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Article ; Online: Advances in direct detection of lysine methylation and acetylation by nuclear magnetic resonance using

Fraser, Olivia A / Namitz, Kevin E W / Showalter, Scott A

2023 Volume 218, Page(s) 72–83

Abstract: Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of ... ...

Abstract	Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [
MeSH term(s)	Methylation ; Acetylation ; Lysine/metabolism ; Protein Processing, Post-Translational ; Magnetic Resonance Spectroscopy
Chemical Substances	Lysine (K3Z4F929H6)
Language	English
Publishing date	2023-07-29
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Research Support, N.I.H., Extramural
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2023.07.010
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Article ; Online: Phase separation promotes a highly active oligomeric scaffold of the MLL1 core complex for regulation of histone H3K4 methylation.

Namitz, Kevin E W / Showalter, Scott A / Cosgrove, Michael S

The Journal of biological chemistry

2023 Volume 299, Issue 10, Page(s) 105204

Abstract: Enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation are crucial for proper cellular differentiation and are frequently mutated in cancer. The Mixed lineage leukemia (MLL) family of enzymes deposit H3K4 mono-, di-, or trimethylation ...

Abstract	Enzymes that regulate the degree of histone H3 lysine 4 (H3K4) methylation are crucial for proper cellular differentiation and are frequently mutated in cancer. The Mixed lineage leukemia (MLL) family of enzymes deposit H3K4 mono-, di-, or trimethylation at distinct genomic locations, requiring precise spatial and temporal control. Despite evidence that the degree of H3K4 methylation is controlled in part by a hierarchical assembly pathway with key subcomplex components, we previously found that the assembled state of the MLL1 core complex is not favored at physiological temperature. To better understand this paradox, we tested the hypothesis that increasing the concentration of subunits in a biomolecular condensate overcomes this thermodynamic barrier via mass action. Here, we demonstrate that MLL1 core complex phase separation stimulates enzymatic activity up to 60-fold but not primarily by concentrating subunits into droplets. Instead, we found that stimulated activity is largely due to the formation of an altered oligomeric scaffold that greatly reduces substrate K
MeSH term(s)	Humans ; Histones/metabolism ; Methylation ; Myeloid-Lymphoid Leukemia Protein/metabolism ; Phase Separation ; Protein Subunits/chemistry ; Protein Subunits/metabolism ; Protein Structure, Quaternary ; Models, Molecular ; Thermodynamics ; Enzyme Activation
Chemical Substances	Histones ; Myeloid-Lymphoid Leukemia Protein (149025-06-9) ; Protein Subunits ; KMT2A protein, human
Language	English
Publishing date	2023-09-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2023.105204
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Article ; Online: Transient Electrostatic Interactions between Fcp1 and Rap74 Bias the Conformational Ensemble of the Complex with Minimal Impact on Binding Affinity.

Prieto, Victor A / Namitz, Kevin E W / Showalter, Scott A

The journal of physical chemistry. B

2021 Volume 125, Issue 39, Page(s) 10917–10927

Abstract: Intrinsically disordered protein (IDP) sequences often contain a high proportion of charged residues in conjunction with their high degree of hydrophilicity and solvation. For high net charge IDPs, long-range electrostatic interactions are thought to ... ...

Abstract	Intrinsically disordered protein (IDP) sequences often contain a high proportion of charged residues in conjunction with their high degree of hydrophilicity and solvation. For high net charge IDPs, long-range electrostatic interactions are thought to play a role in modulating the strength or kinetics of protein-protein interactions. In this work, we examined intramolecular interactions mediated by charged regions of a model IDP, the C-terminal tail of the phosphatase Fcp1. Specifically, this work focuses on intermolecular interactions between acidic and basic patches in the primary structure of Fcp1 and their contributions to binding its predominantly basic partner, the winged helix domain of Rap74. We observe both intramolecular and intermolecular interactions through paramagnetic relaxation enhancement (PRE) consistent with oppositely charged regions associating with one another, both in unbound Fcp1 and in the Fcp1-Rap74 complex. Formation of this complex is strongly driven by hydrophobic interactions in the minimal binding motif. Here, we test the hypothesis that charged residues in Fcp1 that flank the binding helix also contribute to the strength of binding. Charge inversion mutations in Fcp1 generally support this hypothesis, while PRE data suggest substitution of observed transient interactions in the unbound ensemble for similarly transient interactions with Rap74 in the complex.
MeSH term(s)	Phosphoprotein Phosphatases/metabolism ; Protein Binding ; Protein Structure, Secondary ; Static Electricity ; Transcription Factors, TFII/metabolism
Chemical Substances	Transcription Factors, TFII ; Phosphoprotein Phosphatases (EC 3.1.3.16)
Language	English
Publishing date	2021-09-22
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1520-5207
ISSN (online)	1520-5207
DOI	10.1021/acs.jpcb.1c05131
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Article ; Online: Long-Term Adherence to Adjuvant Endocrine Therapy Following Various Radiotherapy Modalities in Early Stage Hormone Receptor Positive Breast Cancer.

Ward, Kristin A / Muller, Donald A / Dutta, Sunil W / Malhi, Jasmine / Sanders, Jason C / Luminais, Christopher K / Millard, Trish A / Showalter, Timothy N / Showalter, Shayna L / Janowski, Einsley-Marie

Clinical breast cancer

2023 Volume 23, Issue 4, Page(s) 369–377

Abstract: Introduction: We compared the rates of long-term adjuvant endocrine therapy (AET) adherence after various radiation therapy (RT) modalities among patients with early stage breast cancer.: Materials and methods: Medical records from patients with ... ...

Abstract	Introduction: We compared the rates of long-term adjuvant endocrine therapy (AET) adherence after various radiation therapy (RT) modalities among patients with early stage breast cancer. Materials and methods: Medical records from patients with stage 0, I, or IIA (tumors ≤3 cm), hormone receptor (HR) positive breast cancer that received adjuvant radiation therapy (RT) from 2013 to 2015 at a single institution were retrospectively reviewed. All patients received breast conserving surgery (BCS) followed by adjuvant RT via one of the following modalities: whole breast radiotherapy (WBI), partial breast irradiation (PBI) with either external beam radiation therapy (EBRT) or fractionated intracavitary high-dose rate (HDR) brachytherapy, or single fraction HDR-brachytherapy intraoperative-radiation therapy (IORT). Results: One hundred fourteen patients were reviewed. Thirty patients received WBI, 41 PBI, and 43 IORT with a median follow up of 64.2, 72.0, and 58.6 months, respectively. For the entire cohort, AET adherence was approximately 64% at 2 years and 56% at 5 years. Among patients in the IORT clinical trial, adherence to AET was approximately 51% at 2 years and 40% at 5 years. After controlling for additional factors, DCIS histology (vs invasive disease) and IORT (compared to other radiation modalities) were associated with decreased endocrine therapy adherence (P < 0.05). Conclusion: DCIS histology and receipt of IORT were associated with lower rates of adherence to AET at 5 years. Our findings suggest that examination of the efficacy of RT interventions such as PBI and IORT in patients who do not receive AET is warranted.
MeSH term(s)	Humans ; Female ; Breast Neoplasms/radiotherapy ; Breast Neoplasms/pathology ; Carcinoma, Intraductal, Noninfiltrating/pathology ; Retrospective Studies ; Treatment Outcome ; Breast/pathology ; Mastectomy, Segmental ; Radiotherapy, Adjuvant
Language	English
Publishing date	2023-02-01
Publishing country	United States
Document type	Journal Article
ZDB-ID	2106734-X
ISSN	1938-0666 ; 1526-8209
ISSN (online)	1938-0666
ISSN	1526-8209
DOI	10.1016/j.clbc.2023.01.012
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Article ; Online: Stereotactic body radiation therapy induced myonecrosis in a patient with prior gemcitabine administered for leiomyosarcoma.

Facer, Benjin D / Dutta, Sunil W / Showalter, Timothy N

Journal of radiosurgery and SBRT

2020 Volume 7, Issue 1, Page(s) 77–80

Abstract: We present the case of radiation myonecrosis of the iliopsoas muscle, identified five months after stereotactic body radiation therapy (SBRT, 21 Gy in three fractions) to a metastatic lesion in the right iliac bone of a patient with leiomyosarcoma. The ... ...

Abstract	We present the case of radiation myonecrosis of the iliopsoas muscle, identified five months after stereotactic body radiation therapy (SBRT, 21 Gy in three fractions) to a metastatic lesion in the right iliac bone of a patient with leiomyosarcoma. The patient had been treated with various chemotherapeutic agents, most notably docetaxel and gemcitabine for five cycles 10 months prior to SBRT. As skeletal muscle is a radio-resistant organ, myonecrosis is rare, but previous case reports suggest that the administration of gemcitabine may increase the likelihood of radiation toxicity, including radiation myonecrosis. Physicians may consider conventional fractionation, rather than a hypofractionated course, in patients who have received or will receive gemcitabine.
Language	English
Publishing date	2020-07-17
Publishing country	United States
Document type	Journal Article
ISSN	2156-4647
ISSN (online)	2156-4647
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Article ; Online: Low-temperature chemotaxis, halotaxis and chemohalotaxis by the psychrophilic marine bacterium Colwellia psychrerythraea 34H.

Showalter, G M / Deming, J W

Environmental microbiology reports

2017 Volume 10, Issue 1, Page(s) 92–101

Abstract: A variety of ecologically important processes are driven by bacterial motility and taxis, yet these basic bacterial behaviours remain understudied in cold habitats. Here, we present a series of experiments designed to test the chemotactic ability of the ... ...

Abstract	A variety of ecologically important processes are driven by bacterial motility and taxis, yet these basic bacterial behaviours remain understudied in cold habitats. Here, we present a series of experiments designed to test the chemotactic ability of the model marine psychrophilic bacterium Colwellia psychrerythraea 34H, when grown at optimal temperature and salinity (8°C, 35 ppt) or its original isolation conditions (-1°C, 35 ppt), towards serine and mannose at temperatures from -8°C to 27°C (above its upper growth temperature of 18°C), and at salinities of 15, 35 and 55 ppt (at 8°C and -1°C). Results indicate that C. psychrerythraea 34H is capable of chemotaxis at all temperatures tested, with strongest chemotaxis at the temperature at which it was first grown, whether 8°C or -1°C. This model marine psychrophile also showed significant halotaxis towards 15 and 55 ppt solutions, as well as strong substrate-specific chemohalotaxis. We suggest that such patterns of taxis may enable bacteria to colonize sea ice, position themselves optimally within its extremely cold, hypersaline and temporally fluctuating microenvironments, and respond to various chemical signals therein.
MeSH term(s)	Adaptation, Physiological ; Alteromonadaceae/physiology ; Aquatic Organisms/physiology ; Chemotaxis/physiology ; Cold Temperature ; Mannose/metabolism ; Models, Biological ; Salinity ; Seawater/microbiology ; Serine/metabolism ; Substrate Specificity
Chemical Substances	Serine (452VLY9402) ; Mannose (PHA4727WTP)
Language	English
Publishing date	2017-12-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1758-2229
ISSN (online)	1758-2229
DOI	10.1111/1758-2229.12610
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Article ; Online: A comparative study using time-driven activity-based costing in single-fraction breast high-dose rate brachytherapy: An integrated brachytherapy suite vs. decentralized workflow.

Squeo, Gabriella C / Lattimore, Courtney M / Simone, Nicole L / Suralik, Greg / Dutta, Sunil W / Schad, Michael D / Su, Lucy / Libby, Bruce / Janowski, Einsley-Marie / Showalter, Shayna L / Lobo, Jennifer M / Showalter, Timothy N

Brachytherapy

2022 Volume 21, Issue 3, Page(s) 334–340

Abstract: Introduction: Precision breast intraoperative radiation therapy (PB-IORT) is a novel approach to adjuvant radiation therapy for early-stage breast cancer performed as part of a phase II clinical trial at two institutions. One institution performs the ... ...

Abstract	Introduction: Precision breast intraoperative radiation therapy (PB-IORT) is a novel approach to adjuvant radiation therapy for early-stage breast cancer performed as part of a phase II clinical trial at two institutions. One institution performs the entire procedure in an integrated brachytherapy suite which contains a CT-on-rails imaging unit and full anesthesia capabilities. At the other, breast conserving surgery and radiation therapy take place in two separate locations. Here, we utilize time-driven activity-based costing (TDABC) to compare these two models for the delivery of PB-IORT. Methods: Process maps were created to describe each step required to deliver PB-IORT at each institution, including personnel, equipment, and supplies. Time investment was estimated for each step. The capacity cost rate was determined for each resource, and total costs of care were then calculated by multiplying the capacity cost rates by the time estimate for the process step and adding any additional product costs. Results: PB-IORT costs less to deliver at a distributed facility, as is more commonly available, than an integrated brachytherapy suite ($3,262.22 vs. $3,996.01). The largest source of costs in both settings ($2,400) was consumable supplies, including the brachytherapy balloon applicator. The difference in costs for the two facility types was driven by personnel costs ($1,263.41 vs. $764.89). In the integrated facility, increased time required by radiation oncology nursing and the anesthesia attending translated to the greatest increases in cost. Equipment costs were also slightly higher in the integrated suite setting ($332.60 vs. $97.33). Conclusions: The overall cost of care is higher when utilizing an integrated brachytherapy suite to deliver PB-IORT. This was primarily driven by additional personnel costs from nursing and anesthesia, although the greatest cost of delivery in both settings was the disposable brachytherapy applicator. These differences in cost must be balanced against the potential impact on patient experience with these approaches.
MeSH term(s)	Brachytherapy/methods ; Breast Neoplasms/radiotherapy ; Breast Neoplasms/surgery ; Female ; Humans ; Mastectomy, Segmental ; Workflow
Language	English
Publishing date	2022-02-04
Publishing country	United States
Document type	Clinical Trial, Phase II ; Journal Article ; Multicenter Study ; Research Support, N.I.H., Extramural
ZDB-ID	2098608-7
ISSN	1873-1449 ; 1538-4721
ISSN (online)	1873-1449
ISSN	1538-4721
DOI	10.1016/j.brachy.2021.12.006
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Article ; Online: Advances in direct detection of lysine methylation and acetylation by nuclear magnetic resonance using 13C-enriched cofactors

Fraser, Olivia A. / Namitz, Kevin E.W. / Showalter, Scott A.

Methods. 2023 Oct., v. 218 p.72-83

2023

Abstract	Post-translational modifications (PTMs) are reversible chemical modifications that can modulate protein structure and function. Methylation and acetylation are two such PTMs with integral and well-characterized biological roles, including modulation of chromatin structure; and unknown or poorly understood roles, exemplified by the influence of these PTMs on transcription factor structure and function. The need for biological insights into the function of these PTMs motivates the development of a nondestructive and label-free method that enables pursuit of molecular mechanisms. Here, we present a protocol for implementing nuclear magnetic resonance (NMR) methods that allow for unambiguous detection of methylation and acetylation events and demonstrate their utility by observing these marks on histone H3 tail as a model system. We leverage strategic isotopic enrichment of cofactor and peptide for visualization by [¹H, ¹³C]-HSQC and ¹³C direct-detect NMR measurements. Finally, we present ¹³C-labeling schemes that facilitate one-dimensional NMR experiments, which combine reduced measurement time relative to two-dimensional spectroscopy with robust filtering of background signals that would otherwise create spectral crowding or limit detection of low-abundance analytes.
Keywords	acetylation ; chemical species ; chromatin ; histones ; isotopic enrichment ; lysine ; methylation ; nuclear magnetic resonance spectroscopy ; peptides ; protein structure ; transcription factors ; Nuclear magnetic resonance ; Histone ; Post-translational modification
Language	English
Dates of publication	2023-10
Size	p. 72-83.
Publishing place	Elsevier Inc.
Document type	Article ; Online
ZDB-ID	1066584-5
ISSN	1095-9130 ; 1046-2023
ISSN (online)	1095-9130
ISSN	1046-2023
DOI	10.1016/j.ymeth.2023.07.010
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Probing multiple enzymatic methylation events in real time with NMR spectroscopy.

Usher, Emery T / Namitz, Kevin E W / Cosgrove, Michael S / Showalter, Scott A

Biophysical journal

2021 Volume 120, Issue 21, Page(s) 4710–4721

Abstract: Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a ... ...

Abstract	Post-translational modification (PTM) of proteins is of critical importance to the regulation of many cellular processes in eukaryotic organisms. One of the most well-studied protein PTMs is methylation, wherein an enzyme catalyzes the transfer of a methyl group from a cofactor to a lysine or arginine side chain. Lysine methylation is especially abundant in the histone tails and is an important marker for denoting active or repressed genes. Given their relevance to transcriptional regulation, the study of methyltransferase function through in vitro experiments is an important stepping stone toward understanding the complex mechanisms of regulated gene expression. To date, most methyltransferase characterization strategies rely on the use of radioactive cofactors, detection of a methyl transfer byproduct, or discontinuous-type assays. Although such methods are suitable for some applications, information about multiple methylation events and kinetic intermediates is often lost. Herein, we describe the use of two-dimensional NMR to monitor mono-, di-, and trimethylation in a single reaction tube. To do so, we incorporated
MeSH term(s)	Histone-Lysine N-Methyltransferase/genetics ; Histone-Lysine N-Methyltransferase/metabolism ; Histones/metabolism ; Magnetic Resonance Spectroscopy ; Methylation ; Protein Processing, Post-Translational
Chemical Substances	Histones ; Histone-Lysine N-Methyltransferase (EC 2.1.1.43)
Language	English
Publishing date	2021-09-28
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2021.09.034
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Article ; Online: Protons versus photons for the treatment of chordoma.

El Sayed, Iman / Trifiletti, Daniel M / Lehrer, Eric J / Showalter, Timothy N / Dutta, Sunil W

The Cochrane database of systematic reviews

2021 Volume 7, Page(s) CD013224

Abstract: Background: Chordoma is a rare primary bone tumour with a high propensity for local recurrence. Surgical resection is the mainstay of treatment, but complete resection is often morbid due to tumour location. Similarly, the dose of radiotherapy (RT) that ...

Abstract	Background: Chordoma is a rare primary bone tumour with a high propensity for local recurrence. Surgical resection is the mainstay of treatment, but complete resection is often morbid due to tumour location. Similarly, the dose of radiotherapy (RT) that surrounding healthy organs can tolerate is frequently below that required to provide effective tumour control. Therefore, clinicians have investigated different radiation delivery techniques, often in combination with surgery, aimed to improve the therapeutic ratio. Objectives: To assess the effects and toxicity of proton and photon adjuvant radiotherapy (RT) in people with biopsy-confirmed chordoma. Search methods: We searched CENTRAL (2021, Issue 4); MEDLINE Ovid (1946 to April 2021); Embase Ovid (1980 to April 2021) and online registers of clinical trials, and abstracts of scientific meetings up until April 2021. Selection criteria: We included adults with pathologically confirmed primary chordoma, who were irradiated with curative intent, with protons or photons in the form of fractionated RT, SRS (stereotactic radiosurgery), SBRT (stereotactic body radiotherapy), or IMRT (intensity modulated radiation therapy). We limited analysis to studies that included outcomes of participants treated with both protons and photons. Data collection and analysis: The primary outcomes were local control, mortality, recurrence, and treatment-related toxicity. We followed current standard Cochrane methodological procedures for data extraction, management, and analysis. We used the ROBINS-I tool to assess risk of bias, and GRADE to assess the certainty of the evidence. Main results: We included six observational studies with 187 adult participants. We judged all studies to be at high risk of bias. Four studies were included in meta-analysis. We are uncertain if proton compared to photon therapy worsens or has no effect on local control (hazard ratio (HR) 5.34, 95% confidence interval (CI) 0.66 to 43.43; 2 observational studies, 39 participants; very low-certainty evidence). Median survival time ranged between 45.5 months and 66 months. We are uncertain if proton compared to photon therapy reduces or has no effect on mortality (HR 0.44, 95% CI 0.13 to 1.57; 4 observational studies, 65 participants; very low-certainty evidence). Median recurrence-free survival ranged between 3 and 10 years. We are uncertain whether proton compared to photon therapy reduces or has no effect on recurrence (HR 0.34, 95% CI 0.10 to 1.17; 4 observational studies, 94 participants; very low-certainty evidence). One study assessed treatment-related toxicity and reported that four participants on proton therapy developed radiation-induced necrosis in the temporal bone, radiation-induced damage to the brainstem, and chronic mastoiditis; one participant on photon therapy developed hearing loss, worsening of the seventh cranial nerve paresis, and ulcerative keratitis (risk ratio (RR) 1.28, 95% CI 0.17 to 9.86; 1 observational study, 33 participants; very low-certainty evidence). There is no evidence that protons led to reduced toxicity. There is very low-certainty evidence to show an advantage for proton therapy in comparison to photon therapy with respect to local control, mortality, recurrence, and treatment related toxicity. Authors' conclusions: There is a lack of published evidence to confirm a clinical difference in effect with either proton or photon therapy for the treatment of chordoma. As radiation techniques evolve, multi-institutional data should be collected prospectively and published, to help identify persons that would most benefit from the available radiation treatment techniques.
MeSH term(s)	Adult ; Bias ; Bone Neoplasms/mortality ; Bone Neoplasms/radiotherapy ; Chordoma/mortality ; Chordoma/radiotherapy ; Disease-Free Survival ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Recurrence, Local/prevention & control ; Observational Studies as Topic ; Photons/adverse effects ; Photons/therapeutic use ; Progression-Free Survival ; Proton Therapy/adverse effects ; Proton Therapy/methods ; Radiosurgery/methods ; Radiotherapy, Adjuvant ; Radiotherapy, Intensity-Modulated/methods ; Time Factors
Language	English
Publishing date	2021-07-01
Publishing country	England
Document type	Journal Article ; Meta-Analysis ; Systematic Review
ISSN	1469-493X
ISSN (online)	1469-493X
DOI	10.1002/14651858.CD013224.pub2
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