Spread-out Bragg peak FLASH: quantifying normal tissue toxicity in a murine model.

Objective: A favorable effect of ultra-high dose rate (FLASH) radiation on normal tissue-sparing has been indicated in several preclinical studies. In these studies, the adverse effects of radiation damage were reduced without compromising tumor control. Most studies of proton FLASH investigate these effects within the entrance of a proton beam. However, the real advantage of proton therapy lies in the Spread-out Bragg Peak (SOBP), which allows for giving a high dose to a target with a limited dose to healthy tissue at the entrance of the beam. Therefore, a clinically relevant investigation of the FLASH effect would be of healthy tissues within a SOBP. Our study quantified the tissue-sparing effect of FLASH radiation on acute and late toxicity within an SOBP in a murine model. Material/Methods: Radiation-induced damage was assessed for acute and late toxicity in the same mice following irradiation with FLASH (Field dose rate of 60 Gy/s) or conventional (CONV, 0.34 Gy/s) dose rates. The right hindleg of unanesthetized female CDF1 mice was irradiated with single-fraction doses between 19.9-49.7 Gy for CONV and 30.4-65.9 Gy for FLASH with 5-8 mice per dose. The leg was placed in the middle of a 5 cm SOBP generated from a mono-energetic beam using a 2D range modulator. Acute skin toxicity quantified by hair loss, moist desquamation and toe separation was monitored daily within 29 days post-treatment. Late toxicity of fibrotic development measured by leg extendibility was monitored biweekly until 30 weeks post-treatment. Results: Comparison of acute skin toxicity following radiation indicated a tissue-sparing effect of FLASH compared to conventional single-fraction radiation with a mean protection ratio of 1.40 (1.35-1.46). Fibrotic development similarly indicated normal tissue sparing with a 1.18 (1.17-1.18) protection ratio. The acute skin toxicity tissue sparing was similar to data from entrance-beam irradiations of Sørensen et al. (4). Conclusion: Full dose-response curves for acute and late toxicity after CONV and FLASH radiation were obtained. Radiation within the SOBP retains the normal-tissue-sparing effect of FLASH with a dose-modifying factor of 40% for acute skin damage and 18% for fibrotic development.

Proton FLASH: Impact of Dose Rate and Split Dose on Acute Skin Toxicity in a Murine Model.

PURPOSE: Preclinical studies have shown a preferential normal tissue sparing effect of FLASH radiation therapy with ultra-high dose rates. The aim of the present study was to use a murine model of acute skin toxicity to investigate the biologic effect of varying dose rates, time structure, and introducing pauses in the dose delivery. METHODS AND MATERIALS: The right hind limbs of nonanaesthetized mice were irradiated in the entrance plateau of a pencil beam scanning proton beam with 39.3 Gy. Experiment 1 was with varying field dose rates (0.7-80 Gy/s) without repainting, experiment 2 was with varying field dose rates (0.37-80 Gy/s) with repainting, and in experiment 3, the dose was split into 2, 3, 4, or 6 identical deliveries with 2-minute pauses. In total, 320 mice were included, with 6 to 25 mice per group. The endpoints were skin toxicity of different levels up to 25 days after irradiation. RESULTS: The dose rate50, which is the dose rate to induce a response in 50% of the animals, depended on the level of skin toxicity, with the higher toxicity levels displaying a FLASH effect at 0.7-2 Gy/s. Repainting resulted in higher toxicity for the same field dose rate. Splitting the dose into 2 deliveries reduced the FLASH effect, and for 3 or more deliveries, the FLASH effect was almost abolished for lower grades of toxicity. CONCLUSIONS: The dose rate that induced a FLASH effect varied for different skin toxicity levels, which are characterized by a differing degree of sensitivity to radiation dosage. Conclusions on a threshold for the dose rate needed to obtain a FLASH effect can therefore be influenced by the dose sensitivity of the used endpoint. Splitting the total dose into more deliveries compromised the FLASH effect. This can have an impact for fractionation as well as for regions where 2 or more FLASH fields overlap within the same treatment session.

Two-dimensional time-resolved scintillating sheet monitoring of proton pencil beam scanning FLASH mouse irradiations.

BACKGROUND: Dosimetry in pre-clinical FLASH studies is essential for understanding the beam delivery conditions that trigger the FLASH effect. Resolving the spatial and temporal characteristics of proton pencil beam scanning (PBS) irradiations with ultra-high dose rates (UHDR) requires a detector with high spatial and temporal resolution. PURPOSE: To implement a novel camera-based system for time-resolved two-dimensional (2D) monitoring and apply it in vivo during pre-clinical proton PBS mouse irradiations. METHODS: Time-resolved 2D beam monitoring was performed with a scintillation imaging system consisting of a 1 mm thick transparent scintillating sheet, imaged by a CMOS camera. The sheet was placed in a water bath perpendicular to a horizontal PBS proton beam axis. The scintillation light was reflected through a system of mirrors and captured by the camera with 500 frames per second (fps) for UHDR and 4 fps for conventional dose rates. The raw images were background subtracted, geometrically transformed, flat field corrected, and spatially filtered. The system was used for 2D spot and field profile measurements and compared to radiochromic films. Furthermore, spot positions were measured for UHDR irradiations. The measured spot positions were compared to the planned positions and the relative instantaneous dose rate to equivalent fiber-coupled point scintillator measurements. For in vivo application, the scintillating sheet was placed 1 cm upstream the right hind leg of non-anaesthetized mice submerged in the water bath. The mouse leg and sheet were both placed in a 5 cm wide spread-out Bragg peak formed from the mono-energetic proton beam by a 2D range modulator. The mouse leg position within the field was identified for both conventional and FLASH irradiations. For the conventional irradiations, the mouse foot position was tracked throughout the beam delivery, which took place through repainting. For FLASH irradiations, the delivered spot positions and relative instantaneous dose rate were measured. RESULTS: The pixel size was 0.1 mm for all measurements. The spot and field profiles measured with the scintillating sheet agreed with radiochromic films within 0.4 mm. The standard deviation between measured and planned spot positions was 0.26 mm and 0.35 mm in the horizontal and vertical direction, respectively. The measured relative instantaneous dose rate showed a linear relation with the fiber-coupled scintillator measurements. For in vivo use, the leg position within the field varied between mice, and leg movement up to 3 mm was detected during the prolonged conventional irradiations. CONCLUSIONS: The scintillation imaging system allowed for monitoring of UHDR proton PBS delivery in vivo with 0.1 mm pixel size and 2 ms temporal resolution. The feasibility of instantaneous dose rate measurements was demonstrated, and the system was used for validation of the mouse leg position within the field.

Oxygen Enhancement Ratio-Weighted Dose Quantitatively Describes Acute Skin Toxicity Variations in Mice After Pencil Beam Scanning Proton FLASH Irradiation With Changing Doses and Time Structures.

PURPOSE: The aim of this work was to investigate the ability of a biological oxygen enhancement ratio-weighted dose, DOER, to describe acute skin toxicity variations observed in mice after proton pencil beam scanning irradiations with changing doses and beam time structures. METHODS AND MATERIALS: In five independent experiments, the right hind leg of a total of 621 CDF1 mice was irradiated previously in the entrance plateau of a pencil beam scanning proton beam. The incidence of acute skin toxicity (of level 1.5-2.0-2.5-3.0-3.5) was scored for 47 different mouse groups that mapped toxicity as function of dose for conventional and FLASH dose rate, toxicity as function of field dose rate with and without repainting, and toxicity when splitting the treatment into 1 to 6 identical deliveries separated by 2 minutes. DOER was calculated for all mouse groups using a simple oxygen kinetics model to describe oxygen depletion. The three independent model parameters (oxygen-depletion rate, oxygen-recovery rate, oxygen level without irradiation) were fitted to the experimental data. The ability of DOER to describe the toxicity variations across all experiments was investigated by comparing DOER-response curves across the five independent experiments. RESULTS: After conversion from the independent variable tested in each experiment to DOER, all five experiments had similar MDDOER50 (DOER giving 50% toxicity incidence) with standard deviations of 0.45 - 1.6 Gy for the five toxicity levels. DOER could thus describe the observed toxicity variations across all experiments. CONCLUSIONS: DOER described the varying FLASH-sparing effect observed for a wide range of conditions. Calculation of DOER for other irradiation conditions can quantitatively estimate the FLASH-sparing effect for arbitrary irradiations for the investigated murine model. With appropriate fitting parameters DOER also may be able to describe FLASH effect variations with dose and dose rate for other assays and endpoints.

The Role of Non-Selective TNF Inhibitors in Demyelinating Events.

The use of non-selective tumor necrosis factor (TNF) inhibitors is well known in the treatment of inflammatory diseases such as rheumatoid arthritis, Crohn's disease, and psoriasis. Its use in neurological disorders is limited however, due to rare adverse events of demyelination, even in patients without preceding demyelinating disease. We review here the molecular and cellular aspects of this neuroinflammatory process in light of a case of severe monophasic demyelination caused by treatment with infliximab. Focusing on the role of TNF, we review the links between CNS inflammation, demyelination, and neurodegenerative changes leading to permanent neurological deficits in a young woman, and we discuss the growing evidence for selective soluble TNF inhibitors as a new treatment approach in inflammatory and neurological diseases.

Peptides Derived from Histone 3 and Modified at Position 18 Inhibit Histone Demethylase KDM6 Enzymes.

The KDM6 subfamily of histone lysine demethylases has recently been implicated as a putative target in the treatment of a number of diseases; this makes the availability of potent and selective inhibitors important. Due to high sequence similarity of the catalytic domain of Jumonji C histone demethylases, the development of small-molecule, family-specific inhibitors has, however, proven challenging. One approach to achieve the selective inhibition of these enzymes is the use of peptides derived from the substrate, the histone 3 C terminus. Here we used computational methods to optimize such inhibitors of the KDM6 family. Through natural amino acid substitution, it is shown that a K18I variant of a histone H3 derived peptide significantly increases affinity towards the KDM6 enzymes. The crystal structure of KDM6B in complex with a histone 3 derived K18I peptide reveals a tighter fit of the isoleucine side chain, compared with that of the arginine. As a consequence, the peptide R17 residue also has increased hydrophilic interactions. These interactions of the optimized peptide are likely to be responsible for the increased affinity to the KDM6 enzymes.

Mass spectrometry analyses of normal and polyglutamine expanded ataxin-3 reveal novel interaction partners involved in mitochondrial function.

Deubiquitinating enzymes (DUBs) play important roles in a variety of cellular processes, including regulation of protein homeostasis. The DUB ataxin-3 is an enzyme implicated in protein quality control mechanisms. In the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3), ataxin-3 contains an expanded polyglutamine (polyQ) stretch that leads to aggregation of the protein and neuronal dysfunction. Increasing the understanding of ataxin-3 protein interaction partners could help to elucidate disease mechanisms. Hence, we analyzed the repertoire of proteins interacting with normal and polyQ expanded ataxin-3 by mass spectrometry. This showed that both normal and polyQ expanded ataxin-3 interacted with components of the protein quality control system and mitochondria. Five proteins showed increased interaction with polyQ expanded ataxin-3 relative to normal and three of these were mitochondrial proteins. The analyses underline the role of ataxin-3 in ubiquitin biology and point towards a role in mitochondrial biology.

Polyglutamine expansion of ataxin-3 alters its degree of ubiquitination and phosphorylation at specific sites.

Ubiquitination and phosphorylation of proteins represent post translational modifications (PTMs) capable of regulating a variety of cellular processes. In the neurodegenerative disorder spinocerebellar ataxia type 3 (SCA3), the disease causing protein ataxin-3 carries an expanded polyglutamine (polyQ) stretch causing it to aggregate in nuclear inclusions. These inclusions are decorated with ubiquitin suggestive of a malfunction in the clearance of the mutant protein. Differences in ubiquitin chain topology between normal and polyQ expanded ataxin-3 could be involved in the differential clearance of the two proteins and play a role in SCA3 pathogenesis. Likewise, changes in phosphorylation patterns between the two variants could contribute to pathogenic processes involved in SCA3. We therefore determined the ubiquitination and phosphorylation patterns, together with the ubiquitin-linkage types associated with normal and polyQ expanded ataxin-3 by mass spectrometry (MS). This analysis revealed a similar ubiquitin linkage pattern on normal and expanded ataxin-3. However, the distribution of ubiquitinated lysine residues was altered in polyQ expanded ataxin-3, with increased ubiquitination at the new identified ubiquitination site lysine-8. MS analysis of phosphorylation also revealed novel phosphorylation sites in ataxin-3, and an increase in phosphorylation of expanded ataxin-3 at several positions. The study suggests that differences in clearance of normal and expanded ataxin-3 are not attributed to differences in ubiquitin-linkage pattern. However, the observed differences between the normal and polyQ expanded ataxin-3 with respect to the degree of ubiquitination and phosphorylation on specific sites may have an impact on ataxin-3 function and SCA3 pathogenesis.

Structure and binding properties of a cameloid nanobody raised against KDM5B.

The histone demethylase KDM5B is considered to be a promising target for anticancer therapy. Single-chain antibodies from llama (nanobodies) have been raised to aid in crystallization and structure determination of this enzyme. The antigen-binding properties of 15 of these nanobodies have been characterized. The crystal structure of one of these (NB17) has been determined to a resolution of 1.85 Å. NB17 crystallizes in space group P4322 with six molecules in the asymmetric unit. The six molecules in the asymmetric unit pack as an entity with approximate D3 symmetry with interactions mediated by the CDR loops, which could act as a crystallization nucleus. NB17 does not bind to monomeric KDM5B residues 1-820, but is found to bind to aggregates formed after incubation at 310 K.

Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells.

The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the ß1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

Adaptive evolution of Escherichia coli to an α-peptide/ß-peptoid peptidomimetic induces stable resistance.

Antimicrobial peptides (AMPs) and synthetic analogues thereof target conserved structures of bacterial cell envelopes and hence, development of resistance has been considered an unlikely event. However, recently bacterial resistance to AMPs has been observed, and the aim of the present study was to determine whether bacterial resistance may also evolve against synthetic AMP analogues, e.g. α-peptide/ß-peptoid peptidomimetics. E. coli ATCC 25922 was exposed to increasing concentrations of a peptidomimetic (10 lineages), polymyxin B (10 lineages), or MilliQ water (4 lineages) in a re-inoculation culturing setup covering approx. 500 generations. All 10 lineages exposed to the peptidomimetic adapted to 32 × MIC while this occurred for 8 out of 10 of the polymyxin B-exposed lineages. All lineages exposed to 32 × MIC of either the peptidomimetic or polymyxin B had a significantly increased MIC (16-32 ×) to the selection agent. Five transfers (≈ 35 generations) in unsupplemented media did not abolish resistance indicating that resistance was heritable. Single isolates from peptidomimetic-exposed lineage populations displayed MICs against the peptidomimetic from wild-type MIC to 32 × MIC revealing heterogeneous populations. Resistant isolates showed no cross-resistance against a panel of membrane-active AMPs. These isolates were highly susceptible to blood plasma antibacterial activity and were killed when plasma concentrations exceeded ≈ 30%. Notably, MIC of the peptidomimetic against resistant isolates returned to wild-type level upon addition of 25% plasma. Whole-genome sequencing of twenty isolates from four resistant lineages revealed mutations, in murein transglycosylase D (mltD) and outer-membrane proteins, which were conserved within and between lineages. However, no common resistance-conferring mutation was identified. We hypothesise that alterations in cell envelope structure result in peptidomimetic resistance, and that this may occur via several distinct mechanisms. Interestingly, this type of resistance result in a concomitant high susceptibility towards plasma, and therefore the present study does not infer additional concern for peptidomimetics as future therapeutics.

Identification of catechols as histone-lysine demethylase inhibitors.

Identification of inhibitors of histone-lysine demethylase (HDM) enzymes is important because of their involvement in the development of cancer. An ELISA-based assay was developed for identification of inhibitors of the HDM KDM4C in a natural products library. Based on one of the hits with affinity in the low µM range (1, a catechol), a subset of structurally related compounds was selected and tested against a panel of HDMs. In this subset, two inhibitors (2 and 10) had comparable affinities towards KDM4C and KDM6A but no effect on PHF8. One inhibitor restored H3K9me3 levels in KDM4C transfected U2-OS cells.

Studies of H3K4me3 demethylation by KDM5B/Jarid1B/PLU1 reveals strong substrate recognition in vitro and identifies 2,4-pyridine-dicarboxylic acid as an in vitro and in cell inhibitor.

Dynamic methylations and demethylations of histone lysine residues are important for gene regulation and are facilitated by histone methyltransferases and histone demethylases (HDMs). KDM5B/Jarid1B/PLU1 is an H3K4me3/me2-specific lysine demethylase belonging to the JmjC domain-containing family of histone demethylases (JHDMs). Several studies have linked KDM5B to breast, prostate and skin cancer, highlighting its potential as a drug target. However, most inhibitor studies have focused on other JHDMs, and inhibitors for KDM5B remain to be explored. Here, we report the expression, purification and characterization of the catalytic core of recombinant KDM5B (ccKDM5B, residues 1-769). We show that ccKDM5B, recombinantly expressed in insect cells, demethylates H3K4me3 and H3K4me2 in vitro. The kinetic characterization showed that ccKDM5B has an apparent Michaelis constant (K(m) (app) ) value of 0.5 µm for its trimethylated substrate H3(1-15)K4me3, a considerably increased apparent substrate affinity than reported for related HDMs. Despite the presence of a PHD domain, the catalytic activity was not affected by additional methylation at the H3K9 position, suggesting that in vitro chromatin cross-talk between H3K4 and H3K9 does not occur for ccKDM5B. Inhibition studies of ccKDM5B showed both in vitro and in cell inhibition of ccKDM5B by 2,4-pyridinedicarboxylic acid (2,4-PDCA) with a potency similar to that reported for the HDM KDM4C. Structure-guided sequence alignment indicated that the binding mode of 2,4-PDCA is conserved between KDM4A/C and KDM5B.

Inhibitors of histone demethylases.

Methylated lysines are important epigenetic marks. The enzymes involved in demethylation have recently been discovered and found to be involved in cancer development and progression. Despite the relative recent discovery of these enzymes a number of inhibitors have already appeared. Most of the inhibitors are either previously reported inhibitors of related enzymes or compounds derived from these. Development in terms of selectivity and potency is still pertinent. Several reports on the development of functional assays have been published.

Enzyme kinetic studies of histone demethylases KDM4C and KDM6A: towards understanding selectivity of inhibitors targeting oncogenic histone demethylases.

To investigate ligand selectivity between the oncogenic KDM4C and tumor repressor protein KDM6A histone demethylases, KDM4C and KDM6A were enzymatically characterized, and subsequently, four compounds were tested for inhibitory effects. 2,4-dicarboxypyridine and (R)-N-oxalyl-O-benzyltyrosine (3) are both known to bind to a close KDM4C homolog and 3 binds in the part of the cavity that accommodates the side chain in position 11 of histone 3. The inhibition measurements showed significant selectivity between KDM4C and KDM6A. This demonstrates that despite very similar active site topologies, selectivity between Jumonji family histone demethylases can be obtained even with small molecule ligands.