The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63-branched ubiquitin chains.

The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.

Exploring the pH dependence of an improved PETase.

Enzymatic recycling of plastic and especially of polyethylene terephthalate (PET) has shown great potential to reduce its negative impact on our society. PET hydrolases (PETases) have been optimized using rational design and machine learning, but the mechanistic details of the PET depolymerization process remain unclear. Belonging to the carboxylic-ester hydrolase family with a canonical Ser-His-Asp catalytic triad, their observed alkaline pH optimum is generally thought to be related to the protonation state of the catalytic His. Here, we explore this aspect in the context of LCCICCG, an optimized PETase, derived from the leaf-branch compost cutinase enzyme. We use NMR to identify the dominant tautomeric structure of the six histidines. Five show surprisingly low pKa values below 4.0, whereas the catalytic H242 in the active enzyme displays a pKa value that varies from 4.9 to 4.7 when temperatures increase from 30°C to 50°C. Whereas the hydrolytic activity of the enzyme toward a soluble substrate can be modeled by the corresponding protonation/deprotonation curve, an important discrepancy is found when the substrate is the solid plastic. This opens the way to further mechanistic understanding of the PETase activity and underscores the importance of studying the enzyme at the liquid-solid interface.

The CUE Domain of Cue1 Aligns Growing Ubiquitin Chains with Ubc7 for Rapid Elongation.

Ubiquitin conjugation is an essential process modulating protein function in eukaryotic cells. Surprisingly, little is known about how the progressive assembly of ubiquitin chains is managed by the responsible enzymes. Only recently has ubiquitin binding activity emerged as an important factor in chain formation. The Ubc7 activator Cue1 carries a ubiquitin binding CUE domain that substantially stimulates K48-linked polyubiquitination mediated by Ubc7. Our results from NMR-based analysis and in vitro ubiquitination reactions point out that two parameters accelerate ubiquitin chain assembly: the increasing number of CUE binding sites and the position of CUE binding within a growing chain. In particular, interactions with a ubiquitin moiety adjacent to the acceptor ubiquitin facilitate chain elongation. These data indicate a mechanism for ubiquitin binding in which Cue1 positions Ubc7 and the distal acceptor ubiquitin for rapid polyubiquitination. Disrupting this mechanism results in dysfunction of the ERAD pathway by a delayed turnover of substrates.

E. coli "Stablelabel" S30 lysate for optimized cell-free NMR sample preparation.

Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15N labels of the amino acids L-Asp, L-Asn, L-Gln, L-Glu and L-Ala, resulting in ambiguous NMR signals as well as in label dilution. Specific inhibitor cocktails suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by engineering corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative "Stablelabel" containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE yielded the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in "Stablelabel" lysates. By taking advantage of ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.

A systematic review and meta-analysis on oncological radiotherapy in patients with a cardiac implantable electronic device: Prevalence and predictors of device malfunction in 3121 patients.

BACKGROUND: The number of patients with cardiac implantable electronic devices (CIEDs) undergoing radiotherapy (RT) for cancer treatment is growing. At present, prevalence and predictors of RT-induced CIEDs malfunctions are not defined. METHODS: Systematic review and meta-analysis conducted following the PRISMA recommendations. PubMed, Scopus and Google Scholar were searched from inception to 31/01/2022 for studies reporting RT-induced malfunctions in CIEDs patients. Aim was to assess the prevalence of RT-induced CIEDs malfunctions and identify potential predictors. RESULTS: Thirty-two out of 3962 records matched the inclusion criteria and were included in the meta-analysis. A total of 135 CIEDs malfunctions were detected among 3121 patients (6.6%, 95% confidence interval [CI]: 5.1%-8.4%). The pooled prevalence increased moving from pacemaker (PM) to implantable cardioverter defibrillator (ICD), and cardiac resynchronization therapy and defibrillator (CRT-D) groups (4.1%, 95% CI: 2.9-5.8; 8.2% 95% CI: 5.9-11.3; and 19.8%, 95% CI: 11.4-32.2 respectively). A higher risk ratio (RR) of malfunctions was found when neutron-producing energies were used as compared to non-neutron-producing energies (RR 9.98, 95% CI: 5.09-19.60) and in patients with ICD/CRT-D as compared to patients with PM/CRT-P (RR 2.07, 95% CI: 1.40-3.06). On the contrary, no association was found between maximal radiation dose at CIED >2 Gy and CIEDs malfunctions (RR 0.93; 95% CI: 0.31-2.76). CONCLUSIONS: Radiotherapy related CIEDs malfunction had a prevalence ranging from 4% to 20%. The use of neutron-producing energies and more complex devices (ICD/CRT-D) were associated with higher risk of device malfunction, while the radiation dose at CIED did not significantly impact on the risk unless higher doses (>10 Gy) were used.

Long-term characterization of MRI-morphologic alterations after active motion-compensated liver SBRT: a multi-institutional pooled analysis.

BACKGROUND: Stereotactic body radiotherapy (SBRT) is an effective therapeutic approach in patients with liver metastases. However, long-term changes in hepatic normal tissue have to be taken into account in multimodal treatment regimes. Magnetic-resonance-imaging (MRI) based morphologic liver alterations (MMA) after liver SBRT have been analyzed longitudinally. MATERIAL AND METHODS: 57 patients treated with gantry-based or robotic-based SBRT of 69 treatment volumes of liver metastases, who had long-term follow-up (FU) ≥6 months were included in this retrospective analysis. Post-SBRT MMAs were contoured on each contrast-enhanced-T1-weighted (T1w) MRI-sequence. Morphologic/volumetric data of the liver and MMAs were evaluated longitudinally, including the dependency on treatment-related factors of the planning target volume (PTV) and liver. RESULTS: The median FU time was 1 year [6-48 months]. 66 of 69 treatment volumes developed MMAs (mean 143.8 ± 135.1 ccm at first appearance). 31.8% of MMAs resolved completely during FU. Of the persisting MMAs 82.2%/13.3% decreased/increased in size until last available FU. Morphological characterization of the MMAs at first appearance included 75% hypointense and 25% hyperintense T1w-MRI-based appearances. Hypointense as compared to hyperintense appearance was significantly associated with a higher mean liver dose EQD2α/ß=3 Gy (p = 0.0212) and non-significantly greater MMA size. Variance analysis demonstrated a significant reduction of MMA and total liver volume after SBRT (p < 0.0001). The volume reduction decelerated longitudinally for both MMA (p < 0.0001) and liver size (p = 0.0033). Radiation doses (PTV-BEDα/ß=3 Gy and 10 Gy) were not significantly associated with MMA volume reduction. SBRT of liver metastases with mean liver dose EQD2α/ß=3 Gy > 18 Gy were characterized by greater MMA volumes (p = 0.0826) and steeper MMA reduction gradients during FU than those with EQD2α/ß=3 Gy ≤ 18 Gy (p < 0.0001). CONCLUSION: Radiogenic MMAs either completely resolve or usually decrease in volume with pronounced reduction during short-term FU. This course was independent of the MMA's morphological appearance. Further, increased mean liver dose was associated with greater MMA size and a greater gradient of MMA size reduction during FU.

Oligorecurrent Non-Small-Cell Lung Cancer Treated by Chemo-Radiation Followed by Immunotherapy and Intracranial Radiosurgery: A Case Report and Mini Review of Literature.

Locally advanced non-small-cell lung cancer still represents a "grey zone" in terms of the best treatment choice and optimal clinical outcomes. Indeed, most patients may be suitable to receive different treatments with similar outcomes such as chemo-radiotherapy (CHT-RT) followed by immunotherapy (IO) or surgery followed by adjuvant local/systemic therapies. We report a clinical case of a patient submitted to primary thoracic surgery who developed a mediastinal nodal recurrence successfully treated by CHT-RT-IO. Subsequently, a single brain lesion was found to have been successfully treated by single fraction stereotactic ablative radiotherapy. The patient is still on follow-up and she is free from disease having a good quality of life. In this report, we also perform a mini review about the role of CHT-RT followed by IO in treating loco-regional relapse after surgery. The role of SABR after IO is also evaluated, finding that it is safe and well tolerated. More robust and larger clinical data are needed in this particular setting to better define the role of the combination of systemic and local treatments in the management of intrathoracic and intracranial relapse for patients already submitted to CHT-RT followed by immunotherapy.

p63 uses a switch-like mechanism to set the threshold for induction of apoptosis.

The p53 homolog TAp63α is the transcriptional key regulator of genome integrity in oocytes. After DNA damage, TAp63α is activated by multistep phosphorylation involving multiple phosphorylation events by the kinase CK1, which triggers the transition from a dimeric and inactive conformation to an open and active tetramer that initiates apoptosis. By measuring activation kinetics in ovaries and single-site phosphorylation kinetics in vitro with peptides and full-length protein, we show that TAp63α phosphorylation follows a biphasic behavior. Although the first two CK1 phosphorylation events are fast, the third one, which constitutes the decisive step to form the active conformation, is slow. Structure determination of CK1 in complex with differently phosphorylated peptides reveals the structural mechanism for the difference in the kinetic behavior based on an unusual CK1/TAp63α substrate interaction in which the product of one phosphorylation step acts as an inhibitor for the following one.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Stereotactic or conventional radiotherapy for macroscopic prostate bed recurrence: a propensity score analysis.

PURPOSE: To assess outcomes between salvage radiation therapy (SRT) with curative intent and stereotactic radiotherapy for macroscopic prostate recurrence (SSRT) after radical prostatectomy (RP). In order to compare these two different options, we compared their outcomes with a propensity score-based matched analysis. METHODS: Data from 185 patients in seven Italian centres treated for macroscopic prostate bed recurrence after RP were retrospectively collected. To make a comparison between the two treatment groups, propensity matching was applied to create comparable cohorts. RESULTS: After matching, 90 patients in the SRT and SSRT groups were selected (45 in each arm). Kaplan-Meier analysis did not show any significant differences in terms of BRFS and PFS between matched populations (p = 0.08 and p = 0.8, respectively). Multivariate models show that treatment was not associated with BRFS, neither in the whole or matched cohort, with HR of 2.15 (95%CI 0.63-7.25, p = 0.21) and 2.65 (95%CI 0.59-11.97, p = 0.21), respectively. In the matched cohort, lower rate of toxicity was confirmed for patients undergoing SSRT, with acute GI and GU adverse events reported in 4.4 versus 44.4% (p < 0.001) and 28.9 versus 46.7% (p = 0.08) of patients, and late GI and GU adverse events reported in 0 versus 13.3% (p = 0.04) and 6.7 versus 22.2% (p = 0.03) of patients, respectively. CONCLUSION: Considering the favourable therapeutic ratio of this approach and the lower number of fractions needed, SSRT should be considered as an attractive alternative to conventional SRT in this setting.

Sequential chemo-hypofractionated RT versus concurrent standard CRT for locally advanced NSCLC: GRADE recommendation by the Italian Association of Radiotherapy and Clinical Oncology (AIRO).

INTRODUCTION: Almost 30% of non-small cell lung cancer (NSCLC) patients have locally advanced-stage disease. In this setting, definitive radiotherapy concurrent to chemotherapy plus adjuvant immunotherapy (cCRT + IO) is the standard of care, although only 40% of these patients are eligible for this approach. AIMS: A comparison between cCRT and hypofractionated radiotherapy regimens (hypo-fx RT) with the addition of sequential chemotherapy (sCHT) could be useful for future combinations with immunotherapy. We developed a recommendation about the clinical question of whether CHT and moderately hypo-fx RT are comparable to cCRT for locally advanced NSCLC MATERIALS AND METHODS: The panel used GRADE methodology and the Evidence to Decision (EtD) framework. After a systematic literature search, five studies were eligible. We identified the following outcomes: progression-free survival (PFS), overall survival (OS), freedom from locoregional recurrence (FFLR), deterioration of quality of life (QoL), treatment-related deaths, severe G3-G4 toxicity, late pulmonary toxicity G3-G4, and acute esophageal toxicity G3-G4. RESULTS: The probability of OS and G3-G4 late lung toxicity seems to be worse in patients submitted to sCHT and hypo-fx RT. The panel judged unfavorable the balance benefits/harms. CONCLUSIONS: The final recommendation was that sCHT followed by moderately hypo-fx RT should not be considered as an alternative to cCRT in unresectable stage III NSCLC patients.

A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1.

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

Site-Specific Detection of Arginine Methylation in Highly Repetitive Protein Motifs of Low Sequence Complexity by NMR.

Post-translational modifications of proteins are widespread in eukaryotes. To elucidate the functional role of these modifications, detection methods need to be developed that provide information at atomic resolution. Here, we report on the development of a novel Arg-specific NMR experiment that detects the methylation status and symmetry of each arginine side chain even in highly repetitive RGG amino acid sequence motifs found in numerous proteins within intrinsically disordered regions. The experiment relies on the excellent resolution of the backbone H,N correlation spectra even in these low complexity sequences. It requires 13C, 15N labeled samples.

Backbone and methyl assignment of bacteriorhodopsin incorporated into nanodiscs.

Resonance assignments are challenging for membrane proteins due to the size of the lipid/detergent-protein complex and the presence of line-broadening from conformational exchange. As a consequence, many correlations are missing in the triple-resonance NMR experiments typically used for assignments. Herein, we present an approach in which correlations from these solution-state NMR experiments are supplemented by data from 13C unlabeling, single-amino acid type labeling, 4D NOESY data and proximity of moieties to lipids or water in combination with a structure of the protein. These additional data are used to edit the expected peaklists for the automated assignment protocol FLYA, a module of the program package CYANA. We demonstrate application of the protocol to the 262-residue proton pump from archaeal bacteriorhodopsin (bR) in lipid nanodiscs. The lipid-protein assembly is characterized by an overall correlation time of 44 ns. The protocol yielded assignments for 62% of all backbone (H, N, Cα, Cß, C') resonances of bR, corresponding to 74% of all observed backbone spin systems, and 60% of the Ala, Met, Ile (δ1), Leu and Val methyl groups, thus enabling to assign a large fraction of the protein without mutagenesis data. Most missing resonances stem from the extracellular half, likely due intermediate exchange line-broadening. Further analysis revealed that missing information of the amino acid type of the preceding residue is the largest problem, and that 4D NOESY experiments are particularly helpful to compensate for that information loss.

Ubiquitination in the ERAD Process.

In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent the accumulation of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation.

Dynamics of Bacteriorhodopsin in the Dark-Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy.

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.

Structural and functional analysis of the GABARAP interaction motif (GIM).

Through the canonical LC3 interaction motif (LIR), [W/F/Y]-X1-X2-[I/L/V], protein complexes are recruited to autophagosomes to perform their functions as either autophagy adaptors or receptors. How these adaptors/receptors selectively interact with either LC3 or GABARAP families remains unclear. Herein, we determine the range of selectivity of 30 known core LIR motifs towards individual LC3s and GABARAPs. From these, we define a G ABARAP I nteraction M otif (GIM) sequence ([W/F]-[V/I]-X2-V) that the adaptor protein PLEKHM1 tightly conforms to. Using biophysical and structural approaches, we show that the PLEKHM1-LIR is indeed 11-fold more specific for GABARAP than LC3B. Selective mutation of the X1 and X2 positions either completely abolished the interaction with all LC3 and GABARAPs or increased PLEKHM1-GIM selectivity 20-fold towards LC3B. Finally, we show that conversion of p62/SQSTM1, FUNDC1 and FIP200 LIRs into our newly defined GIM, by introducing two valine residues, enhances their interaction with endogenous GABARAP over LC3B. The identification of a GABARAP-specific interaction motif will aid the identification and characterization of the expanding array of autophagy receptor and adaptor proteins and their in vivo functions.

Lipid Conversion by Cell-Free Synthesized Phospholipid Methyltransferase Opi3 in Defined Nanodisc Membranes Supports an in Trans Mechanism.

Biomembranes composed of lipids and proteins play central roles in physiological processes, and the precise balance between different lipid species is crucial for maintaining membrane function. One pathway for the biosynthesis of the abundant lipid phosphatidylcholine in eukaryotes involves a membrane-integrated phospholipid methyltransferase named Opi3 in yeast. A still unanswered question is whether Opi3 can catalyze phosphatidylcholine synthesis in trans, at membrane contact sites. While evidence for this activity was obtained from studies with complex in vitro-reconstituted systems based on endoplasmic reticulum membranes, isolated and purified Opi3 could not be analyzed. We present new insights into Opi3 activity by characterizing the in vitro-synthesized enzyme in defined hydrophobic environments. Saccharomyces cerevisiae Opi3 was cell-free synthesized and either solubilized in detergent micelles or co-translationally inserted into preformed nanodisc membranes of different lipid compositions. While detergent-solubilized Opi3 was inactive, the enzyme inserted into nanodisc membranes showed activity and stayed monomeric as revealed by native mass spectrometry. The methylation of its lipid substrate dioleoylphosphatidylmonomethylethanolamine to phosphatidylcholine was monitored by one-dimensional 31P nuclear magnetic resonance. Phosphatidylcholine formation was observed not only in nanodiscs containing inserted Opi3 but also in nanodiscs devoid of the enzyme containing the lipid substrate. This result gives a clear indication for in trans catalysis by Opi3; i.e., it acts on the substrate in juxtaposed membranes, while in cis lipid conversion may also contribute. Our established system for the characterization of pure Opi3 in defined lipid environments may be applicable to other lipid biosynthetic enzymes and help in understanding the subcellular organization of lipid synthesis.

Structural investigation of glycan recognition by the ERAD quality control lectin Yos9.

Yos9 is an essential component of the endoplasmic reticulum associated protein degradation (ERAD) system that is responsible for removing terminally misfolded proteins from the ER lumen and mediating proteasomal degradation in the cytosol. Glycoproteins that fail to attain their native conformation in the ER expose a distinct oligosaccharide structure, a terminal α1,6-linked mannose residue, that is specifically recognized by the mannose 6-phoshate receptor homology (MRH) domain of Yos9. We have determined the structure of the MRH domain of Yos9 in its free form and complexed with 3α, 6α-mannopentaose. We show that binding is achieved by loops between ß-strands performing an inward movement and that this movement also affects the entire ß-barrel leading to a twist. These rearrangements may facilitate the processing of client proteins by downstream acting factors. In contrast, other oligosaccharides such as 2α-mannobiose bind weakly with only locally occurring chemical shift changes underscoring the specificity of this substrate selection process within ERAD.

An offline technique to evaluate residual motion of the diaphragm during deep inspiratory breath-hold from cone-beam CT datasets.

PURPOSE: In radiation therapy, the computer-assisted deep inspiration breath-hold (DIBH) technique is one approach to deal with respiratory motion of tumors in the lung, liver, or upper abdomen. However, inter- and intra-breath-hold deviations from an optimal static tumor position might occur. A novel method is presented to noninvasively measure the diaphragm position and thus estimate its residual deviation (as surrogate for the tumor position) based on cone-beam computed tomography (CBCT) projection data using active breathing control during acquisition. METHODS: The diaphragm dome (DD) position relative to the isocenter of a linear accelerator is known from the static (DIBH) planning CT. A ball-bearing phantom (BB) is placed at this position, a CBCT dataset is acquired, and in each projection the position of the projected BB is determined automatically based on thresholding. The position of the DD is determined manually in CBCT projections of a patient. The distance between DD and BB (ideal static setting) in craniocaudal direction is calculated for a given angle based on the distance in the projection plane and the relative position of the BB referring to the source and the detector. An angle-dependent correction factor is introduced which takes this geometrical setting into account. The accuracy of the method is assessed. RESULTS: The method allows a CBCT projection-based estimation of the deviation between the DD and its optimal position as defined in the planning CT, i.e., the residual motion of the DD can be assessed. The error of this estimation is 2.2 mm in craniocaudal direction. CONCLUSIONS: The developed method allows an offline estimation of the inspiration depth (inter- and intra-breath-hold) over time. It will be useful as a reference for comparison to other methods of residual motion estimation, e.g., surface scanning.