High Initial Sputter Rate Found for Vaccinia Virions Using Isotopic Labeling, NanoSIMS, and AFM

Gates, Sean D.; Condit, Richard C.; Moussatché, Nissin; Stewart, Benjamin; Malkin, Alexander J.; Weber, Peter K.

doi:10.1021/acs.analchem.7b02786

Cited by 13 publications

(12 citation statements)

References 23 publications

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“…We applied stable isotope-assisted metabolic labeling to elucidate cancer metabolism-specific volatiles. , Specifically, we used [U– 13 C]glucose, which is a major source of carbon via the Warburg effect of cancer cells, as a labeling agent . To determine when 13 C from glucose was enriched completely in every volatile candidate, we defined the moment when the cells reached the isotopic steady state using nanoscale secondary ion mass spectrometry (nanoSIMS), as it offers an outstanding spatial resolution adequate for cell samples. , At the steady state, cancer metabolism-derived volatiles were sorted from uncertain volatiles by isotopic influx, which was detected by headspace–solid phase microextraction (HS–SPME) and gas chromatography–mass spectrometry (GC–MS). , …”

Section: Introductionmentioning

confidence: 99%

In Vitro Tracking of Intracellular Metabolism-Derived Cancer Volatiles via Isotope Labeling

Lee

Park

et al. 2018

ACS Cent. Sci.

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Cancer detection relying on the release of volatile biomarkers has been extensively studied, but the individual biochemical processes of the cells from which biogenic volatiles originate have not been thoroughly elucidated to date. Inadequate determination of the metabolic origin of the volatile biomarkers has limited the progress of the scientific and practical applications of volatile biomarkers. To overcome the current limitations, we developed a metabolism tracking approach combining stable isotope labeling and flux analysis of volatiles to trace the intracellular metabolism-derived volatiles and to reveal their relation to cancer metabolic pathways. Specifically, after the 13C labeling of lung cancer cell, the isotopic ratio of whole cellular carbon was measured by nanoscale secondary ion mass spectrometry-based imaging. The kinetic modeling with the time-dependent isotopic ratio determined the period during which cancer cells reach the metabolic steady state, at which time all of the potential volatiles derived from intracellular metabolism were fully enriched isotopically. By measuring the isotopic enrichment of volatiles at the end-stage of isotopic flux, we found that 2-pentadecanone appeared to be derived from the metabolic cascade starting from glucose to fatty acid synthesis. Furthermore, this biosynthetic pathway was determined to be distinct in cancer, as it was upregulated in colon, breast, and pancreatic cancer cells but not in normal cells. The investigation of the metabolic footprint of 2-pentadecanone demonstrates that our novel approach could be applied to trace the metabolic origin of biogenic volatile organic compounds. This analytical strategy represents a potential cutting-edge tool in elucidating the biochemical authenticity of cancer volatiles and further expanding our understanding of the metabolic network of airborne metabolites in vitro.

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Section: Introductionmentioning

confidence: 99%

In Vitro Tracking of Intracellular Metabolism-Derived Cancer Volatiles via Isotope Labeling

Lee

Park

et al. 2018

ACS Cent. Sci.

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“…In a chemically homogeneous material like the epoxy, the depth sputtered away can be controlled by adjusting the sputter time and primary current of a given measurement and knowing the sputtering rate(s) of the material. The sputtering rate of the epoxy is measured here in the steady state regime, where it is constant (it varies by orders of magnitude in the transient state 37 ). As a composite parameter, the sputtering rate is expressed as the depth excavated into the sample in nanometers for a given unit primary ion current and time within a unit area.…”

mentioning

confidence: 99%

Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles

et al. 2020

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Mass spectrometry imaging is a field that promises to become a mainstream bioanalysis technology by allowing the combination of single-cell imaging and subcellular quantitative analysis. The frontier of single-cell imaging has advanced to the point where it is now possible to compare the chemical contents of individual organelles in terms of raw or normalized ion signal. However, to realize the full potential of this technology, it is necessary to move beyond this concept of relative quantification. Here we present a nanoSIMS imaging method that directly measures the absolute concentration of an organelle-associated, isotopically labeled, pro-drug directly from a mass spectrometry image. This is validated with a recently developed nanoelectrochemistry method for single organelles. We establish a limit of detection based on the number of isotopic labels used and the volume of the organelle of interest, also offering this calculation as a web application. This approach allows subcellular quantification of drugs and metabolites, an overarching and previously unmet goal in cell science and pharmaceutical development.

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“…Next, inferences of viral impacts on microbial communities and C cycling will require predicting hosts both in silico (Edwards et al, 2015; Paez-Espino et al, 2017) and in vitro (Deng et al, 2014; Brum & Sullivan, 2015; Cenens et al, 2015), approaches to which are emerging. Finally, identification of the active viral community and characterization of their roles in biogeochemical processes can be better resolved with techniques like stable isotope-based approaches linked with nanoscale secondary ion mass spectrometry (NanoSIP; Pacton et al, 2014; Pasulka et al, 2018; Gates et al, 2018). Application of these and other approaches to soil viromics will increase and diversify publicly available viral datasets, advance our understanding of soil viral ecology, and improve our knowledge of viral roles in soil ecosystems.…”

Section: Discussionmentioning

confidence: 99%

Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils

Trubl

Roux

Solonenko

et al. 2019

PeerJ

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Soils impact global carbon cycling and their resident microbes are critical to their biogeochemical processing and ecosystem outputs. Based on studies in marine systems, viruses infecting soil microbes likely modulate host activities via mortality, horizontal gene transfer, and metabolic control. However, their roles remain largely unexplored due to technical challenges with separating, isolating, and extracting DNA from viruses in soils. Some of these challenges have been overcome by using whole genome amplification methods and while these have allowed insights into the identities of soil viruses and their genomes, their inherit biases have prevented meaningful ecological interpretations. Here we experimentally optimized steps for generating quantitatively-amplified viral metagenomes to better capture both ssDNA and dsDNA viruses across three distinct soil habitats along a permafrost thaw gradient. First, we assessed differing DNA extraction methods (PowerSoil, Wizard mini columns, and cetyl trimethylammonium bromide) for quantity and quality of viral DNA. This established PowerSoil as best for yield and quality of DNA from our samples, though ∼1/3 of the viral populations captured by each extraction kit were unique, suggesting appreciable differential biases among DNA extraction kits. Second, we evaluated the impact of purifying viral particles after resuspension (by cesium chloride gradients; CsCl) and of viral lysis method (heat vs bead-beating) on the resultant viromes. DNA yields after CsCl particle-purification were largely non-detectable, while unpurified samples yielded 1–2-fold more DNA after lysis by heat than by bead-beating. Virome quality was assessed by the number and size of metagenome-assembled viral contigs, which showed no increase after CsCl-purification, but did from heat lysis relative to bead-beating. We also evaluated sample preparation protocols for ssDNA virus recovery. In both CsCl-purified and non-purified samples, ssDNA viruses were successfully recovered by using the Accel-NGS 1S Plus Library Kit. While ssDNA viruses were identified in all three soil types, none were identified in the samples that used bead-beating, suggesting this lysis method may impact recovery. Further, 13 ssDNA vOTUs were identified compared to 582 dsDNA vOTUs, and the ssDNA vOTUs only accounted for ∼4% of the assembled reads, implying dsDNA viruses were dominant in these samples. This optimized approach was combined with the previously published viral resuspension protocol into a sample-to-virome protocol for soils now available at protocols.io, where community feedback creates ‘living’ protocols. This collective approach will be particularly valuable given the high physicochemical variability of soils, which will may require considerable soil type-specific optimization. This optimized protocol provides a starting place for developing quantitatively-amplified viromic datasets and will help enable viral ecogenomic studies on organic-rich soils.

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High Initial Sputter Rate Found for Vaccinia Virions Using Isotopic Labeling, NanoSIMS, and AFM

Cited by 13 publications

References 23 publications

In Vitro Tracking of Intracellular Metabolism-Derived Cancer Volatiles via Isotope Labeling

In Vitro Tracking of Intracellular Metabolism-Derived Cancer Volatiles via Isotope Labeling

Subcellular Mass Spectrometry Imaging and Absolute Quantitative Analysis across Organelles

Towards optimized viral metagenomes for double-stranded and single-stranded DNA viruses from challenging soils

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