Stable Isotope Mass Spectrometry Research Articles

Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].

Extracellular domains of CARs reprogramme T cell metabolism without antigen stimulation.

Metabolism is an indispensable part of T cell proliferation, activation and exhaustion, yet the metabolism of chimeric antigen receptor (CAR)-T cells remains incompletely understood. CARs are composed of extracellular domains-often single-chain variable fragments (scFvs)-that determine ligand specificity and intracellular domains that trigger signalling following antigen binding. Here, we show that CARs differing only in the scFv variously reprogramme T cell metabolism. Even without exposure to antigens, some CARs increase proliferation and nutrient uptake in T cells. Using stable isotope tracers and mass spectrometry, we observed basal metabolic fluxes through glycolysis doubling and amino acid uptake overtaking anaplerosis in CAR-T cells harbouring a rituximab scFv, unlike other similar anti-CD20 scFvs. Disparate rituximab and 14G2a-based anti-GD2 CAR-T cells are similarly hypermetabolic and channel excess nutrients to nitrogen overflow metabolism. Modest overflow metabolism of CAR-T cells and metabolic compatibility between cancer cells and CAR-T cells are identified as features of efficacious CAR-T cell therapy.

Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs invitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs invitro and in a C. elegans model invivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.

FAMetA: a mass isotopologue-based tool for the comprehensive analysis of fatty acid metabolism.

The use of stable isotope tracers and mass spectrometry (MS) is the gold standard method for the analysis of fatty acid (FA) metabolism. Yet, current state-of-the-art tools provide limited and difficult-to-interpret information about FA biosynthetic routes. Here we present FAMetA, an R package and a web-based application (www.fameta.es) that uses 13C mass isotopologue profiles to estimate FA import, de novo lipogenesis, elongation and desaturation in a user-friendly platform. The FAMetA workflow covers the required functionalities needed for MS data analyses. To illustrate its utility, different in vitro and in vivo experimental settings are used in which FA metabolism is modified. Thanks to the comprehensive characterization of FA biosynthesis and the easy-to-interpret graphical representations compared to previous tools, FAMetA discloses unnoticed insights into how cells reprogram their FA metabolism and, when combined with FASN, SCD1 and FADS2 inhibitors, it enables the identification of new FAs by the metabolic reconstruction of their synthesis route.

Study on origin traceability of amur cork-tree bark based on stable isotopes and multielements combined chemometrics

Amur cork-tree bark is a natural medicine in China with a large market, whereas confusion about its origin limits its cultivation and commercial value. To trace the origin, the stable isotope ratios (δ13C, δ15N, δ2H, and δ18O) and multiple elements (As, Hg, Al, Ca, Fe, K, Mg, Na, Cr, Cd, Pb, Be, Cu, Zn, Sr, and Ba) of 83 batches of Amur cork-tree bark samples from 7 habitats were determined by stable isotope mass spectrometry and inductively coupled plasmamass spectrometry. The results showed that the two-dimensional combination of δ13C and δ15N in the scatter diagram of stable isotopes could obviously distinguish Amur cork-tree bark from Sichuan Province from other producing areas, and multielement radar map analysis could also be used to distinguish Amur cork-tree bark from different geographical sources. In this article, the data of stable isotope ratios and multielement content were combined for principal component analysis (PCA), linear discriminant analysis (LDA) and orthogonal partial least square discriminant analysis (OPLS-DA). The results of principal component analysis showed that all samples could be preliminarily distinguished. The prediction model established and verified by linear discriminant analysis realized 100% accurate classification for known samples. The correct rate of analysis and prediction for unknown samples was 94.5%. The effective feature discriminant markers of Amur cork-tree bark caused by VIP > 1 (Ca, K, Pb, Mg, Cr, As) were found by orthogonal partial least square discriminant analysis. It could accurately identify Amur cork-tree bark from different producing areas.

A New Bacterial Adenosine-Derived Nucleoside as an Example of RNA Modification Damage.

The fields of RNA modification and RNA damage both exhibit a plethora of non-canonical nucleoside structures. While RNA modifications have evolved to improve RNA function, the term RNA damage implies detrimental effects. Based on stable isotope labelling and mass spectrometry, we report the identification and characterisation of 2-methylthio-1,N6-ethenoadenosine (ms2 ϵA), which is related to 1,N6-ethenoadenine, a lesion resulting from exposure of nucleic acids to alkylating chemicals in vivo. In contrast, a sophisticated isoprene labelling scheme revealed that ms2 ϵA biogenesis involves cleavage of a prenyl moiety in the known transfer RNA (tRNA) modification 2-methylthio-N6-isopentenyladenosine (ms2 i6 A). The relative abundance of ms2 ϵA in tRNAs from translating ribosomes suggests reduced function in comparison to its parent RNA modification, establishing the nature of the new structure in a newly perceived overlap of the two previously separate fields, namely an RNA modification damage.

Stable isotope characterization of tobacco products: A determination of synthetic or natural nicotine authenticity.

"Tobacco-free" or synthetic nicotine products have appeared in some markets, increasing potential health risks and regulatory compliance challenges. Currently, there are few reliable methods for the determination of authenticity of natural and synthetic nicotine. Analytical techniques based on stable isotopes have broad application prospects in the traceability and identification of agricultural products. Tobacco leaves from four main tobacco production regions in China and different types of tobacco products were extracted with n-hexane and 5% sodium hydroxide to obtain nicotine extracts. Subsequent stable isotope mass spectrometry was performed by analyzing δ2 H, δ13 C, and δ15 N values of nicotine. Firstly, results from a batch of 233 samples indicated stable isotopes were closely related to climate and geographical locations and provide a basis for a determination of the origin of tobacco leaves. In addition, the δ2 H values had significant differences between natural and synthetic nicotine and the results indicate a δ2 H value of -163.0‰ could be the threshold for assessing synthetic and natural nicotine. Finally, a total of 239 results further validated the δ2 H value as a metric for source authentication of commercial tobacco products. Synthetic (S)-(-)-nicotine could be accurately and quickly identified using the method developed by measuring δ2 H values in a qualitative manner. To our knowledge, this is the first time a stable isotope mass spectrometry technique has been used for distinguishing the source of nicotine. This technique will aid in the accurate identification, labelling, and regulation of synthetic nicotine-based tobacco products.

N2O production and emission pathways in anammox biofilter for treating wastewater with low nitrogen concentrations

Although anaerobic ammonia oxidation (anammox) is a cost-effective nitrogen removal process, nitrous oxide (N2O) production will greatly reduce the advantages of this process. It is important to identify the N2O emission pathways and then reduce the N2O production in anammox system. To date, very limited research has been done to investigate the N2O production and N2O emission pathways in anammox biofilter. In this study, N2O production were investigated under different filtration rates in anammox biofilter for treating wastewater with low nitrogen concentrations, and N2O emission pathways were analyzed with batch tests using N2O microsensor and stable isotope mass spectrometry. The results showed N2O production increased with the increase of filtration rates in anammox biofilter, where the N2O emission factor increased from 0.012 % at 1.0 m/h to 0.496 % at 3.0 m/h. And the optimal operation condition was at filtration rate of 1.5 m/h, where NH4+-N and NO2−-N removal efficiencies reached 99 % and N2O concentration was the lowest. qPCR showed that anammox bacteria, nitrifying bacteria and denitrifying bacteria were all present in anammox biofilter, with anammox bacteria in the highest abundance. And nitrifying bacteria and denitrifying bacteria provided the possibility of N2O production. The batch tests and stable isotope mass spectrometry analysis indicated that nitrifier denitrification, hydroxylamine oxidation and endogenous heterotrophic denitrification were N2O production pathways in aerobic zone and anoxic zone of anammox biofilter, respectively. In addition, batch tests under different conditions showed no oxygen environment could reduce N2O production. Therefore, the production of N2O in anammox system is a problem that cannot be ignored and should be paid more attention to.

MicroRNA-27b regulates hepatic lipase enzyme LIPC and reduces triglyceride degradation during hepatitis C virus infection

miRNAs are short, noncoding RNAs that negatively and specifically regulate protein expression, the cumulative effects of which can result in broad changes to cell systems and architecture. The miRNA miR-27b is known to regulate lipid regulatory pathways in the human liver and is also induced by the hepatitis C virus (HCV). However, the functional targets of miR-27b are not well established. Herein, an activity-based protein profiling method using a serine hydrolase probe, coupled with stable isotope labeling and mass spectrometry identified direct and indirect targets of miR-27b. The hepatic lipase C (LIPC) stood out as both highly dependent on miR-27b and as a major modulator of lipid pathway misregulation. Modulation of miR-27b using both exogenous miRNA mimics and inhibitors demonstrated that transcription factors Jun, PPARα, and HNF4α, all of which also influence LIPC levels and activity, are regulated by miR-27b. LIPC was furthermore shown to affect the progress of the life cycle of HCV and to decrease levels of intracellular triglycerides, upon which HCV is known to depend. In summary, this work has demonstrated that miR-27b mediates HCV infection by downregulating LIPC, thereby reducing triglyceride degradation, which in turn increases cellular lipid levels.

Influence of life history variation and habitat on mercury bioaccumulation in a high-order predatory fish in tropical Australia

Mercury distribution and bioaccumulation in aquatic ecosystems of tropical Australia is poorly characterised. Barramundi (Lates calcarifer), a widespread high-order predator in both fresh and coastal marine waters of the region, fulfils requirements for a bio-indicator of mercury contamination. In a study of the Mary River system of the Northern Territory, total mercury in the muscle tissue of 300 specimens gathered over four years (2013–2017, across both wet and dry seasons) was determined by direct combustion–atomic absorption spectrometry. Source of nutrition and trophic position of barramundi in the food web was also estimated via carbon and nitrogen isotopes (δ13C and δ15N), respectively, in tissue by stable isotope mass spectrometry, and determination of strontium isotopes (87Sr/86Sr) in otoliths by laser ablation–ICPMS differentiated between freshwater and saltwater residence. Results showed that fish moving into freshwater floodplain wetlands concentrated mercury in muscle tissue at approximately twice the level of those that remained in saline habitats. Resolving life histories through otolith analyses demonstrated diversity in mercury bioaccumulation for individual fish of the same migratory contingent on the floodplains. Although trophic level (δ15N), capture location, source of nutrition (δ13C), and age or size partly predicted mercury concentrations in barramundi, our results suggest that individual variability in diets, migration patterns and potentially metabolism are also influential. Using a migratory fish as a bio-indicator, and tracking its life history and use of resources, proved valuable as a tool to discern hot spots in a coastal waterway for a contaminant, such as mercury.

Linking post-translational modifications and protein turnover by site-resolved protein turnover profiling

Proteome-wide measurements of protein turnover have largely ignored the impact of post-translational modifications (PTMs). To address this gap, we employ stable isotope labeling and mass spectrometry to measure the turnover of >120,000 peptidoforms including >33,000 phosphorylated, acetylated, and ubiquitinated peptides for >9,000 native proteins. This site-resolved protein turnover (SPOT) profiling discloses global and site-specific differences in turnover associated with the presence or absence of PTMs. While causal relationships may not always be immediately apparent, we speculate that PTMs with diverging turnover may distinguish states of differential protein stability, structure, localization, enzymatic activity, or protein-protein interactions. We show examples of how the turnover data may give insights into unknown functions of PTMs and provide a freely accessible online tool that allows interrogation and visualisation of all turnover data. The SPOT methodology is applicable to many cell types and modifications, offering the potential to prioritize PTMs for future functional investigations.

A Review of Stable Isotope Labeling and Mass Spectrometry Methods to Distinguish Exogenous from Endogenous DNA Adducts and Improve Dose-Response Assessments.

Cancer remains the second most frequent cause of death in human populations worldwide, which has been reflected in the emphasis placed on management of risk from environmental chemicals considered to be potential human carcinogens. The formation of DNA adducts has been considered as one of the key events of cancer, and persistence and/or failure of repair of these adducts may lead to mutation, thus initiating cancer. Some chemical carcinogens can produce DNA adducts, and DNA adducts have been used as biomarkers of exposure. However, DNA adducts of various types are also produced endogenously in the course of normal metabolism. Since both endogenous physiological processes and exogenous exposure to xenobiotics can cause DNA adducts, the differentiation of the sources of DNA adducts can be highly informative for cancer risk assessment. This review summarizes a highly applicable methodology, termed stable isotope labeling and mass spectrometry (SILMS), that is superior to previous methods, as it not only provides absolute quantitation of DNA adducts but also differentiates the exogenous and endogenous origins of DNA adducts. SILMS uses stable isotope-labeled substances for exposure, followed by DNA adduct measurement with highly sensitive mass spectrometry. Herein, the utilities and advantage of SILMS have been demonstrated by the rich data sets generated over the last two decades in improving the risk assessment of chemicals with DNA adducts being induced by both endogenous and exogenous sources, such as formaldehyde, vinyl acetate, vinyl chloride, and ethylene oxide.

An atlas of protein turnover rates in mouse tissues

Protein turnover is critical to cellular physiology as well as to the growth and maintenance of tissues. The unique synthesis and degradation rates of each protein help to define tissue phenotype, and knowledge of tissue- and protein-specific half-lives is directly relevant to protein-related drug development as well as the administration of medical therapies. Using stable isotope labeling and mass spectrometry, we determine the in vivo turnover rates of thousands of proteins—including those of the extracellular matrix—in a set of biologically important mouse tissues. We additionally develop a data visualization platform, named ApplE Turnover, that enables facile searching for any protein of interest in a tissue of interest and then displays its half-life, confidence interval, and supporting measurements. This extensive dataset and the corresponding visualization software provide a reference to guide future studies of mammalian protein turnover in response to physiologic perturbation, disease, or therapeutic intervention.

Altitudinal variation of leaf carbon isotope for Dendrosenecio keniensis and Lobelia gregoriana in Mount Kenya alpine zone

AbstractAbiotic factors vary along altitudinal gradients, and this may influence plant morphology, physiology and function. This study aimed to test the hypothesis that leaf δ13C—a common proxy for water use efficiency—was indirectly influenced by morphological adjustments with changing climatic factors along an altitudinal gradient on Mount Kenya. We sampled leaves of Dendrosenecio keniensis and Lobelia gregoriana using seventy‐two 10 × 10 m plots situated every 100 m starting from 3600 to 4300 m. We determined leaf δ13C using stable isotope mass spectrometry. We also quantified the following morphological factors; leaf area, leaf mass per area, specific leaf area and leaf thickness. Climate data included mean annual temperature and precipitation, diurnal temperature range and water vapor pressure. Our results revealed that there was a leaf δ13C enrichment of 1.76 ‰ km−1 and 1.62 ‰ km−1 with altitude for D. keniensis and L. gregoriana, respectively. Leaf δ13C was enrichment by 0.01 ‰ mm−1 with mean annual precipitation along the altitude gradient for D. keniensis and 0.02 ‰ mm−1 for L. gregoriana. D. keniensis and L. gregoriana have high‐water use efficiency, an adaptation for surviving near freezing alpine temperatures and high‐diurnal range. Leaf δ13C exhibited a depletion of −0.37 ‰ per °C increase of mean annual temperature along the altitude gradient for D. keniensis and −0.34 ‰ per °C increase for L. gregoriana. Our results also showed a negative relationship between pCO2 and leaf δ13C and positive relationship between pCO2 and ∆13C for both species. Low temperatures led to the increase in leaf thickness and specific leaf area for these two species, factors that influenced leaf δ13C and ∆13C.Abstract in Chinese is available with online material.

Hydrocarbon transformation pathways and soil organic carbon stability in the biostimulation of oil-contaminated soil: Implications of 13C natural abundance

Mineralization, assimilation, and humification are key processes to detoxify oil-contaminated soil by biostimulation remediation strategies, and these processes are affected by stimulants. In this study, we investigated the effects of either inorganic salts or organic stimulants (organic compost and sawdust) on hydrocarbon transformation. Total petroleum hydrocarbons (TPH) and hydrocarbon components were determined by gravimetry and gas chromatography, and the 13C of CO2, microbial biomass carbon (MBC), and humus were measured by stable isotope mass spectrometry. The results showed that organic compost was the most beneficial for the dissipation of hydrocarbons. After 60 days of remediation, the removal rates of TPH, saturates, aromatics, C7-C30 n-alkanes, and 16 PAHs were 35.7%, 39.6%, 15.9%, 80.5%, and 8.8%, respectively. A total of 84.7%–88.5% of the removed hydrocarbons were mineralized in all the treatments. The hydrocarbon degradation pathway in the control soil (without stimulant addition) was “assimilation → humification → mineralization”. The hydrocarbon transformation pathways in the biostimulation treatments were “assimilation → mineralization → humification”. The soil organic carbon (SOC) stability decreased during remediation, which was attributed to the enhanced microbial activity and the removal of recalcitrant hydrocarbons.

Three Gorges Reservoir construction induced dissolved organic matter chemistry variation between the reservoir and non-reservoir areas along the Xiangxi tributary

As one of the most dynamic and reactive compound pools, DOM plays a crucial part in various biochemical processes such as element cycling and nutrient export. Although the reservoir DOM has been investigated extensively, the variation of DOM between reservoir area and non-reservoir area induced by reservoir construction is not comprehensively assessed. By the combination of a series of complementary techniques including stable carbon isotope, optical spectroscopy, and ultrahigh-resolution mass spectrometry, here we show that hydrological alterations induced by Three Gorges Reservoir (TGR) construction were responsible for the variation in DOM molecular composition between reservoir area and non-reservoir area in Xiangxi tributary. With water intrusion from mainstream induced by reservoir construction and operation, reservoir area had relatively higher terrestrial input and more recalcitrant DOM than those in the non-reservoir area with limited influence of reservoir operation, whereas, relatively more autochthonous source and higher molecular lability of DOM were observed in the non-reservoir area. The water intrusion from mainstream to tributaries induced by reservoir construction is likely the main factor controlling DOM variation between reservoir area and non-reservoir area, and might devote to the organic carbon burial in the reservoir. This research provides new insight into spatial variations of riverine DOM composition induced by reservoir construction, underlining the important role of the reservoir in DOM cycling.

Petrographic and geochemical characterization of the Middle\u2012Upper Jurassic Fe\u2013Mn crusts and mineralizations from Monte Inici (north-western Sicily): genetic implications

Fe–Mn concretions and mineralizations, associated with condensed horizons and hardground, are significant archives in ancient carbonate rocks. Their petro-chemical study allows an assessment of the palaeoenvironmental context in which they were formed also connected to their biotic or abiotic origin. At the western side of the Monte Inici (Fornazzo section, north-western Sicily) a well exposed outcrop of condensed pelagic limestones (Rosso Ammonitico facies: Middle‒Upper Jurassic) is well-known and thoroughly studied. In this section, the base of the Rosso Ammonitico facies consists of a very condensed level rich in fossils with a variable thickness deposited from the early Bathonian to the early/middle Callovian. It is characterized, at the top, by the noticeable presence of Fe–Mn concretions, typical of the Tethyan Jurassic and related to very low sedimentation rates. For this study, Fe–Mn crusts and mineralizations from the Fornazzo section were investigated using X-ray diffraction, scanning electron microscopy, X-ray fluorescence, ICP and stable-isotope mass spectrometry. The collected samples, deposited in submarine conditions (as testified by stable oxygen and carbon isotopes), have been subdivided in two typologies with different macroscopic and mineralogical features. The Fe–Mn crusts consist of hematite, todorokite and birnessite and are characterized by a relatively low Mn/Fe ratio. Their content in trace elements, rare earths and yttrium (REY) is compatible with a hydrogenetic origin involving the oxy-hydroxides colloids precipitation directly from seawater. Microbially mediated processes are here testified by the recognition of filamentous and coccoid-shaped microstructures referable to coexistence of chemosynthetic fungi and photosynthetic cyanobacteria and accounting for a deposition in the deep euphotic zone. An average growth rate of ~ 8.5 mm/Myr for the Fe–Mn crusts, estimated by cobalt concentrations, suggests a time elapsed for deposition of ~ 3.5 ± 1 Myr. This value is compatible with the stratigraphic gap embracing the time span from the early/middle Callovian to the middle Oxfordian. In the neighbouring pelagic limestones, Fe–Mn deposits are present in the form of micro-dendrites mainly consisting of pyrolusite, sometimes associated with carbonato-fluorapatite. The geochemical composition gives evidence of a prevalent early diagenetic origin with precipitation, at the sediment/water interface or in the first centimeters of sediments, of metals diffused from the crusts as consequence of fluctuating redox conditions. Although the well-preserved Frutexites texture is commonly related to a microbial activity, other bacterial microstructures have not been recognized, having probably been obliterated during the growth of the dendrites. Nevertheless, it is possible to suppose a deepening in the bathymetry consistent with the involvement of chemosynthetic microorganisms in the formation of Frutexites structures.

Radiocarbon measurements using new automated graphite preparation laboratory coupled with stable isotope mass-spectrometry at Birbal Sahni Institute of Palaeosciences, Lucknow (India)

Conventional beta counting technique based radiocarbon dating facility at Birbal Sahni Institute of Palaeosciences- Lucknow was established in 1974 (Rajagopalan,1978). In 2017–18, BSIP received an upgrade with installation of an Automated Graphitization Equipment (AGE) coupled with an Elemental Analyser, a Carbonate Handling System (CHS) along with an in-line stable isotope mass-spectrometer (IRMS). Using this combo, stable Carbon (C), Nitrogen (N) and Sulfur (S) isotopic measurements could be carried out in both organic and inorganic type samples followed by graphite preparation (~1 mg) for 14C measurement by Accelerator Mass Spectrometry (AMS). This communication addresses details of pre-processing, processing, and quality checks adopted for achieving acceptable and demonstrable accuracy and precision of measured Δ14C, δ13C, δ15N, and δ34S measurements. Information regarding chemical preparation of samples for aforesaid stable and radio isotopic analysis is provided in succinct manner. Overall, average coefficient of variation determining precision of our graphite powders for 14C measurements is ~2.4%. The mean age of blank (anthracite) processed using established EA-IRMS-AGE unit comes as 42,100 ± 300 years.

Fluid properties and sources of Sixiangchang carbonate-associated mercury deposit, southwest China

Mercury mines in Guizhou province are the main base of mercury production and the most important resource base in China. The San-Dan mercury belt in Guizhou province contains a series of important mercury deposits. However, the source of metallogenic materials and the properties of metallogenic fluid of these mercury deposits have long been a controversial issue. In this study, we used cathode luminescence techniques to distinguish different stages of dolomite and calcite, laser ablation inductively coupled plasma mass spectrometry to analyze the trace elements, and stable isotope mass spectrometry techniques to analyze C–O isotopes compositions of dolomite and calcite in the Sixiangchang mercury deposit in San-Dan mercury belt. We also measured the sulfur isotope composition of cinnabar. Our study showed that dolomite can be divided into two stages, the lumpy dolomite associated with cinnabar in Dol 1 stage and dolomite vein in Dol 2 stage, which is associated with Cal 2 stage calcite vein. With the progress of mineralization, Al, As, Mo, Sb, and Sr elements were gradually enriched in the ore-forming fluid. The rare earth element (REE) partition curve of Dol 1 stage dolomite showed a trend of light REE enrichment. Cal 2 stage calcite and Dol 2 stage dolomite exhibited a flat-type REE partition curve, and Dol 2 stage dolomite showed a strong negative anomaly for Eu. δ13C of carbonate mineral variation ranges from − 6.89 to − 2.16 ‰, while δ18O variation ranges from 13.80 to 23.09 ‰, and the δ34S variation range of cinnabar is 16.51–24.28 ‰. Carbonate mineral trace elements and C–O isotopes compositions suggested that early ore-forming fluid was reduced, and late ore-forming fluid was oxidized. The ore-forming fluid of the Sixiangchang mercury deposit is a mixture of deep crustal fluid and meteoric water in deep thermal circulation, and involved in the oxidation of organic matter. The cinnabar δ34S results showed that sulfur mainly came from seawater sulfate with the participation of microbial reduction. Sulfur is sedimentary in origin and was derived mainly from the host-rock strata.

Quantifying Intermediary Metabolism and Lipogenesis in Cultured Mammalian Cells Using Stable Isotope Tracing and Mass Spectrometry.

Metabolism plays a central role in virtually all diseases, including diabetes, cancer, and neurodegeneration. Detailed analysis is required to identify the specific metabolic pathways dysregulated in the context of a given disease or biological perturbation. Measurement of metabolite concentrations can provide some insights into altered pathway activity or enzyme function, but since most biochemicals are metabolized by various enzymes in distinct pathways within cells and tissues, these approaches are somewhat limited. By applying metabolic tracers to a biological system, one can visualize pathway-specific information depending on the tracer used and analytes measured. To this end, stable isotope tracers and mass spectrometry are emerging as important tools for the examination of metabolic pathways and fluxes in cultured mammalian cells and other systems. Here, we describe a detailed workflow for quantifying metabolic processes in mammalian cell cultures using stable isotopes and gas chromatography coupled to mass spectrometry (GC-MS). As a case study, we apply 13C isotopic labeled glucose and glutamine to a cancer cell line to quantify substrate utilization for TCA metabolism and lipogenesis. Guidelines are also provided for interpretation of data and considerations for application to other cell systems. Ultimately, this approach provides a robust and precise method for quantifying stable isotope labeling in metabolite pools that can be applied to diverse biological systems.