Problems, principles and progress in computational annotation of NMR metabolomics data.

BACKGROUND: Compound identification remains a critical bottleneck in the process of exploiting Nuclear Magnetic Resonance (NMR) metabolomics data, especially for 1H 1-dimensional (1H 1D) data. As databases of reference compound spectra have grown, workflows have evolved to rely heavily on their search functions to facilitate this process by generating lists of potential metabolites found in complex mixture data, facilitating annotation and identification. However, approaches for validating and communicating annotations are most often guided by expert knowledge, and therefore are highly variable despite repeated efforts to align practices and define community standards. AIM OF REVIEW: This review is aimed at broadening the application of automated annotation tools by discussing the key ideas of spectral matching and beginning to describe a set of terms to classify this information, thus advancing standards for communicating annotation confidence. Additionally, we hope that this review will facilitate the growing collaboration between chemical data scientists, software developers and the NMR metabolomics community aiding development of long-term software solutions. KEY SCIENTIFIC CONCEPTS OF REVIEW: We begin with a brief discussion of the typical untargeted NMR identification workflow. We differentiate between annotation (hypothesis generation, filtering), and identification (hypothesis testing, verification), and note the utility of different NMR data features for annotation. We then touch on three parts of annotation: (1) generation of queries, (2) matching queries to reference data, and (3) scoring and confidence estimation of potential matches for verification. In doing so, we highlight existing approaches to automated and semi-automated annotation from the perspective of the structural information they utilize, as well as how this information can be represented computationally.

Impact of prescription isodose level and collimator selection on dose homogeneity and plan quality in robotic radiosurgery.

PURPOSE: In stereotactic radiosurgery (SRS), prescription isodoses and resulting dose homogeneities vary widely across different platforms and clinical entities. Our goal was to investigate the physical limitations of generating dose distributions with an intended level of homogeneity in robotic SRS. METHODS: Treatment plans for non-isocentric irradiation of 4 spherical phantom targets (volume 0.27-7.70 ml) and 4 clinical targets (volume 0.50-5.70 ml) were calculated using Sequential (phantom) or VOLOTM (clinical) optimizers (Accuray, Sunnyvale, CA, USA). Dose conformity, volume of 12 Gy isodose (V12Gy) as a measure for dose gradient, and treatment time were recorded for different prescribed isodose levels (PILs) and collimator settings. In addition, isocentric irradiation of phantom targets was examined, with dose homogeneity modified by using different collimator sizes. RESULTS: Dose conformity was generally high (nCI ≤ 1.25) and varied little with PIL. For all targets and collimator sets, V12Gy was highest for PIL ≥ 80% and lowest for PIL ≤ 65%. The impact of PIL on V12Gy was highest for isocentric irradiation and lowest for clinical targets (VOLOTM optimization). The variability of V12Gy as a function of collimator selection was significantly higher than that of PIL. V12Gy and treatment time were negatively correlated. Plans utilizing a single collimator with a diameter in the range of 70-80% of the target diameter were fastest, but showed the strongest dependence on PIL. CONCLUSION: Inhomogeneous dose distributions with PIL ≤ 70% can be used to minimize dose to normal tissue. PIL ≥ 90% is associated with a marked and significant increase in off-target dose exposure. Careful selection of collimators during planning is even more important.

RTExtract: time-series NMR spectra quantification based on 3D surface ridge tracking.

MOTIVATION: Time-series nuclear magnetic resonance (NMR) has advanced our knowledge about metabolic dynamics. Before analyzing compounds through modeling or statistical methods, chemical features need to be tracked and quantified. However, because of peak overlap and peak shifting, the available protocols are time consuming at best or even impossible for some regions in NMR spectra. RESULTS: We introduce Ridge Tracking-based Extract (RTExtract), a computer vision-based algorithm, to quantify time-series NMR spectra. The NMR spectra of multiple time points were formulated as a 3D surface. Candidate points were first filtered using local curvature and optima, then connected into ridges by a greedy algorithm. Interactive steps were implemented to refine results. Among 173 simulated ridges, 115 can be tracked (RMSD < 0.001). For reproducing previous results, RTExtract took less than 2 h instead of ∼48 h, and two instead of seven parameters need tuning. Multiple regions with overlapping and changing chemical shifts are accurately tracked. AVAILABILITY AND IMPLEMENTATION: Source code is freely available within Metabolomics toolbox GitHub repository (https://github.com/artedison/Edison_Lab_Shared_Metabolomics_UGA/tree/master/metabolomics_toolbox/code/ridge_tracking) and is implemented in MATLAB and R. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Intertidal habitat complexity influences the density of the non-native crab Hemigrapsus sanguineus.

Habitat structural complexity can provide protection from predators, potentially affecting population density of native and non-native prey. The invasive Asian shore crab, Hemigrapsus sanguineus, occurs in variable densities in the rocky intertidal zone of eastern North America and northern Europe, often in densities greater than in its native range. The present study examined the influence of habitat complexity on the density of H. sanguineus. Artificial shelters of concrete pavers with stones arranged in increasing complexity were deployed in the intertidal zone along a rocky shore in southeastern Massachusetts, USA, for 21 consecutive weekly intervals in 2020. Crabs consistently reached the highest densities in the most complex shelters despite their lower internal surface area. In addition, crabs exhibited shelter selectivity based on body size, with large crabs occupying artificial shelters in greater numbers than adjacent natural substrate. In a subsequent lab study, crab activity over 1 h was observed in the presence of the same artificial shelters, under simulated tidal conditions. Shelter complexity had little influence on the number of crabs under the pavers although crabs were more active when submerged in water than exposed to air. These results show that crab density increases as habitat complexity increases, and complexity may serve as a predictor of H. sanguineus density but not short-term behavior.

Efficacy and Toxicity of Robotic Stereotactic Body Radiotherapy of Lung Metastases in Patients With Oligometastatic Disease.

BACKGROUND: Stereotactic body radiotherapy is a locally effective treatment for lung metastases in patients with oligometastatic disease, a modern variant of which is robotic (rSBRT). Since it is unclear which factors determine the success of rSBRT, we investigated a cohort of patients with lung metastases treated with rSBRT. PATIENTS AND METHODS: In our retrospective single-center analysis, we included patients with oligometastatic disease of different cancer types who underwent SBRT of lung metastases using an Accuray Cyberknife® device between 2012 and 2019. We evaluated local control rate (LC), progression-free (PFS) and overall (OS) survival, and toxicity. Multivariate analysis was performed to identify independent factors associated with the efficacy and toxicity of rSBRT. RESULTS: A total of 70 lung metastases of 54 patients were evaluated. The 4-year Kaplan-Meier estimate for LC, PFS and OS were 72.0%, 12.4% and 49.7%, respectively. Cox regression showed that LC of metastases of colorectal carcinoma and metastases treated with a biological effective dose at an α/ß-ratio of 10 (BED10) of <100 Gy was significantly worse than for other metastases. Patients suffered from grade I-II pneumonitis in 21.4% of cases treated with rSBRT (grade I: 20.0%; grade II: 1.4%). CONCLUSION: rSBRT is an effective and safe therapy for lung metastases. A BED10 of >100 Gy should be aimed for, especially for potentially radioresistant histologies such as colorectal carcinoma.

Use of dose-area product to assess plan quality in robotic radiosurgery.

PURPOSE: In robotic stereotactic radiosurgery (SRS), optimal selection of collimators from a set of fixed cones must be determined manually by trial and error. A unique and uniformly scaled metric to characterize plan quality could help identify Pareto-efficient treatment plans. METHODS: The concept of dose-area product (DAP) was used to define a measure (DAPratio) of the targeting efficiency of a set of beams by relating the integral DAP of the beams to the mean dose achieved in the target volume. In a retrospective study of five clinical cases of brain metastases with representative target volumes (range: 0.5-5.68 ml) and 121 treatment plans with all possible collimator choices, the DAPratio was determined along with other plan metrics (conformity index CI, gradient index R50%, treatment time, total number of monitor units TotalMU, radiotoxicity index f12, and energy efficiency index η50%), and the respective Spearman's rank correlation coefficients were calculated. The ability of DAPratio to determine Pareto efficiency for collimator selection at DAPratio < 1 and DAPratio < 0.9 was tested using scatter plots. RESULTS: The DAPratio for all plans was on average 0.95 ±â€¯0.13 (range: 0.61-1.31). Only the variance of the DAPratio was strongly dependent on the number of collimators. For each target, there was a strong or very strong correlation of DAPratio with all other metrics of plan quality. Only for R50% and η50% was there a moderate correlation with DAPratio for the plans of all targets combined, as R50% and η50% strongly depended on target size. Optimal treatment plans with CI, R50%, f12, and η50% close to 1 were clearly associated with DAPratio < 1, and plans with DAPratio < 0.9 were even superior, but at the cost of longer treatment times and higher total monitor units. CONCLUSIONS: The newly defined DAPratio has been demonstrated to be a metric that characterizes the target efficiency of a set of beams in robotic SRS in one single and uniformly scaled number. A DAPratio < 1 indicates Pareto efficiency. The trade-off between plan quality on the one hand and short treatment time or low total monitor units on the other hand is also represented by DAPratio.

Uncovering in vivo biochemical patterns from time-series metabolic dynamics.

System biology relies on holistic biomolecule measurements, and untangling biochemical networks requires time-series metabolomics profiling. With current metabolomic approaches, time-series measurements can be taken for hundreds of metabolic features, which decode underlying metabolic regulation. Such a metabolomic dataset is untargeted with most features unannotated and inaccessible to statistical analysis and computational modeling. The high dimensionality of the metabolic space also causes mechanistic modeling to be rather cumbersome computationally. We implemented a faster exploratory workflow to visualize and extract chemical and biochemical dependencies. Time-series metabolic features (about 300 for each dataset) were extracted by Ridge Tracking-based Extract (RTExtract) on measurements from continuous in vivo monitoring of metabolism by NMR (CIVM-NMR) in Neurospora crassa under different conditions. The metabolic profiles were then smoothed and projected into lower dimensions, enabling a comparison of metabolic trends in the cultures. Next, we expanded incomplete metabolite annotation using a correlation network. Lastly, we uncovered meaningful metabolic clusters by estimating dependencies between smoothed metabolic profiles. We thus sidestepped the processes of time-consuming mechanistic modeling, difficult global optimization, and labor-intensive annotation. Multiple clusters guided insights into central energy metabolism and membrane synthesis. Dense connections with glucose 1-phosphate indicated its central position in metabolism in N. crassa. Our approach was benchmarked on simulated random network dynamics and provides a novel exploratory approach to analyzing high-dimensional metabolic dynamics.

Chronic arsenate exposure affects amphipod size distribution and reproduction.

Arsenate (As V) is the predominant inorganic species of arsenic in oxic aquatic environments. Chronic water quality criteria for arsenate have not been established due to the scarcity of relevant studies on its impact on aquatic biota. We examined the acute and chronic effects of arsenate on the benthic amphipod Leptocheirus plumulosus, an important test organism for ecotoxicological studies. We determined that a concentration of 1,971 µg/L arsenate induced 50% mortality (LC50) in juveniles in 96-hr water only exposure. Subsequently, we tested the efficacy of a 42-day sediment bioassay to determine chronic population-level effects. Specifically, we analyzed the survivorship, size distribution, reproduction and offspring sex ratio of this amphipod to a sublethal concentration of arsenate. The sublethal concentration was determined based on the results of the acute tests. Arsenate (500 µg/L) was added to seawater (20 psu) overlying natural sediments (particle sizes < 250 µm). Fifteen replicate chambers per treatment were employed, each stocked with 20 stage-sorted juvenile amphipods (500-600 µm). Five replicates per treatment were destructively sampled on week 3 and ten replicates were sampled on week 6. Arsenate exposure did not affect the survivorship of parental amphipods, as expected, but it decreased significantly the number of offspring in the largest size classes. By week 6, arsenate-exposed replicates had statistically fewer sexually mature offspring compared to controls, likely because the overall reproduction was suppressed. Arsenate exposed amphipods had significantly fewer offspring than controls by week 6 (9.3 vs. 19.1 per parent), but the sex ratio of the offspring was not altered. Our results indicate that size distribution and reproduction may be more sensitive endpoints than survivorship for the chronic effects of arsenate in oxic systems. The extended 42-day bioassay with Leptocheirus plumulosus is a very promising tool to study the effects of toxicants on population dynamics.

Continuous in vivo Metabolism by NMR.

Dense time-series metabolomics data are essential for unraveling the underlying dynamic properties of metabolism. Here we extend high-resolution-magic angle spinning (HR-MAS) to enable continuous in vivo monitoring of metabolism by NMR (CIVM-NMR) and provide analysis tools for these data. First, we reproduced a result in human chronic lymphoid leukemia cells by using isotope-edited CIVM-NMR to rapidly and unambiguously demonstrate unidirectional flux in branched-chain amino acid metabolism. We then collected untargeted CIVM-NMR datasets for Neurospora crassa, a classic multicellular model organism, and uncovered dynamics between central carbon metabolism, amino acid metabolism, energy storage molecules, and lipid and cell wall precursors. Virtually no sample preparation was required to yield a dynamic metabolic fingerprint over hours to days at ~4-min temporal resolution with little noise. CIVM-NMR is simple and readily adapted to different types of cells and microorganisms, offering an experimental complement to kinetic models of metabolism for diverse biological systems.

Correlations Between LC-MS/MS-Detected Glycomics and NMR-Detected Metabolomics in Caenorhabditis elegans Development.

This study examined the relationship between glycans, metabolites, and development in C. elegans. Samples of N2 animals were synchronized and grown to five different time points ranging from L1 to a mixed population of adults, gravid adults, and offspring. Each time point was replicated seven times. The samples were each assayed by a large particle flow cytometer (Biosorter) for size distribution data, LC-MS/MS for targeted N- and O-linked glycans, and NMR for metabolites. The same samples were utilized for all measurements, which allowed for statistical correlations between the data. A new protocol was developed to correlate Biosorter developmental data with LC-MS/MS data to obtain stage-specific information of glycans. From the five time points, four distinct sizes of worms were observed from the Biosorter distributions, ranging from the smallest corresponding to L1 to adult animals. A network model was constructed using the four binned sizes of worms as starting nodes and adding glycans and metabolites that had correlations with r ≥ 0.5 to those nodes. The emerging structure of the network showed distinct patterns of N- and O-linked glycans that were consistent with previous studies. Furthermore, some metabolites that were correlated to these glycans and worm sizes showed interesting interactions. Of note, UDP-GlcNAc had strong positive correlations with many O-glycans that were expressed in the largest animals. Similarly, phosphorylcholine correlated with many N-glycans that were expressed in L1 animals.

Abundance of non-native crabs in intertidal habitats of New England with natural and artificial structure.

Marine habitats containing complex physical structure (e.g., crevices) can provide shelter from predation for benthic invertebrates. To examine effects of natural and artificial structure on the abundance of intertidal juvenile crabs, 2 experiments were conducted in Kingston Bay, Massachusetts, USA, from July to September, 2012. In the first experiment, structure was manipulated in a two-factor design that was placed in the high intertidal for 3 one-week periods to test for both substrate type (sand vs. rock) and the presence or absence of artificial structure (mesh grow-out bags used in aquaculture, ∼0.5 m(2) with 62 mm(2) mesh openings). The Asian shore crab, Hemigrapsus sanguineus, and small individuals of the green crab, Carcinus maenas, were observed only in the treatments of rocks and mesh bag plus rocks. Most green crabs were small (<6 mm in carapace width) whereas H. sanguineus occurred in a wide range of sizes. In the second experiment, 3 levels of oyster-shell treatments were established using grow-out bags placed on a muddy sand substrate in the low intertidal zone: mesh grow-out bags without shells, grow-out bags with oyster shells, and grow-out bags containing live oysters. Replicate bags were deployed weekly for 7 weeks in a randomized complete block design. All crabs collected in the bags were juvenile C. maenas (1-15 mm carapace width), and numbers of crabs differed 6-fold among treatments, with most crabs present in bags with live oysters (29.5 ± 10.6 m(-2) [mean ± S.D.]) and fewest in bags without shells (4.9 ± 3.7 m(-2)). Both C. maenas and H. sanguineus occurred in habitats with natural structure (cobble rocks). The attraction of juvenile C. maenas to artificial structure consisting of plastic mesh bags containing both oyster shells and living oysters could potentially impact oyster aquaculture operations.

The application of near-infrared spectroscopy for the quality control analysis of rocket propellant fuel pre-mixes.

The viability of near-infrared (NIR) spectroscopy as a technique for the quality control analysis of ingredient concentrations in a rocket propellant fuel liquid pre-mix was investigated. The pre-mix analyzed consisted of a polybutadiene pre-polymer, a plasticizer and two antioxidants. It was determined that NIR spectroscopy offered a fast and convenient method of verifying the percentage level of all four ingredients while requiring no sample preparation. The NIR methodology exhibited a high level of accuracy and precision. There was also a clear indication that the technique allowed monitoring of antioxidant depletion in the pre-mix on ageing.