role of H2A.J was investigated in radiation dermatitis. METHODS AND MATERIALS: H2A.J wild-type (WT) and knock-out (KO) mice were exposed to moderate or high IR doses (≤20 Gy, skinfold IR). Radiation-induced skin reactions were investigated up to 2 weeks post-IR at macroscopic and microscopic levels. H2A.J and other senescence markers, as well as DNA damage and proliferation markers, were studied by immunohistochemistry, immunofluorescence, and electron microscopy. After high-dose IR, protein-coding transcriptomes were analyzed by RNA sequencing, immune cell infiltration by flow cytometry, and gene expression by reverse transcription polymerase chain reaction in (non-) irradiated WT versus KO skin. RESULTS: In WT skin, epidermal keratinocytes showed time- and dose-dependent H2A.J accumulation after IR exposure. Unexpectedly, stronger inflammatory reactions with increased epidermal thickness and progressive hair follicle loss were observed in irradiated KO versus WT skin. Clearly more radiation-induced senescence was observed in keratinocyte populations of KO skin after moderate and high doses, with hair follicle stem cells being particularly badly damaged, leading to follicle atrophy. After high-dose IR, transcriptomic analysis revealed enhanced senescence-associated signatures in irradiated KO skin, with intensified release of SASP factors. Flow cytometric analysis indicated increased immune cell infiltration in both WT and KO skin; however, specific chemokine-mediated signaling in irradiated KO skin led to more neutrophil recruitment, thereby aggravating radiation toxicities. Increased skin damage in irradiated KO skin led to hyperproliferation, abnormal differentiation, and cornification of keratinocytes, accompanied by increased upregulation of transcription-factor JunB. CONCLUSIONS: Lack of radiation-induced H2A.J expression in keratinocytes is associated with increased senescence induction, modulation of SASP expression, and exacerbated inflammatory skin reactions. Hence, epigenetic H2A.J-mediated gene expression in response to IR regulates keratinocyte immune functions and plays an essential role in balancing the inflammatory response during radiation dermatitis.

Heme protonation affects iron-NO binding in the NO transport protein nitrophorin.

The nitrophorins (NPs) comprise an unusual group of heme proteins with stable ferric heme iron nitric oxide (Fe-NO) complexes. They are found in the salivary glands of the blood-sucking kissing bug Rhodnius prolixus, which uses the NPs to transport the highly reactive signaling molecule NO. Nuclear resonance vibrational spectroscopy (NRVS) of both isoform NP2 and a mutant NP2(Leu132Val) show, after addition of NO, a strong structured vibrational band at around 600 cm-1, which is due to modes with significant Fe-NO bending and stretching contribution. Based on a hybrid calculation method, which uses density functional theory and molecular mechanics, it is demonstrated that protonation of the heme carboxyl groups does influence both the vibrational properties of the Fe-NO entity and its electronic ground state. Moreover, heme protonation causes a significant increase of the gap between the highest occupied and lowest unoccupied molecular orbital by almost one order of magnitude leading to a stabilization of the Fe-NO bond.

Measuring out-of-field dose to the hippocampus in common radiotherapy indications.

BACKGROUND: The high susceptibility of the hippocampus region to radiation injury is likely the causal factor of neurocognitive dysfunctions after exposure to ionizing radiation. Repetitive exposures with even low doses have been shown to impact adult neurogenesis and induce neuroinflammation. We address the question whether the out-of-field doses during radiotherapy of common tumour entities may pose a risk for the neuronal stem cell compartment in the hippocampus. METHODS: The dose to the hippocampus was determined for a single fraction according to different treatment plans for the selected tumor entities: Point dose measurements were performed in an anthropomorphic Alderson phantom and the out-of-field dose to the hippocampus was measured using thermoluminescence dosimeters. RESULTS: For carcinomas in the head and neck region the dose exposure to the hippocampal region for a single fraction ranged from to 37.4 to 154.8 mGy. The hippocampal dose was clearly different for naso-, oro- and hypopharynx, with maximal values for nasopharynx carcinoma. In contrast, hippocampal dose levels for breast and prostate cancer ranged between 2.7 and 4.1 mGy, and therefore significantly exceeded the background irradiation level. CONCLUSION: The mean dose to hippocampus for treatment of carcinomas in the head and neck region is high enough to reduce neurocognitive functions. In addition, care must be taken regarding the out of field doses. The mean dose is mainly related to scattering effects, as is confirmed by the data from breast or prostate treatments, with a very different geometrical set-up but similar dosimetric results.

Imaging doses for different CBCT protocols on the Halcyon 3.0 linear accelerator - TLD measurements in an anthropomorphic phantom.

INTRODUCTION: Image guided radiotherapy allows for particularly conformal tumour irradiation through precise patient positioning. Becoming the standard for radiotherapy, this increases imaging doses to the patient. The Halcyon 3.0 linear accelerator (Varian Medical Systems, Palo Alto, CA) requires daily imaging due to its geometry. For this reason, the accelerator is equipped with on-line kV and MV imaging. However, daily CBCT images required for irradiation apply additional radiation, which increases the dose to normal tissue and therefore can affect the patient's secondary cancer risk. In this study, actual organ doses were measured for the kV system, and a comparison of normal tissue doses for all available kV CBCT protocols was presented to demonstrate differences in imaging doses across entities and protocols. In addition, effective dose and secondary cancer risk from imaging are evaluated. MATERIAL AND METHODS: Measurements were performed with thermoluminescent dosimeters in an anthropomorphic phantom positioned according to each entity (brain, head and neck, breast, lung, pelvis). CBCT images were obtained, using all available pre-set protocols without further adjustment of the parameters. Measured doses for each position and each protocol were then compared and secondary cancer risk of relevant and specifically radiosensitive organs was calculated. RESULTS: It was found that imaging doses for protocols such as Pelvis and Head could be reduced by up to half using the corresponding Fast and Low Dose modes, respectively. On the other hand, larger field sizes or the Large mode yielded higher doses than their initial protocols. Image Gently was found to spare normal tissue best, however it is not suitable for certain entities due to low image quality or insufficient projection data. DISCUSSION: By using appropriate kV-CBCT protocols, it is possible to reduce imaging doses to a significant extent and therefore spare healthy tissue. Combined with studies of image quality, the results of this study could lead to adjustments in workflow regarding the choice of protocols used in daily routine. This could prevent unnecessary radiation exposure and reduce secondary cancer risk.

Comparison of Breast Cancer Radiotherapy Techniques Regarding Secondary Cancer Risk and Normal Tissue Complication Probability - Modelling and Measurements Using a 3D-Printed Phantom.

Background: Radiotherapy after breast-conserving therapy is a standard postoperative treatment of breast cancer, which can be carried out with a variety of irradiation techniques. The treatment planning must take into consideration detrimental effects on the neighbouring organs at risk-the lung, the heart, and the contralateral breast, which can include both short- and long-term effects represented by the normal tissue complication probability and secondary cancer risk. Patients and Methods: In this planning study, we investigate intensity-modulated (IMRT) and three-dimensional conformal (3D-CRT) radiotherapy techniques including sequential or simultaneously integrated boosts as well as interstitial multicatheter brachytherapy boost techniques of 38 patients with breast-conserving surgery retrospectively. We furthermore develop a 3D-printed breast phantom add-on to allow for catheter placement and to measure the out-of-field dose using thermoluminescent dosimeters placed inside an anthropomorphic phantom. Finally, we estimate normal tissue complication probabilities using the Lyman-Kutcher-Burman model and secondary cancer risks using the linear non-threshold model (out-of-field) and the model by Schneider et al. (in-field). Results: The results depend on the combination of primary whole-breast irradiation and boost technique. The normal tissue complication probabilities for various endpoints are of the following order: 1%-2% (symptomatic pneumonitis, ipsilateral lung), 2%-3% (symptomatic pneumonitis, whole lung), and 1%-2% (radiation pneumonitis grade ≥ 2, whole lung). The additional relative risk of ischemic heart disease ranges from +25% to +35%. In-field secondary cancer risk of the ipsilateral lung in left-sided treatment is around 50 per 10,000 person-years for 20 years after exposure at age 55. Out-of-field estimation of secondary cancer risk results in approximately 5 per 10,000 person-years each for the contralateral lung and breast. Conclusions: In general, 3D-CRT shows the best risk reduction in contrast to IMRT. Regarding the boost concepts, brachytherapy is the most effective method in order to minimise normal tissue complication probability and secondary cancer risk compared to teletherapy boost concepts. Hence, the 3D-CRT technique in combination with an interstitial multicatheter brachytherapy boost is most suitable in terms of risk avoidance for treating breast cancer with techniques including boost concepts.

Apd1 and Aim32 Are Prototypes of Bishistidinyl-Coordinated Non-Rieske [2Fe-2S] Proteins.

Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxin-like ferredoxin (TLF) domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi, and unicellular pathogenic eukaryotes but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability of yeast cells upon treatment with the redox mediators gallobenzophenone or pyrogallol, respectively. UV-vis, EPR, and Mössbauer spectroscopy of purified wild-type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion, and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S]2+/1+ cluster. The [2Fe-2S] center of Apd1 ( Em,7 = -164 ± 5 mV, p Kox1,2 = 7.9 ± 0.1 and 9.7 ± 0.1) differs from both dioxygenase ( Em,7 ≈ -150 mV, p Kox1,2 = 9.8 and 11.5) and cytochrome bc1/ b6 f Rieske clusters ( Em,7 ≈ +300 mV, p Kox1,2= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands, or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of the [2Fe-2S] cluster containing proteins with bishistidinyl coordination.

Iron uptake and storage in the HAB dinoflagellate Lingulodinium polyedrum.

The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood with two basic strategies being distinguished: Strategy I involves the induction of an Fe(III)-chelate reductase (ferrireductase) along with Fe(II) or Fe(III) transporter proteins while strategy II plants have evolved sophisticated systems based on high-affinity, iron specific, binding compounds called phytosiderophores. In contrast, there is little knowledge about the corresponding systems in marine, plant-like lineages. Herein we report a study of the iron uptake and storage mechanisms in the harmful algal bloom dinoflagellate Lingulodinium polyedrum. L. polyedrum is an armored dinoflagellate with a mixotrophic lifestyle and one of the most common bloom species on Southern California coast widely noted for its bioluminescent properties and as a producer of yessotoxins. Short term radio-iron uptake studies indicate that iron is taken up by L. polyedrum in a time dependent manner consistent with an active transport process. Based on inhibitor and other studies it appears that a reductive-oxidative pathway such as that found in yeast and the green alga Chlamydomonas reinhardtii is likely. Of the various iron sources tested vibrioferrin, a photoactive and relatively weak siderophore produced by potentially mutualistic Marinobacter bacterial species, was the most efficient. Other more stable and non-photoactive siderophores such as ferrioxamine E were ineffective. Several pieces of data including long term exposure to 57Fe using Mössbauer spectroscopy suggest that L. polyedrum does not possess an iron storage system but rather presumably relies on an efficient iron uptake system, perhaps mediated by mutualistic interactions with bacteria.

Mössbauer Spectroscopic Characterization of Iron(III)-Polysaccharide Coordination Complexes: Photochemistry, Biological, and Photoresponsive Materials Implications.

While polycarboxylates and hydroxyl-acid complexes have long been known to be photoactive, simple carboxylate complexes which lack a significant LMCT band are not typically strongly photoactive. Hence, it was somewhat surprising that a series of reports demonstrated that materials synthesized from iron(III) and polysaccharides such as alginate (poly[guluronan-co-mannuronan]) or pectate (poly[galacturonan]) formed photoresponsive materials that convert from hydrogels to sols under the influence of visible light. These materials have numerous potential applications in areas such as photopatternable materials, materials for controlled drug delivery, and tissue engineering. Despite the near-identity of the functional units in the polysaccharide ligands, the reactivity of iron(III) hydrogels can depend on the configuration of some chiral centers in the sugar units and in the case of alginate the guluronate to mannuronate block composition, as well as pH. Here, using temperature- and field-dependent transmission Mössbauer spectroscopy, we show that the dominant iron compound detected for both the alginate and pectate gels displays features typical of a polymeric (Fe3+O6) system. The Mössbauer spectra of such systems are strongly dependent on temperature, field, size, and crystallinity, indicative of superparamagnetic relaxation of magnetically ordered nanoparticles. Pectate and alginate hydrogels differ in the size distribution of the iron oxyhydroxy nanoparticles, suggesting that in general smaller nanoparticles are more reactive. Potential biological implications of these results are also discussed.

ATP-Dependent Electron Activation Module of Benzoyl-Coenzyme A Reductase from the Hyperthermophilic Archaeon Ferroglobus placidus.

The class I benzoyl-coenzyme A (BzCoA) reductases (BCRs) are key enzymes in the anaerobic degradation of aromatic compounds that catalyze the ATP-dependent dearomatization of their substrate to a cyclic dienoyl-CoA. The phylogenetically distinct Thauera- and Azoarcus-type BCR subclasses are iron-sulfur enzymes and consist of an ATP-hydrolyzing electron activation module and a BzCoA reduction module. More than 20 years after their initial identification, all biochemical information about class I BCRs derives from studies of the wild-type enzyme from the denitrifying bacterium Thauera aromatica (BCRTaro). Here, we describe the first heterologous production and purification of the ATP-hydrolyzing, electron-activating module of an Azoarcus-type BCR from the hyperthermophilic archaeon Ferroglobus placidus, BzdPQFpla. The Fe content, UV/vis spectroscopic, and Mössbauer spectroscopic properties of the 57Fe-enriched enzyme clearly identified a [4Fe-4S]+/2+ cluster with a redox potential (E°') of -376 mV as a cofactor. ATP hydrolysis is required to overcome a redox barrier of ∼250 mV for stoichiometric electron transfer from the [4Fe-4S]+ cluster to the substrate benzene ring (E°'BzCoA/dienoyl-CoA = -622 mV). BzdPQFpla exhibited ATPase activity (15 nmol min-1 mg-1; Km = 270 µM) at 75 °C, which was relatively stable in air in contrast to BCRTaro. The results obtained revealed high levels of functional and molecular similarity between Azoarcus-type BCRs and the homologous ATP-dependent activator components of 2-hydroxyacyl-CoA dehydratases involved in amino acid fermentations. Insights into the diversity and evolution of ATP-dependent electron-activating modules for catalytic or stoichiometric low-potential electron transfer processes are presented.

Correction: Surface binding, localization and storage of iron in the giant kelp Macrocystis pyrifera.

Correction for 'Surface binding, localization and storage of iron in the giant kelp Macrocystis pyrifera' by Eric P. Miller et al., Metallomics, 2016, 8, 403-411.

Surface binding, localization and storage of iron in the giant kelp Macrocystis pyrifera.

Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. Although the iron uptake and storage mechanisms of terrestrial/higher plants have been well-studied, the corresponding systems in marine algae have received far less attention. While the iron many marine algae take up from the environment, irrespective of its detailed internalization mechanism, arrives at the cell surface by diffusion, there is growing evidence for more "active" means of concentrating this element prior to uptake. It has been well established in both laboratory and environmentally derived samples, that a large amount of iron can be "non-specifically" adsorbed to the surface of marine algae. While this phenomenon is widely recognized and has prompted the development of experimental protocols to eliminate its contribution to iron uptake studies, its potential biological significance as a concentrated iron storage source for marine algae is only now being recognized. In this study, using an interdisciplinary array of techniques, we show that the giant kelp Macrocystis pyrifera also displays significant cell surface bound iron although less than that seen with the related brown alga Ectocarpus siliculosus. The iron on the surface is likely bound to carboxylate groups and once inside the iron is found to localize differently depending on cell type. Iron appears to be stored in an as yet undefined mineral phase.