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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

RNA Crossing Membranes: Systems and Mechanisms Contextualizing Extracellular RNA and Cell Surface GlycoRNAs.

The subcellular localization of a biopolymer often informs its function. RNA is traditionally confined to the cytosolic and nuclear spaces, where it plays critical and conserved roles across nearly all biochemical processes. Our recent observation of cell surface glycoRNAs may further explain the extracellular role of RNA. While cellular membranes are efficient gatekeepers of charged polymers such as RNAs, a large body of research has demonstrated the accumulation of specific RNA species outside of the cell, termed extracellular RNAs (exRNAs). Across various species and forms of life, protein pores have evolved to transport RNA across membranes, thus providing a mechanistic path for exRNAs to achieve their extracellular topology. Here, we review types of exRNAs and the pores capable of RNA transport to provide a logical and testable path toward understanding the biogenesis and regulation of cell surface glycoRNAs.

LINE-associated cryptic splicing induces dsRNA-mediated interferon response and tumor immunity.

RNA splicing plays a critical role in post-transcriptional gene regulation. Exponential expansion of intron length poses a challenge for accurate splicing. Little is known about how cells prevent inadvertent and often deleterious expression of intronic elements due to cryptic splicing. In this study, we identify hnRNPM as an essential RNA binding protein that suppresses cryptic splicing through binding to deep introns, preserving transcriptome integrity. Long interspersed nuclear elements (LINEs) harbor large amounts of pseudo splice sites in introns. hnRNPM preferentially binds at intronic LINEs and represses LINE-containing pseudo splice site usage for cryptic splicing. Remarkably, a subgroup of the cryptic exons can form long dsRNAs through base-pairing of inverted Alu transposable elements scattered in between LINEs and trigger interferon immune response, a well-known antiviral defense mechanism. Notably, these interferon-associated pathways are found to be upregulated in hnRNPM-deficient tumors, which also exhibit elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity. Targeting hnRNPM in tumors may be used to trigger an inflammatory immune response thereby boosting cancer surveillance.

Glucose dissociates DDX21 dimers to regulate mRNA splicing and tissue differentiation.

Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.

Nuclear accumulation of host transcripts during Zika Virus Infection.

Zika virus (ZIKV) infects fetal neural progenitor cells (NPCs) causing severe neurodevelopmental disorders in utero. Multiple pathways involved in normal brain development are dysfunctional in infected NPCs but how ZIKV centrally reprograms these pathways remains unknown. Here we show that ZIKV infection disrupts subcellular partitioning of host transcripts critical for neurodevelopment in NPCs and functionally link this process to the up-frameshift protein 1 (UPF1). UPF1 is an RNA-binding protein known to regulate decay of cellular and viral RNAs and is less expressed in ZIKV-infected cells. Using infrared crosslinking immunoprecipitation and RNA sequencing (irCLIP-Seq), we show that a subset of mRNAs loses UPF1 binding in ZIKV-infected NPCs, consistent with UPF1's diminished expression. UPF1 target transcripts, however, are not altered in abundance but in subcellular localization, with mRNAs accumulating in the nucleus of infected or UPF1 knockdown cells. This leads to diminished protein expression of FREM2, a protein required for maintenance of NPC identity. Our results newly link UPF1 to the regulation of mRNA transport in NPCs, a process perturbed during ZIKV infection.

Dosimetric delivery validation of dynamically collimated pencil beam scanning proton therapy.

Objective. Pencil beam scanning (PBS) proton therapy target dose conformity can be improved with energy layer-specific collimation. One such collimator is the dynamic collimation system (DCS), which consists of four nickel trimmer blades that intercept the scanning beam as it approaches the lateral extent of the target. While the dosimetric benefits of the DCS have been demonstrated through computational treatment planning studies, there has yet to be experimental verification of these benefits for composite multi-energy layer fields. The objective of this work is to dosimetrically characterize and experimentally validate the delivery of dynamically collimated proton therapy with the DCS equipped to a clinical PBS system.Approach. Optimized single field, uniform dose treatment plans for 3 × 3 × 3 cm3target volumes were generated using Monte Carlo dose calculations with depths ranging from 5 to 15 cm, trimmer-to-surface distances ranging from 5 to 18.15 cm, with and without a 4 cm thick polyethylene range shifter. Treatment plans were then delivered to a water phantom using a prototype DCS and an IBA dedicated nozzle system and measured with a Zebra multilayer ionization chamber, a MatriXX PT ionization chamber array, and Gafchromic™ EBT3 film.Main results. For measurements made within the SOBPs, average 2D gamma pass rates exceeded 98.5% for the MatriXX PT and 96.5% for film at the 2%/2 mm criterion across all measured uncollimated and collimated plans, respectively. For verification of the penumbra width reduction with collimation, film agreed with Monte Carlo with differences within 0.3 mm on average compared to 0.9 mm for the MatriXX PT.Significance. We have experimentally verified the delivery of DCS-collimated fields using a clinical PBS system and commonly available dosimeters and have also identified potential weaknesses for dosimeters subject to steep dose gradients.

A novel optimization algorithm for enabling dynamically collimated proton arc therapy.

The advent of energy-specific collimation in pencil beam scanning (PBS) proton therapy has led to an improved lateral dose conformity for a variety of treatment sites, resulting in better healthy tissue sparing. Arc PBS delivery has also been proposed to enhance high-dose conformity about the intended target, reduce skin toxicity, and improve plan robustness. The goal of this work was to determine if the combination of proton arc and energy-specific collimation can generate better dose distributions as a logical next step to maximize the dosimetric advantages of proton therapy. Plans were optimized using a novel DyNamically collimated proton Arc (DNA) genetic optimization algorithm that was designed specifically for the application of proton arc therapy. A treatment planning comparison study was performed by generating an uncollimated two-field intensity modulated proton therapy and partial arc treatments and then replanning these treatments using energy-specific collimation as delivered by a dynamic collimation system, which is a novel collimation technology for PBS. As such, we refer to this novel treatment paradigm as Dynamically Collimated Proton Arc Therapy (DC-PAT). Arc deliveries achieved a superior target conformity and improved organ at risk (OAR) sparing relative to their two-field counterparts at the cost of an increase to the low-dose, high-volume region of the healthy brain. The incorporation of DC-PAT using the DNA optimizer was shown to further improve the tumor dose conformity. When compared to the uncollimated proton arc treatments, the mean dose to the 10mm of surrounding healthy tissue was reduced by 11.4% with the addition of collimation without meaningfully affecting the maximum skin dose (less than 1% change) relative to a multi-field treatment. In this case study, DC-PAT could better spare specific OARs while maintaining better target coverage compared to uncollimated proton arc treatments. While this work presents a proof-of-concept integration of two emerging technologies, the results are promising and suggest that the addition of these two techniques can lead to superior treatment plans warranting further development.