irCLIP-RNP and Re-CLIP reveal patterns of dynamic protein associations on RNA.

RNA binding proteins ( RBPs ) control varied processes, including RNA splicing, stability, transport, and translation 1-3 . Dysfunctional RNA-RBP interactions contribute to the pathogenesis of human disease 1,4,5 , however, characterizing the nature and dynamics of multiprotein assemblies on RNA has been challenging. To address this, non-isotopic ligation-based ultraviolet crosslinking immunoprecipitation 6 was combined with mass spectrometry ( irCLIP-RNP ) to identify RNA-dependent associated proteins ( RDAPs ) co-bound to RNA with any RBP of interest. irCLIP-RNP defined landscapes of multimeric protein assemblies on RNA, uncovering previously unknown patterns of RBP-RNA associations, including cell-type-selective combinatorial relationships between RDAPs and primary RBPs. irCLIP-RNP also defined dynamic RDAP remodeling in response to epidermal growth factor ( EGF ), uncovering EGF-induced recruitment of UPF1 adjacent to HNRNPC to effect splicing surveillance of cell proliferation mRNAs. To identify the RNAs simultaneously co-bound by multiple studied RBPs, a sequential immunoprecipitation irCLIP ( Re-CLIP ) method was also developed. Re-CLIP confirmed binding relationships seen in irCLIP-RNP and detected simultaneous HNRNPC and UPF1 co-binding on RND3 and DDX3X mRNAs. irCLIP-RNP and Re-CLIP provide a framework to identify and characterize dynamic RNA-protein assemblies in living cells.

O-glycosylation contributes to mammalian glycoRNA biogenesis.

There is an increasing appreciation for the role of cell surface glycans in modulating interactions with extracellular ligands and participating in intercellular communication. We recently reported the existence of sialoglycoRNAs, where mammalian small RNAs are covalently linked to N-glycans through the modified base acp3U and trafficked to the cell surface. However, little is currently known about the role for O-glycosylation, another major class of carbohydrate polymer modifications. Here, we use parallel genetic, enzymatic, and mass spectrometry approaches to demonstrate that O-linked glycan biosynthesis is responsible for the majority of sialoglycoRNA levels. By examining the O-glycans associated with RNA from cell lines and colon organoids we find known and previously unreported O-linked glycan structures. Further, we find that O-linked glycans released from small RNA from organoids derived from ulcerative colitis patients exhibit higher levels of sialylation than glycans from healthy organoids. Together, our work provides flexible tools to interrogate O-linked glycoRNAs (O-glycoRNA) and suggests that they may be modulated in human disease.

The modified RNA base acp3U is an attachment site for N-glycans in glycoRNA.

GlycoRNA consists of RNAs modified with secretory N-glycans that are presented on the cell surface. Although previous work supported a covalent linkage between RNA and glycans, the direct chemical nature of the RNA-glycan connection was not described. Here, we develop a sensitive and scalable protocol to detect and characterize native glycoRNAs. Leveraging RNA-optimized periodate oxidation and aldehyde ligation (rPAL) and sequential window acquisition of all theoretical mass spectra (SWATH-MS), we identified the modified RNA base 3-(3-amino-3-carboxypropyl)uridine (acp3U) as a site of attachment of N-glycans in glycoRNA. rPAL offers sensitivity and robustness as an approach for characterizing direct glycan-RNA linkages occurring in cells, and its flexibility will enable further exploration of glycoRNA biology.

Patient-specific quality assurance of dynamically-collimated proton therapy treatment plans.

BACKGROUND: The dynamic collimation system (DCS) provides energy layer-specific collimation for pencil beam scanning (PBS) proton therapy using two pairs of orthogonal nickel trimmer blades. While excellent measurement-to-calculation agreement has been demonstrated for simple cube-shaped DCS-trimmed dose distributions, no comparison of measurement and dose calculation has been made for patient-specific treatment plans. PURPOSE: To validate a patient-specific quality assurance (PSQA) process for DCS-trimmed PBS treatment plans and evaluate the agreement between measured and calculated dose distributions. METHODS: Three intracranial patient cases were considered. Standard uncollimated PBS and DCS-collimated treatment plans were generated for each patient using the Astroid treatment planning system (TPS). Plans were recalculated in a water phantom and delivered at the Miami Cancer Institute (MCI) using an Ion Beam Applications (IBA) dedicated nozzle system and prototype DCS. Planar dose measurements were acquired at two depths within low-gradient regions of the target volume using an IBA MatriXX ion chamber array. RESULTS: Measured and calculated dose distributions were compared using 2D gamma analysis with 3%/3 mm criteria and low dose threshold of 10% of the maximum dose. Median gamma pass rates across all plans and measurement depths were 99.0% (PBS) and 98.3% (DCS), with a minimum gamma pass rate of 88.5% (PBS) and 91.2% (DCS). CONCLUSIONS: The PSQA process has been validated and experimentally verified for DCS-collimated PBS. Dosimetric agreement between the measured and calculated doses was demonstrated to be similar for DCS-collimated PBS to that achievable with noncollimated PBS.

Meet the authors: Lauren Kageler, Jonathan Perr, and Ryan A. Flynn.

In an interview with Dr. Mishtu Dey, editor-in-chief of Cell Chemical Biology, the authors of the review titled "Tools to investigate the cell surface: Proximity as a central concept in glycoRNA biology"-Lauren Kageler, Jonathan Perr, and Dr. Ryan A. Flynn-tell us more about their career paths and what excites them about science.

Pharmacokinetics and pharmacodynamics of intravenously and subcutaneously administered pantoprazole in sheep (Ovis aries).

Abomasal ulcers are recognized in sheep of all ages, but research regarding therapeutic interventions is limited. Proton Pump Inhibitors (PPIs) such as pantoprazole, are clinically used with a paucity of evidence regarding efficacy in mature sheep. Intravenous and subcutaneously administered pantoprazole dosed at 1.0 mg/kg in adult sheep will increase the pH of abomasal fluid compared to pre-administration baseline. The objectives were to assess the effect of pantoprazole, after single and multiple administration, on abomasal fluid pH in adult sheep. A third objective was to describe the pharmacokinetic parameters of IV and SC pantoprazole. Four clinically healthy adult Southdown ewes previously fitted with a gastrostomy tube in the abomasum were utilized in this randomized, 2-way cross-over trial. Ewes received pantoprazole (1.0 mg/kg) as a single and 3-dose regimen (every 24 hours). After a 10 day washout period the reverse treatment was applied. Blood for analysis of pantoprazole concentration was collected intermittently for 24 hours, and abomasal fluid pH was measured at intervals for a 96-hour period. The pH of the abomasal fluid was higher in pantoprazole treatments for up to 24 hours after dosing. Following intravenous administration of pantoprazole to study ewes, elimination half-life, volume of distribution, and clearance of pantoprazole was estimated as 3.29 hours, 0.35 L/kg, and 65.26 mL/hr/kg respectively. After subcutaneous dosing, maximum concentration, time to maximum concentration, half-life of elimination, and volume of distribution, were estimated as 2604 ng/mL, 0.55 hours, 2.48 hours, and 0.37 L/kg. Additionally, the bioavailability was estimated as 83.33%. Pantoprazole administered IV or SC may be useful for treatment or prevention of abomasal ulcers in adult sheep.

Tools to investigate the cell surface: Proximity as a central concept in glycoRNA biology.

Proximity is a fundamental concept in chemistry and biology, referring to the convergence of molecules to facilitate new molecular interactions or reactions. Hybrid biopolymers like glycosylphosphatidylinositol (GPI)-anchored proteins, ubiquitinated proteins, glycosylated RNAs (glycoRNAs), and RNAylated proteins exemplify this by covalent bonding of moieties that are often orthogonally active. Hybrid molecules like glycoRNAs are localized to new physical spaces, generating new interfaces for biological functions. To fully investigate the compositional and spatial features of molecules like glycoRNAs, flexible genetic and chemical tools that encompass different encoding and targeting biopolymers are required. Here we discuss concepts of molecular proximity and explore newer proximity labeling technologies that facilitate applications in RNA biology, cell surface biology, and the interface therein with a particular focus on glycoRNA biology. We review the advantages and disadvantages of methods pertaining to cell surface RNA identification and provide insights into the vast opportunities for method development in this area.

hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing.

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

Development and application of GlycanDIA workflow for glycomic analysis.

Glycans modify protein, lipid, and even RNA molecules to form the regulatory outer coat on cells called the glycocalyx. The changes in glycosylation have been linked to the initiation and progression of many diseases. Thus, while the significance of glycosylation is well established, a lack of accessible methods to characterize glycans has hindered the ability to understand their biological functions. Mass spectrometry (MS)-based methods have generally been at the core of most glycan profiling efforts; however, modern data-independent acquisition (DIA), which could increase sensitivity and simplify workflows, has not been benchmarked for analyzing glycans. Herein, we developed a DIA-based glycomic workflow, termed GlycanDIA, to identify and quantify glycans with high sensitivity and accuracy. The GlycanDIA workflow combined higher energy collisional dissociation (HCD)-MS/MS and staggered windows for glycomic analysis, which facilitates the sensitivity in identification and the accuracy in quantification compared to conventional data-dependent acquisition (DDA)-based glycomics. To facilitate its use, we also developed a generic search engine, GlycanDIA Finder, incorporating an iterative decoy searching for confident glycan identification and quantification from DIA data. The results showed that GlycanDIA can distinguish glycan composition and isomers from N-glycans, O-glycans, and human milk oligosaccharides (HMOs), while it also reveals information on low-abundant modified glycans. With the improved sensitivity, we performed experiments to profile N-glycans from RNA samples, which have been underrepresented due to their low abundance. Using this integrative workflow to unravel the N-glycan profile in cellular and tissue glycoRNA samples, we found that RNA-glycans have specific forms as compared to protein-glycans and are also tissue-specific differences, suggesting distinct functions in biological processes. Overall, GlycanDIA can provide comprehensive information for glycan identification and quantification, enabling researchers to obtain in-depth and refined details on the biological roles of glycosylation.

A re-activation model for 169Yb intensity modulated brachytherapy sources accounting for spatiotemporal isotopic composition.

BACKGROUND: Intensity modulated brachytherapy based on partially shielded intracavitary and interstitial applicators is possible with a cost-effective 169Yb production method. 169Yb is a traditionally expensive isotope suitable for this purpose, with an average γ-ray energy of 93 keV. Re-activating a single 169Yb source multiple times in a nuclear reactor between clinical uses was shown to theoretically reduce cost by approximately 75% relative to conventional single-activation sources. With re-activation, substantial spatiotemporal variation in isotopic source composition is expected between activations via 168Yb burnup and 169Yb decay, resulting in time dependent neutron transmission, precursor usage, and reactor time needed per re-activation. PURPOSE: To introduce a generalized model of radioactive source production that accounts for spatiotemporal variation in isotopic source composition to improve the efficiency estimate of the 169Yb production process, with and without re-activation. METHODS AND MATERIALS: A time-dependent thermal neutron transport, isotope transmutation, and decay model was developed. Thermal neutron flux within partitioned sub-volumes of a cylindrical active source was calculated by raytracing through the spatiotemporal dependent isotopic composition throughout the source, accounting for thermal neutron attenuation along each ray. The model was benchmarked, generalized, and applied to a variety of active source dimensions with radii ranging from 0.4 to 1.0 mm, lengths from 2.5 to 10.5 mm, and volumes from 0.31 to 7.85 mm3, at thermal neutron fluxes from 1 × 1014 to 1 × 1015 n cm-2 s-1. The 168Yb-Yb2O3 density was 8.5 g cm-3 with 82% 168Yb-enrichment. As an example, a reference re-activatable 169Yb active source (RRS) constructed of 82%-enriched 168Yb-Yb2O3 precursor was modeled, with 0.6 mm diameter, 10.5 mm length, 3 mm3 volume, 8.5 g cm-3 density, and a thermal neutron activation flux of 4 × 1014 neutrons cm-2 s-1. RESULTS: The average clinical 169Yb activity for a 0.99 versus 0.31 mm3 source dropped from 20.1 to 7.5 Ci for a 4 × 1014 n cm-2 s-1 activation flux and from 20.9 to 8.7 Ci for a 1 × 1015 n cm-2 s-1 activation flux. For thermal neutron fluxes ≥2 × 1014 n cm-2 s-1, total precursor and reactor time per clinic-year were maximized at a source volume of 0.99 mm3 and reached a near minimum at 3 mm3. When the spatiotemporal isotopic composition effect was accounted for, average thermal neutron transmission increased over RRS lifetime from 23.6% to 55.9%. A 28% reduction (42.5 days to 30.6 days) in the reactor time needed per clinic-year for the RRS is predicted relative to a model that does not account for spatiotemporal isotopic composition effects. CONCLUSIONS: Accounting for spatiotemporal isotopic composition effects within the RRS results in a 28% reduction in the reactor time per clinic-year relative to the case in which such changes are not accounted for. Smaller volume sources had a disadvantage in that average clinical 169Yb activity decreased substantially below 20 Ci for source volumes under 1 mm3. Increasing source volume above 3 mm3 adds little value in precursor and reactor time savings and has a geometric disadvantage.

Magnetic Resonance Imaging of Iron Metabolism with T2* Mapping Predicts an Enhanced Clinical Response to Pharmacologic Ascorbate in Patients with GBM.

PURPOSE: Pharmacologic ascorbate (P-AscH-) is hypothesized to be an iron (Fe)-dependent tumor-specific adjuvant to chemoradiation in treating glioblastoma (GBM). This study determined the efficacy of combining P-AscH- with radiation and temozolomide in a phase II clinical trial while simultaneously investigating a mechanism-based, noninvasive biomarker in T2* mapping to predict GBM response to P-AscH- in humans. PATIENTS AND METHODS: The single-arm phase II clinical trial (NCT02344355) enrolled 55 subjects, with analysis performed 12 months following the completion of treatment. Overall survival (OS) and progression-free survival (PFS) were estimated with the Kaplan-Meier method and compared across patient subgroups with log-rank tests. Forty-nine of 55 subjects were evaluated using T2*-based MRI to assess its utility as an Fe-dependent biomarker. RESULTS: Median OS was estimated to be 19.6 months [90% confidence interval (CI), 15.7-26.5 months], a statistically significant increase compared with historic control patients (14.6 months). Subjects with initial T2* relaxation < 50 ms were associated with a significant increase in PFS compared with T2*-high subjects (11.2 months vs. 5.7 months, P < 0.05) and a trend toward increased OS (26.5 months vs. 17.5 months). These results were validated in preclinical in vitro and in vivo model systems. CONCLUSIONS: P-AscH- combined with temozolomide and radiotherapy has the potential to significantly enhance GBM survival. T2*-based MRI assessment of tumor iron content is a prognostic biomarker for GBM clinical outcomes. See related commentary by Nabavizadeh and Bagley, p. 255.

Galectin-3 does not interact with RNA directly.

Galectin-3, well characterized as a glycan binding protein, has been identified as a putative RNA binding protein, possibly through participation in pre-mRNA maturation through interactions with splicosomes. Given recent developments with cell surface RNA biology, the putative dual-function nature of galectin-3 evokes a possible non-classical connection between glycobiology and RNA biology. However, with limited functional evidence of a direct RNA interaction, many molecular-level observations rely on affinity reagents and lack appropriate genetic controls. Thus, evidence of a direct interaction remains elusive. We demonstrate that antibodies raised to endogenous human galectin-3 can isolate RNA-protein crosslinks, but this activity remains insensitive to LGALS3 knock-out. Proteomic characterization of anti-galectin-3 IPs revealed enrichment of galectin-3, but high abundance of hnRNPA2B1, an abundant, well-characterized RNA-binding protein with weak homology to the N-terminal domain of galectin-3, in the isolate. Genetic ablation of HNRNPA2B1, but not LGALS3, eliminates the ability of the anti-galectin-3 antibodies to isolate RNA-protein crosslinks, implying either an indirect interaction or cross-reactivity. To address this, we introduced an epitope tag to the endogenous C-terminal locus of LGALS3. Isolation of the tagged galectin-3 failed to reveal any RNA-protein crosslinks. This result suggests that the galectin-3 does not directly interact with RNA and may be misidentified as an RNA-binding protein, at least in HeLa where the putative RNA associations were first identified. We encourage further investigation of this phenomenon employ gene deletions and, when possible, endogenous epitope tags to achieve the specificity required to evaluate potential interactions.

Nucleolus activity-dependent recruitment and biomolecular condensation by pH sensing.

DEAD-box ATPases are major regulators of biomolecular condensates and orchestrate diverse biochemical processes that are critical for the functioning of cells. How DEAD-box proteins are selectively recruited to their respective biomolecular condensates is unknown. We explored this in the context of the nucleolus and DEAD-box protein DDX21. We find that the pH of the nucleolus is intricately linked to the transcriptional activity of the organelle and facilitates the recruitment and condensation of DDX21. We identify an evolutionarily conserved feature of the C terminus of DDX21 responsible for nucleolar localization. This domain is essential for zebrafish development, and its intrinsically disordered and isoelectric properties are necessary and sufficient for the ability of DDX21 to respond to changes in pH and form condensates. Molecularly, the enzymatic activities of poly(ADP-ribose) polymerases contribute to maintaining the nucleolar pH and, consequently, DDX21 recruitment and nucleolar partitioning. These observations reveal an activity-dependent physicochemical mechanism for the selective recruitment of biochemical activities to biomolecular condensates.

Integration and dosimetric validation of a dynamic collimation system for pencil beam scanning proton therapy.

Objective.To integrate a Dynamic Collimation System (DCS) into a pencil beam scanning (PBS) proton therapy system and validate its dosimetric impact.Approach.Uncollimated and collimated treatment fields were developed for clinically relevant targets using an in-house treatment plan optimizer and an experimentally validated Monte Carlo model of the DCS and IBA dedicated nozzle (DN) system. The dose reduction induced by the DCS was quantified by calculating the mean dose in 10- and 30-mm two-dimensional rinds surrounding the target. A select number of plans were then used to experimentally validate the mechanical integration of the DCS and beam scanning controller system through measurements with the MatriXX-PT ionization chamber array and EBT3 film. Absolute doses were verified at the central axis at various depths using the IBA MatriXX-PT and PPC05 ionization chamber.Main results.Simulations demonstrated a maximum mean dose reduction of 12% for the 10 mm rind region and 45% for the 30 mm rind region when utilizing the DCS. Excellent agreement was observed between Monte Carlo simulations, EBT3 film, and MatriXX-PT measurements, with gamma pass rates exceeding 94.9% for all tested plans at the 3%/2 mm criterion. Absolute central axis doses showed an average verification difference of 1.4% between Monte Carlo and MatriXX-PT/PPC05 measurements.Significance.We have successfully dosimetrically validated the delivery of dynamically collimated proton therapy for clinically relevant delivery patterns and dose distributions with the DCS. Monte Carlo simulations were employed to assess dose reductions and treatment planning considerations associated with the DCS.

The population percentile allowance method for determining systematic spatial error tolerances for temporary intensity modulated brachytherapy.

BACKGROUND: Multiple approaches are under development for delivering temporary intensity modulated brachytherapy (IMBT) using partially shielded applicators wherein the delivered dose distributions are sensitive to spatial uncertainties in both the applicator position and shield orientation, rather than only applicator position as with conventional high-dose-rate brachytherapy (HDR-BT). Sensitivity analyses to spatial uncertainties have been reported as components of publications on these emerging technologies, however, a generalized framework for the rigorous determination of the spatial uncertainty tolerances of dose-volume parameters is needed. PURPOSE: To derive and present the population percentile allowance (PPA) method, a generalized mathematical and statistical framework to evaluate the tolerance of temporary IMBT approaches to spatial uncertainties in applicator position and shield orientation. METHODS: A mathematical formalism describing geometric applicator position and shield orientation shifts was derived that supports straight and curved applicators and applies to serial and helical rotating shield brachytherapy (RSBT) and direction modulated brachytherapy (DMBT). The PPA method entails defining the percentage of a patient population receiving a given therapy that is, allowed to receive dose-volume errors in the target volume and specified organs at risk of a defined percentage or less, then determining what combinations of applicator position and shield orientation systematic errors would be expected to produce that outcome in the population. The PPA method was applied to the use case of multi-shield helical 169 Yb-based RSBT for cervical cancer, with 45° and 180° shield emission angles. A total of 37 cervical cancer patients were considered in the population, with average (± 1 standard deviation) HR-CTV volumes of 79 cm3  ± 37 cm3 and optimized baseline treatment plans (no spatial uncertainties applied) created for each patient to meet dose-volume requirements of 85 GyEQD2 (equivalent uniform dose in 2 Gy fraction), with D2cc tolerance doses of 90 GyEQD2 , 75 GyEQD2 , and 75 GyEQD2 for bladder, rectum, and sigmoid colon, respectively. RESULTS: For the PPA requirement that 90% of cervical cancer patients receiving multi-shield helical RSBT could have a maximum dose-volume uncertainty of 10% for high-risk clinical target volume (HR-CTV) D90 (minimum dose to hottest 90%) and bladder, rectum, and sigmoid colon D2cc (minimum dose to hottest 2 cm3 ), the tolerance systematic applicator position and shield orientation uncertainties were approximately ± 1.0 mm and ± 4.25°, respectively. For ± 1.5 mm and ± 5° systematic applicator position and shield orientation tolerances, 90% of the patients considered would have a maximum dose-volume uncertainty of 12.8% or less. CONCLUSION: The PPA method was formalized to determine the temporary IMBT spatial uncertainty tolerances that would be expected to result in an allowed percentage of a population of patients receiving relative dose-volume errors above a defined percentage. Multi-shield, helical 169 Yb-based RSBT for cervical cancer was evaluated and tolerances determined, which, if applied on each treatment fraction, would represent an extreme situation. The PPA method is applicable to a variety of temporary IMBT approaches and can be used to rigorously determine the design parameters for the delivery systems such as mechanical driver motor accuracy, shield angle backlash, applicator rotation, and applicator fixation stability.

PETRA: A pencil beam trimming algorithm for analytical proton therapy dose calculations with the dynamic collimation system.

BACKGROUND: The Dynamic Collimation System (DCS) has been shown to produce superior treatment plans to uncollimated pencil beam scanning (PBS) proton therapy using an in-house treatment planning system (TPS) designed for research. Clinical implementation of the DCS requires the development and benchmarking of a rigorous dose calculation algorithm that accounts for pencil beam trimming, performs monitor unit calculations to produce deliverable plans at all beam energies, and is ideally implemented with a commercially available TPS. PURPOSE: To present an analytical Pencil bEam TRimming Algorithm (PETRA) for the DCS, with and without its range shifter, implemented in the Astroid TPS (.decimal, Sanford, Florida, USA). MATERIALS: PETRA was derived by generalizing an existing pencil beam dose calculation model to account for the DCS-specific effects of lateral penumbra blurring due to the nickel trimmers in two different planes, integral depth dose variation due to the trimming process, and the presence and absence of the range shifter. Tuning parameters were introduced to enable agreement between PETRA and a measurement-validated Dynamic Collimation Monte Carlo (DCMC) model of the Miami Cancer Institute's IBA Proteus Plus system equipped with the DCS. Trimmer position, spot position, beam energy, and the presence or absence of a range shifter were all used as variables for the characterization of the model. The model was calibrated for pencil beam monitor unit calculations using procedures specified by International Atomic Energy Agency Technical Report Series 398 (IAEA TRS-398). RESULTS: The integral depth dose curves (IDDs) for energies between 70 MeV and 160 MeV among all simulated trimmer combinations, with and without the ranger shifter, agreed between PETRA and DCMC at the 1%/1 mm 1-D gamma criteria for 99.99% of points. For lateral dose profiles, the median 2-D gamma pass rate for all profiles at 1.5%/1.5 mm was 99.99% at the water phantom surface, plateau, and Bragg peak depths without the range shifter and at the surface and Bragg peak depths with the range shifter. The minimum 1.5%/1.5 mm gamma pass rates for the 2-D profiles at the water phantom surface without and with the range shifter were 98.02% and 97.91%, respectively, and, at the Bragg peak, the minimum pass rates were 97.80% and 97.5%, respectively. CONCLUSION: The PETRA model for DCS dose calculations was successfully defined and benchmarked for use in a commercially available TPS.

Design, testing and characterization of a proton central axis alignment device for the dynamic collimation system.

Objective. Proton therapy conformity has improved over the years by evolving from passive scattering to spot scanning delivery technologies with smaller proton beam spot sizes. Ancillary collimation devices, such the Dynamic Collimation System (DCS), further improves high dose conformity by sharpening the lateral penumbra. However, as spot sizes are reduced, collimator positional errors play a significant impact on the dose distributions and hence accurate collimator to radiation field alignment is critical.Approach. The purpose of this work was to develop a system to align and verify coincidence between the center of the DCS and the proton beam central axis. The Central Axis Alignment Device (CAAD) is composed of a camera and scintillating screen-based beam characterization system. Within a light-tight box, a 12.3-megapixel camera monitors a P43/Gadox scintillating screen via a 45° first-surface mirror. When a collimator trimmer of the DCS is placed in the uncalibrated center of the field, the proton radiation beam continuously scans a 7×7 cm2square field across the scintillator and collimator trimmer while a 7 s exposure is acquired. From the relative positioning of the trimmer to the radiation field, the true center of the radiation field can be calculated.Main results.The CAAD can calculate the offset between the proton beam radiation central axis and the DCS central axis within 0.054 mm accuracy and 0.075 mm reproducibility.Significance.Using the CAAD, the DCS is now able to be aligned accurately to the proton radiation beam central axis and no longer relies on an x-ray source in the gantry head which is only validated to within 1.0 mm of the proton beam.

The pharmacokinetics and pharmacodynamics of esomeprazole in sheep after intravenous dosing.

Abomasal (gastric) ulceration is a morbidity in sheep, and currently, there is a paucity of pharmacokinetic and pharmacodynamic data for gastroprotectant drugs reported for this species. The proton pump inhibitor esomeprazole has been used in small animal and human patients for gastroprotection via increasing the gastric pH. The objective of this study was to report the pharmacokinetic parameters and pharmacodynamic effect of esomeprazole in sheep after single intravenous dosing. Four healthy adult Southdown cross ewes had blood collected over a 24 h time period after single intravenous dosing of esomeprazole at 1.0 mg/kg. Abomasal fluid was sampled over 24 h before and after esomeprazole administration. Plasma samples were analyzed for concentrations of esomeprazole and the esomeprazole metabolite, esomeprazole sulfone by high performance liquid chromatography. Pharmacokinetic and pharmacodynamic data were evaluated with specialized software. Esomeprazole was rapidly eliminated after IV administration. Elimination half-life, area under the curve, initial concentration (C0), and clearance were 0.2 h, 1,197 h*ng/mL, 4,321 ng/mL, and 0.83 mL/h/kg, respectively. For the sulfone metabolite elimination half-life, area under the curve and maximum concentration were 0.16 h, 22.5 h*ng/mL, and 65.0 ng/mL, respectively. Abomasal pH was significantly elevated from 1 to 6 h after administration and remained above 4.0 for at least 8 h after administration. No adverse effects were noted in these sheep. Esomeprazole was rapidly eliminated in sheep, similar to goats. Abomasal pH was increased, but future studies will be necessary to develop a clinical management approach to the use of esomeprazole in sheep.

Co-transcriptional genome surveillance by HUSH is coupled to termination machinery.

The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.