Purpose
This report presents the methods and results of the Thoracic Auto‐Segmentation Challenge organized at the 2017 Annual Meeting of American Association of Physicists in Medicine. The purpose of ...the challenge was to provide a benchmark dataset and platform for evaluating performance of autosegmentation methods of organs at risk (OARs) in thoracic CT images.
Methods
Sixty thoracic CT scans provided by three different institutions were separated into 36 training, 12 offline testing, and 12 online testing scans. Eleven participants completed the offline challenge, and seven completed the online challenge. The OARs were left and right lungs, heart, esophagus, and spinal cord. Clinical contours used for treatment planning were quality checked and edited to adhere to the RTOG 1106 contouring guidelines. Algorithms were evaluated using the Dice coefficient, Hausdorff distance, and mean surface distance. A consolidated score was computed by normalizing the metrics against interrater variability and averaging over all patients and structures.
Results
The interrater study revealed highest variability in Dice for the esophagus and spinal cord, and in surface distances for lungs and heart. Five out of seven algorithms that participated in the online challenge employed deep‐learning methods. Although the top three participants using deep learning produced the best segmentation for all structures, there was no significant difference in the performance among them. The fourth place participant used a multi‐atlas‐based approach. The highest Dice scores were produced for lungs, with averages ranging from 0.95 to 0.98, while the lowest Dice scores were produced for esophagus, with a range of 0.55–0.72.
Conclusion
The results of the challenge showed that the lungs and heart can be segmented fairly accurately by various algorithms, while deep‐learning methods performed better on the esophagus. Our dataset together with the manual contours for all training cases continues to be available publicly as an ongoing benchmarking resource.
Top-down proteomics has improved over the past decade despite the significant challenges presented by the analysis of large protein ions. Here, the detection of these high mass species by ...electrospray-based mass spectrometry (MS) is examined from a theoretical perspective to understand the mass-dependent increases in the number of charge states, isotopic peaks, and interfering species present in typical protein mass spectra. Integrating these effects into a quantitative model captures the reduced ability to detect species over 25 kDa with the speed and sensitivity characteristic of proteomics based on <3 kDa peptide ions. The model quantifies the challenge that top-down proteomics faces with respect to current MS instrumentation and projects that depletion of 13C and 15N isotopes can improve detection at high mass by only <2-fold at 100 kDa whereas the effect is up to 5-fold at 10 kDa. Further, we find that supercharging electrosprayed proteins to the point of producing <5 charge states at high mass would improve detection by more than 20-fold.
A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved ...digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.
The use of stereotactic body radiotherapy (SBRT) for early-stage primary non-small cell lung cancer (NSCLC) reported excellent local control rates. But the optimal SBRT dose for oligometastatic lung ...tumors (OLTs) from colorectal cancer (CRC) has not yet been determined. This study aimed to evaluate whether SBRT to a dose of 48-60 Gy in 4-5 fractions could result in similar local outcomes for OLTs from CRC as compared to early-stage NSCLC, and to examine potential dose-response relationships for OLTs from CRC.
OLTs from CRC and primary NSCLCs treated with SBRT to 48-60 Gy in 4-5 fractions at a single institution were evaluated, and a matched-pair analysis was performed. Local recurrence-free survival (LRFS) was estimated by the Kaplan-Meier method. Univariate Cox regression was performed to identify significant predictors.
There were 72 lung lesions in 61 patients (24 OLTs from CRC in 15 patients and 48 NSCLCs in 46 patients) were analyzed with a median follow-up of 30 months. LRFS for OLTs from CRC was significantly worse than that of NSCLC when treated with 48-60 Gy/4-5 fx (p = 0.006). The 1, 3 and 5-year LRFS of OLTs from CRC vs NSCLC were 80.6% vs. 100%, 68.6% vs. 97.2%, and 68.6% vs. 81.0%, respectively. On univariate analysis, OLTs from CRC treated with higher dose (BED
= 132 Gy) exhibited significantly better local recurrence-free survival than those treated to lower doses (BED
≤ 105.6 Gy) (p = 0.0022). The 1 and 3-year LRFS rates for OLTs treated to a higher dose (BED
= 132 Gy) were 88.9% and 81.5%, vs 33.3%, and not achieved for lower doses (BED
≤ 105.6 Gy).
The LRFS of OLTs from CRC after SBRT of 48-60 Gy/4-5 fx was significantly worse than that of primary NSCLC. Lower dose SBRT appeared to have inferior control for OLTs of CRC in this cohort. Further studies with larger sample sizes are needed.
Protein lysine acetylation (LysAc) has recently been demonstrated to be widespread in E. coli and Salmonella, and to broadly regulate bacterial physiology and metabolism. However, LysAc in plant ...pathogenic bacteria is largely unknown. Here we first report the lysine acetylome of Erwinia amylovora, an enterobacterium causing serious fire blight disease of apples and pears. Immunoblots using generic anti-lysine acetylation antibodies demonstrated that growth conditions strongly affected the LysAc profiles in E. amylovora. Differential LysAc profiles were also observed for two E. amylovora strains, known to have differential virulence in plants, indicating translational modification of proteins may be important in determining virulence of bacterial strains. Proteomic analysis of LysAc in two E. amylovora strains identified 141 LysAc sites in 96 proteins that function in a wide range of biological pathways. Consistent with previous reports, 44% of the proteins are involved in metabolic processes, including central metabolism, lipopolysaccharide, nucleotide and amino acid metabolism. Interestingly, for the first time, several proteins involved in E. amylovora virulence, including exopolysaccharide amylovoran biosynthesis- and type III secretion-associated proteins, were found to be lysine acetylated, suggesting that LysAc may play a major role in bacterial virulence. Comparative analysis of LysAc sites in E. amylovora and E. coli further revealed the sequence and structural commonality for LysAc in the two organisms. Collectively, these results reinforce the notion that LysAc of proteins is widespread in bacterial metabolism and virulence.
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► Growth conditions dramatically altered LysAc profiles. ► Differential LysAc profiles were observed for two Erwinia amylovora strains with differential virulence. ► For the first time, virulence-related T3SS and EPS proteins were found to be lys-acetylated in bacteria. ► Conserved residues specific to protein structures were LysAc. ► Consistent with previous findings, 44% LysAc proteins were involved in metabolism.
In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations ...in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, 10 endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.
Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome ...sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a “protein-first” method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach ( Nat. Biotechnol. 2009, 27, 951−956 ), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously “orphan” gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2–4 Mbp genomes) used previously.
Applying high-throughput Top-Down MS to an entire proteome requires a yet-to-be-established model for data processing. Since Top-Down is becoming possible on a large scale, we report our latest ...software pipeline dedicated to capturing the full value of intact protein data in automated fashion. For intact mass detection, we combine algorithms for processing MS1 data from both isotopically resolved (FT) and charge-state resolved (ion trap) LC-MS data, which are then linked to their fragment ions for database searching using ProSight. Automated determination of human keratin and tubulin isoforms is one result. Optimized for the intricacies of whole proteins, new software modules visualize proteome-scale data based on the LC retention time and intensity of intact masses and enable selective detection of PTMs to automatically screen for acetylation, phosphorylation, and methylation. Software functionality was demonstrated using comparative LC-MS data from yeast strains in addition to human cells undergoing chemical stress. We further these advances as a key aspect of realizing Top-Down MS on a proteomic scale.
Understanding how a small brain region, the suprachiasmatic nucleus (SCN), can synchronize the body's circadian rhythms is an ongoing research area. This important time-keeping system requires a ...complex suite of peptide hormones and transmitters that remain incompletely characterized. Here, capillary liquid chromatography and FTMS have been coupled with tailored software for the analysis of endogenous peptides present in the SCN of the rat brain. After ex vivo processing of brain slices, peptide extraction, identification, and characterization from tandem FTMS data with <5-ppm mass accuracy produced a hyperconfident list of 102 endogenous peptides, including 33 previously unidentified peptides, and 12 peptides that were post-translationally modified with amidation, phosphorylation, pyroglutamylation, or acetylation. This characterization of endogenous peptides from the SCN will aid in understanding the molecular mechanisms that mediate rhythmic behaviors in mammals.
The multiple myeloma SET domain (MMSET) protein is overexpressed in multiple myeloma (MM) patients with the translocation t(4;14). Although studies have shown the involvement of MMSET/Wolf-Hirschhorn ...syndrome candidate 1 in development, its mode of action in the pathogenesis of MM is largely unknown. We found that MMSET is a major regulator of chromatin structure and transcription in t(4;14) MM cells. High levels of MMSET correlate with an increase in lysine 36 methylation of histone H3 and a decrease in lysine 27 methylation across the genome, leading to a more open structural state of the chromatin. Loss of MMSET expression alters adhesion properties, suppresses growth, and induces apoptosis in MM cells. Consequently, genes affected by high levels of MMSET are implicated in the p53 pathway, cell cycle regulation, and integrin signaling. Regulation of many of these genes required functional histone methyl-transferase activity of MMSET. These results implicate MMSET as a major epigenetic regulator in t(4;14)+ MM.
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