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Freehand needles can be used with multichannel vaginal cylinders (MCVC) to cover vaginal cancer7 mm thick or with supra-vaginal extension. We report our institutional outcomes using this novel hybrid technique. Patients with vaginal malignancies treated with HDR BT using MCVC plus freehand needles from 2014-2021 at our institution were identified. Clinical characteristics, details of brachytherapy, initial response, and overall local control (LC) outcomes were recorded. LC was analyzed via Kaplan-Meier method. 34 patients were identified with median follow-up 1.9 years. 19 patients had primary endometrial cancer with vaginal recurrence/disease, and remaining had primary vaginal cancer or other primaries. 7 patients had recurrence after previous RT course. 25 patients received EBRT with median dose 45 Gy in 25 fractions, and rest received BT alone. Median HR-CTV D90 for patients treated with EBRT plus BT was 77.4 Gy. 30 patients had complete local response to BT on initial examination and/or follow-up imaging. 1 and 2-year LC rates in patients without prior RT treated with EBRT + BT were 94.1% and 94.1%, respectively. 1 and 2-year LC rates for those without prior RT were 88.1% and 76.4%, respectively. 1 and 2-year LC rates for those with prior RT were 68.6% and 34.3%, respectively. 1 patient had vaginal laceration requiring surgical repair, and 1 patient developed small bowel obstruction 1 month after BT, with no additional acute grade 3+ toxicities identified. Our approach with MCVC plus freehand needles with MRI-based planning was feasible and safe, with excellent initial local response and low rate of serious toxicities.

Sentinel lymph node biopsy has led to an increase in the detection of isolated tumor cells (ITCs) in up to 10% of early stage endometrioid endometrial cancer patients. In addition, the risk of nonsentinel lymph node involvement is approximately 10% or lower in patients with ITCs. In most studies, approximately 60% to 70% of patients with ITCs either underwent completion lymphadenectomy or received adjuvant therapy. Therefore, although multiple studies have shown that the effect of ITCs on disease outcomes is favorable, the true effect of ITCs without additional treatment is not known. In this report we describe our philosophy of relying on extent of surgical nodal staging and presence or absence of adverse intrauterine pathologic factors at the time of adjuvant therapy decision making for endometrioid endometrial carcinoma patients with ITCs.

Chemical cross-linking of proteins in complex samples, cells, or even tissues is emerging to provide unique structural information on proteins and complexes that exist within native or nativelike environments. The public database XLinkDB automatically maps cross-links to available structures based on sequence homology. Structures most likely to reflect protein conformations in the cross-linked sample are routinely identified by having cross-linked residues separated by Euclidean distances within the maximum span of the applied cross-linker. Solvent accessible surface distance (SASD), which considers the accessibility of the cross-linked residues and the path connecting them, is a better predictor of consistency than the Euclidean distance. However, SASDs of structures are not publicly available, and their calculation is computationally intensive. Here, we describe in XLinkDB version 4.0 the automatic calculation of SASDs using Jwalk for all cross-links mapped to structures, both with and without regard to ligands, and derive empirical maximum SASD spans for BDP-NHP and DSSO cross-linkers of 51 and 43 Å, respectively. We document ligands proximal to cross-links in structures and demonstrate how SASDs can be used to help infer sample protein conformations and ligand occupancy, highlighting cross-links sensitive to ADP binding in mitochondria isolated from HEK293 cells.

To evaluate the impact of prophylactic paraortic lymph node (PALN) radiation therapy (RT) on clinical outcomes in patients with International Federation of Obstetrics and Gynecology 2018 stage IIIC1 endometrial cancer (EC).A multi-institutional retrospective study included patients with International Federation of Obstetrics and Gynecology 2018 stage IIIC1 EC lymph node assessment, status postsurgical staging, followed by adjuvant chemotherapy and RT using various sequencing regimens. Overall survival (OS) and recurrence-free survival (RFS) rates were estimated by the Kaplan-Meier method. Univariable and multivariable analysis were performed by Cox proportional hazard models for RFS/OS. In addition, propensity score matching was used to estimate the effect of the radiation field extent on survival outcomes.A total of 378 patients were included, with a median follow-up of 45.8 months. Pelvic RT was delivered to 286 patients, and 92 patients received pelvic and PALN RT. The estimated OS and RFS rates at 5 years for the entire cohort were 80% and 69%, respectively. There was no difference in the 5-year OS (77% vs 87%, P = .47) and RFS rates (67% vs 70%, P = .78) between patients treated with pelvic RT and those treated with pelvic and prophylactic PALN RT, respectively. After propensity score matching, the estimated hazard ratios (HRs) of prophylactic PALN RT versus pelvic RT were 1.50 (95% confidence interval, 0.71-3.19; P = .28) for OS and 1.24 (95% confidence interval, 0.64-2.42; P = .51) for RFS, suggesting that prophylactic PALN RT does not improve survival outcomes. Distant recurrence was the most common site of first recurrence, and the extent of RT field was not associated with the site of first recurrence (P = .79).Prophylactic PALN RT was not significantly associated with improved survival outcomes in stage IIIC1 EC. Distant metastasis remains the most common site of failure despite routine use of systemic chemotherapy. New therapeutic approaches are necessary to optimize the outcomes for women with stage IIIC1 EC.

Ureteral stenosis (US) is an underreported complication of brachytherapy (BT) for cervical cancer (CC), with limited data on toxicity risk reduction. A previous study demonstrated ureter EQD2 D0.1cc77 Gy correlated with US development. We sought to assess feasibility of this constraint while maintaining similar HR-CTV coverage.Patients with locally advanced CC treated with EBRT plus HDR MRI-based brachytherapy boost without hydronephrosis at diagnosis and with ureter dose EQD2 D0.1cc77 Gy were included. Replan was attempted to achieve HR-CTV D90 ≥ 80-85 Gy and ureter dose reduction. Ureter distance from lateral margin of HR-CTV and tandem was recorded. t-test was performed to compare ureteral dose and HR-CTV D90.Of 25 patients were identified. Hundred percent received 45 Gy in 25 fractions to the pelvis ± paraaortic lymph nodes and 80% receiving median additional parametrial dose of 5.4 Gy. Replan meeting ureteral dose of ≤77 Gy was feasible in 18 of 25 patients, with a reduction in median ureter D0.1cc from 82.3 to 76.8 Gy (p0.001). Median HR-CTV D90 was similar (84.7 vs. 85.0 Gy). Replan achieved D0.1cc ≤77 Gy in 56% of patients who experienced US. All unilateral US cases occurred in the ureter closest to HR-CTV.Optimization to reduce ureter dose to ≤77 Gy is feasible when ureters are visible and contoured. Ureters may be considered as potential OAR during MRI-based brachytherapy treatment. Reduced ring to tandem total reference air kerma (TRAK) ratio may provide an additional metric by which to lower US risk.

Emphasis on value-based healthcare has led to increasing use of time-driven activity-based costing (TDABC) across medical departments. When applied to brachytherapy, TDABC provides insight into differences in costs across various modes of therapy, the nuances that drive cost including institutional factors and involved personnel, and discrepancies in reimbursement which influence clinical practice. This is especially important with the new alternative payment model (APM) in radiation oncology which offers fixed reimbursement per 90-day episode of care. The TDABC model can thus be utilized to improve efficiency, optimize the role of ancillary staff in treatment planning and care delivery, and implement shorter fraction schedules when clinically appropriate to promote value-based care. Ultimately, application of this methodology could potentiate changes to practice and incentives to improve patient care. In this review, we discuss the utility and limitations of TDABC in the context of existing studies in brachytherapy which have utilized this methodology.

Advancements in cross-linking mass spectrometry (XL-MS) bridge the gap between purified systems and native tissue environments, allowing the detection of protein structural interactions in their native state. Here we use isobaric quantitative protein interaction reporter technology (iqPIR) to compare the mitochondria protein interactomes in healthy and hypertrophic murine hearts, 4 weeks post-transaortic constriction. The failing heart interactome includes 588 statistically significant cross-linked peptide pairs altered in the disease condition. We observed an increase in the assembly of ketone oxidation oligomers corresponding to an increase in ketone metabolic utilization; remodeling of NDUA4 interaction in Complex IV, likely contributing to impaired mitochondria respiration; and conformational enrichment of ADP/ATP carrier ADT1, which is non-functional for ADP/ATP translocation but likely possesses non-selective conductivity. Our application of quantitative cross-linking technology in cardiac tissue provides molecular-level insights into the complex mitochondria remodeling in heart failure while bringing forth new hypotheses for pathological mechanisms.

Background Conflicting data exists regarding the benefit of prophylactic cranial irradiation (PCI) in patients with extensive-stage small-cell lung cancer (ES-SCLC). We sought to retrospectively review outcomes of patients within our network with ES-SCLC treated with and without PCI between 2009 and 2020. Methods Endpoints assessed using the Kaplan-Meier estimator were overall survival (OS), freedom from death with uncontrolled intracranial disease (UI-DFS), brain metastasis-free survival (BMFS), and symptomatic BMFS (SBMFS). Log-rank test was performed for univariate comparison of outcomes, with Cox regression performed for univariate and multivariable analysis of OS and UI-DFS. Results Some 250 patients were determined to be eligible for PCI based on any response to upfront chemotherapy, with 46 patients excluded owing to lack of negative staging brain magnetic resonance imaging (MRI). Brain MRI was performed both at diagnosis and near completion of chemotherapy in 108 patients, with brain metastases identified near completion of chemotherapy in 17 patients (15.7%), excluding them from further analysis. Median OS in remaining eligible 187 patients was 9.0 months, with 2-year Kaplan-Meier estimate of OS of 21.9%. PCI was associated with improved UI-DFS, BMFS, and SBMFS. However, PCI was not associated with improved OS in the entire cohort or the propensity matched cohort. Conclusion Our study suggests screening with MRI following chemotherapy is important because of the identification of unsuspected brain metastases in nearly 16% of patients with response to chemotherapy. PCI is associated with reduction in brain metastases, without a demonstrable impact on OS in the era of MRI screening.

Our purpose was to evaluate the effect of sequence and type of adjuvant therapy for patients with stage IIIC endometrial carcinoma (EC) on outcomes.In a multi-institutional retrospective cohort study, patients with stage IIIC EC who had surgical staging and received both adjuvant chemotherapy and radiation therapy (RT) were included. Adjuvant treatment regimens were classified as adjuvant chemotherapy followed by sequential RT (upfront chemo), which was predominant sequence; RT with concurrent chemotherapy followed by chemotherapy (concurrent); systemic chemotherapy before and after RT (sandwich); adjuvant RT followed by chemotherapy (upfront RT); or chemotherapy concurrent with vaginal cuff brachytherapy alone (chemo-brachy). Overall survival (OS) and recurrence-free survival (RFS) rates were estimated by the Kaplan-Meier method.A total of 686 eligible patients were included with a median follow-up of 45.3 months. The estimated 5-year OS and RFS rates were 74% and 66%, respectively. The sequence and type of adjuvant therapy were not correlated with OS or RFS (adjusted P = .68 and .84, respectively). On multivariate analysis, black race, nonendometrioid histology, grade 3 tumor, stage IIIC2, and presence of adnexal and cervical involvement were associated with worse OS and RFS (all P.05). Regardless of the sequence of treatment, the most common site of first recurrence was distant metastasis (20.1%). Vaginal only, pelvic only, and paraortic lymph node (PALN) recurrences occurred in 11 (1.6%),15 (2.2 %), and 43 (6.3 %) patients, respectively. Brachytherapy alone was associated with a higher rate of PALN recurrence (15%) compared with external beam radiation therapy (5%) P.0001.The sequence and type of combined adjuvant therapy did not affect OS or RFS rates. Brachytherapy alone was associated with a higher rate of PALN recurrence, emphasizing the role of nodal radiation for stage IIIC EC. The vast proportion of recurrences were distant despite systemic chemotherapy, highlighting the need for novel regimens.

Biological systems have evolved to utilize proteins to accomplish nearly all functional roles needed to sustain life. A majority of biological functions occur within the crowded environment inside cells and subcellular compartments where proteins exist in a densely packed complex network of protein-protein interactions. The structural biology field has experienced a renaissance with recent advances in crystallography, NMR, and CryoEM that now produce stunning models of large and complex structures previously unimaginable. Nevertheless, measurements of such structural detail within cellular environments remain elusive. This review will highlight how advances in mass spectrometry, chemical labeling, and informatics capabilities are merging to provide structural insights on proteins, complexes, and networks that exist inside cells. Because of the molecular detection specificity provided by mass spectrometry and proteomics, these approaches provide systems-level information that not only benefits from conventional structural analysis, but also is highly complementary. Although far from comprehensive in their current form, these approaches are currently providing systems structural biology information that can uniquely reveal how conformations and interactions involving many proteins change inside cells with perturbations such as disease, drug treatment, or phenotypic differences. With continued advancements and more widespread adaptation, systems structural biology based on in-cell labeling and mass spectrometry will provide an even greater wealth of structural knowledge.

Purpose To examine associations between ductal carcinoma in situ (DCIS) patients’ characteristics, treating locations and DCIS treatments received and to pilot assessing quality-of-life (QoL) values among DCIS patients with diverse backgrounds. Methods We performed a retrospective tumor registry review of all patients diagnosed and treated with DCIS from 2018 to 2019 in the UPMC-integrated network throughout central and western Pennsylvania. Demographics, clinical information, and administered treatments were compiled from tumor registry records. We categorized contextual factors such as different hospital setting (academic vs. community), socioeconomic status based on the neighborhood deprivation index (NDI) as well as age and race. QoL survey was administered to DCIS patients with diverse backgrounds via QoL questionnaire breast cancer module 23 and qualitative assessment questions. Results A total of 912 patients were reviewed. There were no treatment differences noted for age, race, or NDI. Mastectomy rate was higher in academic sites than community sites (29 vs. 20.4%; p = 0.0045), while hormone therapy (HT) utilization rate was higher in community sites (74 vs. 62%; p = 0.0012). QoL survey response rate was 32%. Only HT side effects negatively affected in QoL scores and there was no significant difference in QoL domains and decision-making process between races, age, NDI, treatment groups, and treatment locations. Conclusion Our integrated health network did not show chronically noted disparities arising from social determinates of health for DCIS treatments by implementing clinical pathways and system-wide peer review. Also, we demonstrated feasibility in collecting QoL for DCIS women with diverse backgrounds and different socioeconomic statuses.

XLinkDB is a fast-expanding public database now storing more than 100,000 distinct identified cross-linked protein residue pairs acquired by chemical cross-linking with mass spectrometry from samples of 12 species(1). Mapping identified cross-links to protein structures, when available, provides valuable guidance on protein conformations detected in the cross-linked samples. As more and more structures become available in the Protein Data Bank(2), we sought to leverage their utility for cross-link studies by automatically mapping identified cross-links to structures based on sequence homology of the cross-linked proteins with those within structures. This enables use of structures derived from organisms different from those of samples, including large multi-protein complexes and complexes in alternative states. We demonstrate utility of mapping to orthologous structures, highlighting a cross-link between two subunits of mouse mitochondrial Complex I that was mapped to 15 structures derived from five mammals, its distances there of 16.2 ± 0.4Å indicating strong conservation of the protein interaction and proximity of the cross-linked sites. We furthermore show how multimeric structures enable reassessment of cross-links presumed to be intra-protein as potentially homodimeric inter-protein in origin, and how multi-protein structures to which inter-protein cross-links are mapped can be used to evaluate cross-link aided protein-protein docking.

OBJECTIVES The brain is a rare site for sarcoma metastases. Sarcoma's radioresistance also makes standard whole-brain radiotherapy less appealing. We hypothesize that stereotactic radiation techniques (stereotactic radiosurgery [SRS]/stereotactic fractionated radiotherapy [FSRT]) may provide effective local control. MATERIALS AND METHODS This single-institution retrospective analysis evaluated our experience with linear acceleator-based SRS/FSRT for sarcoma brain metastases. Time to event analysis was estimated via Kaplan-Meier. Univariable/multivariable Cox regression analyses followed to assess the impact of patient and disease characteristics on outcomes. RESULTS Between 2003 and 2018, 24 patients were treated with 34 courses of SRS/FSRT to 58 discrete lesions. The median age at first treatment was 57 years (range: 25 to 87 y). Majority of patients had concurrent lung metastases (n=21; 88%), diagnosed spindle cell sarcoma (n=15; 25%) or leiomyosarcoma (n=12; 21%) histology, and were treated with either SRS (n=43; median dose=19 Gy, range: 15 to 24 Gy) or FSRT (n=17; 3/5 fractions, median dose=25 Gy, range: 25 to 35 Gy). With a median follow-up after brain metastasis of 7.3 months, the 6 month/12 month local control, distant brain control, and overall survival of 89%/89%, 59%/34%, and 50%/38%, respectively. All local failures were of primary spindle cell histology (P

Chemical cross-linking with mass spectrometry (XL-MS) has emerged as a useful technique for interrogating protein structures and interactions. When combined with quantitative proteomics strategies, protein conformational and interaction dynamics can be probed. Quantitative XL-MS has been demonstrated with the use of stable isotopes incorporated metabolically, or into the cross-linker molecules. Isotope labeled cross-linkers have primarily utilized deuterium and rely on MS1 based quantitation of precursor ion extracted ion chromatograms. Recently the development and application of isobaric quantitative protein interaction reporter (iqPIR) cross-linkers were reported, which utilize (13)C and (15)N isotope labels. Quantitation is accomplished using relative fragment ion isotope abundances in tandem mass spectra. Here we describe the synthesis and initial evaluation of a multiplexed set of iqPIR molecules, allowing for up to six cross-linked samples to be quantified simultaneously. To analyze data for such cross-linkers, the two-channel mode of iqPIR quantitative analysis was adapted to accommodate any number of channels with defined ion isotope peak mass offsets. The summed ion peak intensities in the overlapping channel isotope envelopes are apportioned among the channels to minimize the difference with respect to the predicted ion isotope envelopes. The result is accurate and reproducible relative quantitation enabling direct comparison among six differentially labeled cross-linked samples. The approach described here is generally extensible for the iqPIR strategy, accommodating future iqPIR reagent design, and enables large-scale in vivo quantitative XL-MS investigation of the interactome

Purpose Halcyon linear accelerators employ intensity‐modulated radiation therapy (IMRT) and stereotactic body radiation therapy (SBRT) techniques. The Halcyon offers translational, but not rotational, couch correction, which only allows a 3 degrees of freedom (3‐DOF) correction. In contrast, the TrueBeam (TB) linear accelerator offers full 6‐DOF corrections. This study aims to evaluate the difference in treatment plan quality for single thoracic or lumbar vertebral segment SBRT between the Halcyon and TB linear accelerators. In addition, this study will also investigate the effect of patient rotational setup errors on the final plan quality. Methods We analyzed 20 patients with a single‐level spine metastasis located between the T7 and L5 vertebrae near the spinal canal. The median planning target volume was 52.0 cm3 (17.9–138.7 cm3). The median tumor diameter in the axial plane was 4.6 cm (range 1.7–6.8 cm), in the sagittal plane was 3.3 cm (range 2–5 cm). The prescription doses were either 12–16 Gy in 1 fraction or 18–24 Gy in 3 fractions. All patients were treated on the TB linear accelerator with a 2.5 mm Multi‐Leaf Collimator (MLC) leaf width. Treatment plans were retrospectively created for the Halcyon, which has a 5 mm effective MLC leaf width. The 20 patients had a total of 50 treatments. Analysis of the 50 cone beam computed tomography (CBCT) scans showed average rotational setup errors of 0.6°, 1.2°, and 0.8° in pitch, yaw, and roll, respectively. Rotational error in roll was not considered in this study, as the original TB plans used a coplanar volumetric modulated arc therapy (VMAT) technique, and each 1° of roll will contribute an error of 1/360. If a plan has 3 arcs, the contribution from errors in roll will be 1° in the absence of 6‐DOF correction capabilities.

Acquisition of the citrus greening bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (CLas) by Asian citrus psyllid (Diaphorina citri) nymphs is required efficient tree-to-tree transmission during the adult stage. Quantitative isotope-labeled protein interaction reporter (PIR) cross-linkers were used in parallel with protein quantification using spectral counting to quantify protein interactions within microbe-enriched cellular fractions of nymph and adult D. citri. Over 100 unique crosslinks were found between five insect histone proteins, and over 30% of these were more abundant in nymph compared to adult insects. Strikingly, some cross-links detected in D. citri proteins are conserved in cross-linking studies on human cells, suggesting these protein interaction topologies were present in the common ancestor (∼750MYA) or are subject to convergent evolution. Analysis of posttranslational modifications of crosslinked histones revealed the presence of acetylated and methylated lysine residues, which may impact psyllid chromatin structure and gene expression. Histone H3 peptides acetylated in the N terminal tail region were found to be more abundant in nymph compared to adult insects in two orthogonal proteomics methods. The insect life stage-specific histone posttranslational modifications and protein interactions represent physical evidence that metamorphosis is associated with changes in chromatin structure that regulate genome-wide transcriptional reprogramming.

To evaluate short-term patient reported urinary quality of life scores in patients with prostate cancer treated at our institution with and without perioperative prednisone following Cesium-131 (We started routinely using a perioperative 7-day course of prednisone at a dose of 5 mg per day, beginning 1 day prior toPretreatment EPIC and AUA scores were available in 197 patients. Less reduction in EPIC US score was noted at 0.5-1.0 month in the group who received prednisone with mean change of -22.9 (SD 15.4) when compared to the group who did not receive prednisone with mean change of -31.7 (SD 19.3), p0.01, with significance lost at 3 months. There was no significant difference in acute urinary retention requiring postoperative urinary catheter placement with perioperative prednisone (OR 1.13, p = 0.71).A short course of perioperative low-dose prednisone was associated with less severe worsening in urinary symptoms by the EPIC questionnaire at the 0.5-1.0-month timepoint suggesting some improvement in acute urinary quality of life, although differences did not remain statistically significant at 3 months.

The methylation of histidine is a post-translational modification whose function is poorly understood. Methyltransferase histidine protein methyltransferase 1 (Hpm1p) monomethylates H243 in the ribosomal protein Rpl3p and represents the only known histidine methyltransferase in Saccharomyces cerevisiae. Interestingly, the hpm1 deletion strain is highly pleiotropic, with many extraribosomal phenotypes including improved growth rates in alternative carbon sources. Here, we investigate how the loss of histidine methyltransferase Hpm1p results in diverse phenotypes, through use of targeted mass spectrometry (MS), growth assays, quantitative proteomics, and differential crosslinking MS. We confirmed the localization and stoichiometry of the H243 methylation site, found unreported sensitivities of Δhpm1 yeast to nonribosomal stressors, and identified differentially abundant proteins upon hpm1 knockout with clear links to the coordination of sugar metabolism. We adapted the emerging technique of quantitative large-scale stable isotope labeling of amino acids in cell culture crosslinking MS for yeast, which resulted in the identification of 1267 unique in vivo lysine-lysine crosslinks. By reproducibly monitoring over 350 of these in WT and Δhpm1, we detected changes to protein structure or protein-protein interactions in the ribosome, membrane proteins, chromatin, and mitochondria. Importantly, these occurred independently of changes in protein abundance and could explain a number of phenotypes of Δhpm1, not addressed by expression analysis. Further to this, some phenotypes were predicted solely from changes in protein structure or interactions and could be validated by orthogonal techniques. Taken together, these studies reveal a broad role for Hpm1p in yeast and illustrate how crosslinking MS will be an essential tool for understanding complex phenotypes.

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial FoF1-ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or α-adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of FoF1-ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized HIF1α, leading to transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives FoF1-ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart.

Advancements of cross-linking mass spectrometry (XL-MS) for structural analysis of proteins bridges the gap between purified systems and native tissue environments. Here, isobaric quantitative protein interaction reporter technology (iqPIR) was utilized to further extend XL-MS to the first system-wide comparative study of mitochondrial proteins from healthy and diseased murine hearts. The failing heart interactome includes 602 statistically significant cross-linked peptide pairs altered in the disease condition. Structural insight into ketone oxidation metabolons, OXPHOS machinery, and nucleotide transporter hybrid-conformations, support mitochondrial remodeling in failing heart while bringing forth new hypotheses for pathological mechanisms. Application of quantitative cross-linking technology in tissue provides molecular-level insight to complex biological systems difficult to model in cell culture, thus providing a valuable resource for study of human diseases.

Molecular interactions between two different classes of β-lactamase enzymes and outer membrane protein A (OmpA) were studied by in vivo chemical cross-linking of a multi-drug-resistant strain of Acinetobacter baumannii AB5075. Class A β-lactamase blaGES-11 and Class D β-lactamase Oxa23, responsible for hydrolysis of different types of β-lactam antibiotics, were found to be cross-linked to similar lysine sites of the periplasmic domain of outer membrane protein OmpA, despite low sequence homology between the two enzymes. The findings from in vivo XL-MS suggest that the interacting surfaces between both β-lactamase enzymes and OmpA are conserved during molecular evolution, and the OmpA C-terminus domain serves an important function of anchoring different types of β-lactamase enzymes in the periplasmic space.

Chemical cross-linking and mass spectrometry is of growing use for establishment of distance constraints on protein conformations and interactions. Whereas intra-protein cross-links can arise from proteins in isolation, inter-protein cross-links reflect proximity of two interacting proteins in the sample. Prediction of expected ratios of the number of inter-protein to intra-protein cross-links is hindered by lacking comprehensive knowledge on the interactome network and global occupancy levels for all interacting complex subunits. Here we determine the theoretical number of possible inter- and intra-protein cross-links in available PDB structures of proteins bound in complexes to predict a maximum expected fraction of inter-protein cross-links in large scale in vivo cross-linking studies. We show how the maximum fraction can guide interpretation of reported inter-protein fractions with respect to the extent of sample protein binding, comparing whole cell and lysate cross-linked samples as an example. We also demonstrate how an observation of inter-protein cross-link fractions greater than the maximum value can result from the presence of false positive cross-links which are predominantly inter-protein, their number estimable from the observed surplus fraction of inter-protein cross-links.

PURPOSE/OBJECTIVE(S) The optimal use of stereotactic body radiotherapy (SBRT) for the diverse group of patients with gynecologic malignancies has not been clearly defined. This multi-institutional pooled analysis aims to characterize the use of SBRT for different primary disease types and indications within the gynecologic cancer population. MATERIALS/METHODS Patients treated with SBRT at three institutions with oligometastatic (OM) disease (5 or fewer sites), oligoprogressive (OP) disease (limited areas of progression with controlled systemic disease), or progression in an eloquent area were included. Descriptive statistics were used to report patient and treatment characteristics and toxicity. Local recurrence-free survival (LRFS), distant progression-free survival (DPFS), salvage chemotherapy-free survival (CFS, excluding patients on systemic therapy at time of SBRT) and overall survival (OS) probabilities following SBRT were calculated using Kaplan-Meier methods. Associations with disease and tumor factors were examined using Cox regression methods. RESULTS One hundred forty-four patients (144) with gynecologic cancers were treated with SBRT to 206 lesions. The median age was 61 (range 46-86) and median follow up was 16.6 months (range 0.5-106.8). Patients had cancers of the uterus (n = 101), ovary (n = 67), cervix (n = 27), and vulva or vagina (n = 8). Lesions were considered OM (n = 124), OP (n = 68), or progressive in an eloquent area (n = 14). Treatment was delivered to lesions in the pelvis (n = 50), lung (n = 40), para-aortic region (n = 37), abdomen or liver (n = 36), bone (n = 31), and distant nodes (n = 10). SBRT dose ranged from 20-60Gy (median 40Gy) in 2-5 fractions. Twenty-three (11.1%) lesions recurred locally after SBRT. Median LRFS was 23.9 months (95% CI 20.2-27.5), DPFS was 10.7 months (95% CI 10.7-16.9), and OS 22.9 months (95% CI 12.8-31.3). LRFS was 73.0% and 47.8%, DPFS was 44.2% and 19.6%, and OS was at 72.9% and 46.6% 1 and 2 years respectively. Twenty-nine percent of patients received salvage systemic therapy, and median CFS was 14.1 months (95% CI 10.4-17.8). On multi-variable analysis, only higher BED (OR 0.98, P = 0.013) was associated with LRFS. Primary disease type, tumor size, dose of radiotherapy, nodal versus non-nodal target, and DFI were not significantly associated with OS or DPFS on MVA. CONCLUSION This large cohort of patients receiving SBRT for oligometastatic and oligoprogressive gynecologic cancers had excellent tumor control, and promising DPFS and OS. Local control appears to be related to SBRT dose. SBRT may have the potential to delay time to initiation of chemotherapy or targeted therapies in select gynecologic cancer populations. Further prospective studies are required to determine which primary disease types benefit most from SBRT, and to establish the optimal sequencing of SBRT with other oncologic treatments.

A hallmark of impaired myocardial energetics in failing hearts is the downregulation of the creatine kinase (CK) system. In heart failure patients and animal models, myocardial phosphocreatine content and the flux of the CK reaction are negatively correlated with the outcome of heart failure. While decreased CK activity is highly reproducible in failing hearts, the underlying mechanisms remains elusive. Here, we report an inverse relationship between the activity and acetylation of CK muscle form (CKM) in human and mouse failing hearts. Hyperacetylation of recombinant CKM disrupted MM homodimer formation and reduced enzymatic activity, which could be reversed by sirtuin 2 treatment. Mass spectrometry analysis identified multiple lysine residues on the MM dimer interface, which were hyperacetylated in the failing hearts. Molecular modeling of CK MM homodimer suggested that hyperacetylation prevented dimer formation through interfering salt bridges within and between the 2 monomers. Deacetylation by sirtuin 2 reduced acetylation of the critical lysine residues, improved dimer formation, and restored CKM activity from failing heart tissue. These findings reveal a potentially novel mechanism in the regulation of CK activity and provide a potential target for improving high-energy phosphoryl transfer in heart failure.

To retrospectively analyse the impact of prophylactic cranial irradiation (PCI) on survival and intracranial progression in patients with limited stage small cell lung cancer (LS-SCLC) in the modern era of widespread magnetic resonance imaging brain screening.Patients with LS-SCLC treated within our network between 2009 and 2020 who responded to initial therapy were stratified by receipt of PCI and stage of disease. A propensity score match analysis was carried out for stage II-III patients. Overall and neurological survival were defined as time to death and presumed death due to uncontrolled intracranial disease, respectively. Brain metastasis-free survival and symptomatic brain metastasis-free survival were defined as freedom from intracranial progression and symptomatic intracranial progression, respectively. The effect of PCI on these outcomes was assessed using Kaplan-Meier and Cox proportional hazards models.In total, 243 (69.6%) of 349 patients received PCI. On multivariate analysis in the propensity matched stage II-III cohort, PCI was a significant predictor of improved neurological survival (hazard ratio 0.23, 95% confidence interval 0.08-0.65; P = 0.01), brain metastasis-free survival (hazard ratio 0.25, 95% confidence interval 0.12-0.51; P0.01) and symptomatic brain metastasis-free survival (hazard ratio 0.21, 95% confidence interval 0.08-0.55; P0.01), but not improved overall survival. Two-year neurological survival estimates within the propensity matched cohort were 96.8% (95% confidence interval 87.6-99.2%) with PCI and 77.2% (95% confidence interval 63.0-86.4%) without PCI and 1- and 2-year estimates of incidence of brain metastases were 3.9% (95% confidence interval 1.3-11.7%) and 11.7% (95% confidence interval 5.6-23.5%) in the PCI group and 31.6% (95% confidence interval 22.1-43.9%) and 40.4% (95% confidence interval 29.2-54.0%) in the no PCI group, respectively.In the modern era of magnetic resonance imaging screening, PCI was associated with reduced incidence of intracranial progression in patients with stage II-III LS-SCLC who respond to initial therapy. This, importantly, translated to a decreased risk of neurological death within our propensity matched cohort, without significant improvement in overall survival.