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Zinc is a ubiquitous contaminant in many buffers, purified products and common labware that has previously been suggested to impact on the results of functional GlyR studies and may inadvertently cause the effectiveness of some GlyR modulators to be over-estimated. This could greatly impact the assessment of potential drug-candidates and contribute to the reduced effectiveness of compounds that reach clinical stages. This is especially true for GlyR modulators being developed for pain therapeutics due to the changes in spinal zinc concentrations that have been observed during chronic pain conditions. In this study we use two-electrode voltage clamp electrophysiology to evaluate the metal chelators tricine and Ca-EDTA, and show that tricine produces inhibitory effects at GlyRα1 that are not mediated by zinc. We also utilized the zinc insensitive W170S mutation as a tool to validate metal chelators and confirm that zinc contamination has not impacted the examination of lipid modulators previously developed by our lab. This study helps to further develop methods to negate the impact of contaminating zinc in functional studies of GlyRs which should be incorporated into future studies that seek to characterize the activity of novel modulators at GlyRs.
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Nanoparticles are produced at accelerating rates, are increasingly integrated into scientific and industrial applications, and are widely discharged into the environment. Analytical techniques are required to characterize parameters such as particle number concentrations, mass and size distributions, molecular and elemental compositions, and particle stability. This is not only relevant to investigate their utility for various industrial or medical applications and for controlling the manufacturing processes but also to assess toxicity and environmental fate. Different analytical strategies aim to characterize certain facets of particles but are difficult to combine to retrieve relevant parameters coherently and to provide a more comprehensive picture. In this work, we demonstrate the first online hyphenation of optofluidic force induction (OF2i) with Raman spectroscopy and inductively coupled plasma-time-of-flight-mass spectrometry (ICP-TOFMS) to harness their complementary technology-specific advantages and to promote comprehensive particle characterizations. We optically trapped individual particles on a weakly focused vortex laser beam by aligning a microfluidic flow antiparallelly to the laser propagation direction. The position of particles in this optical trap depended on the hydrodynamic diameter and therefore enabled size calibration as well as matrix elimination. Additionally, laser light scattered on particles was analyzed in a single particle (SP) Raman spectroscopy setup for the identification of particulate species and phases. Finally, particles were characterized regarding elemental composition and their distributions in mass and size using SP ICP-TOFMS. In a proof of concept, we analyzed polystyrene-based microplastic and TiO2 nanoparticles and demonstrated the opportunities provided through the coupling of OF2i with SP Raman and SP ICP-TOFMS.
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DGet! is an open-source analysis package written in Python for calculating the degree of deuterium enrichment in isotopically labelled molecules using mass spectrometric data. The nuclear properties of deuterium make it a valuable tracer in metabolic studies and an excellent contrast agent in nuclear spectroscopies. Determination of molecular deuteration levels is typically performed using mass spectrometry, however software options to perform these calculations are scarce. The in-house scripts and spreadsheets currently used rarely account for isotopic interferences from 13C or multi-isotopic elements that impact deuteration calculations. DGet! removes isotopic interferences using de-convolution and both the isotopological makeup and overall deuteration level can be accurately recovered. The software is available as command line and web applications that take a molecular formula and mass spectrometry data and output a graphical representation of the degree of deuteration as well as the distribution of partially deuterated analogues. These applications are designed to be easy to use and enable superior characterisation of deuterated molecules for users of all levels of expertise, without the limitations of techniques currently used by the majority of deuteration laboratories and researchers.
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The urgency of responding to climate change for corals necessitates the exploration of innovative methods to swiftly enhance our understanding of crucial processes. In this study, we employ an integrated chemical omics approach, combining elementomics, metabolomics, and volatilomics methodologies to unravel the biochemical pathways associated with the thermal response of the coral symbiont, Symbiodiniaceae Durusdinium trenchii. We outline the complimentary sampling approaches and discuss the standardised data corrections used to allow data integration and comparability. Our findings highlight the efficacy of individual methods in discerning differences in the biochemical response of D. trenchii under both control and stress-inducing temperatures. However, a deeper insight emerges when these methods are integrated, offering a more comprehensive understanding, particularly regarding oxidative stress pathways. Employing correlation network analysis enhanced the interpretation of volatile data, shedding light on the potential metabolic origins of volatiles with undescribed functions and presenting promising candidates for further exploration. Elementomics proves to be less straightforward to integrate, likely due to no net change in elements but rather elements being repurposed across compounds. The independent and integrated data from this study informs future omic profiling studies and recommends candidates for targeted research beyond Symbiodiniaceae biology. This study highlights the pivotal role of omic integration in advancing our knowledge, addressing critical gaps, and guiding future research directions in the context of climate change and coral reef preservation.
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Wildfires that raged across Australia during the 2019-2020 'Black Summer' produced an enormous quantity of particulate matter (PM) pollution, with plumes that cloaked many urban centres and ecosystems along the eastern seaboard. This has motivated a need to understand the magnitude and nature of PM exposure, so that its impact on both built and natural environments can be more accurately assessed. Here we present the potentially toxic fingerprint of PM captured by building heating, ventilation, and air conditioning filters in Sydney, Australia during the peak of the Wildfires, and from ambient urban emissions one year later (Reference period). Atmospheric PM and meteorological monitoring data were also assessed to determine the magnitude and source of high PM exposure. The wildfires were a major source of PM pollution in Sydney, exceeding the national standards on 19 % of days between November-February. Wildfire particles were finer and more spherical compared to Reference PM, with count median diameters of 892.1 ± 23.1 versus 1484.8 ± 96.7 nm (mean ± standard error). On an equal-mass basis, differences in potentially toxic elements were predominantly due to higher SO42--S (median 20.4 vs 4.7 mg g-1) and NO3--N (2.4 vs 1.2 mg g-1) in Wildfire PM, and higher PO43--P (10.4 vs 1.4 mg g-1) in Reference PM. Concentrations of remaining elements were similar or lower than Reference PM, except for enrichments to F-, Cl-, dissolved Mn, and particulate Mn, Co and Sb. Fractional solubilities of trace elements were similar or lower than Reference PM, except for enhanced Hg (12.1 vs 1.0 %) and greater variability in Cd, Hg and Mn solubility, which displayed upper quartiles exceeding that of Reference PM. These findings contribute to our understanding of human and ecosystem exposures to the toxic components of mixed smoke plumes, especially in regions downwind of the source.
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Contaminantes Atmosféricos , Contaminación del Aire , Mercurio , Humanos , Contaminantes Atmosféricos/análisis , Ecosistema , Monitoreo del Ambiente , Material Particulado/análisis , Contaminación del Aire/análisisRESUMEN
Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.
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Terapia por Láser , Metales , Espectrometría de Masas/métodos , Metales/análisis , Terapia por Láser/métodos , Diagnóstico por ImagenRESUMEN
Toxic heavy metals have been the focus of many investigations into chronic kidney disease of unknown aetiology (CKDu) within Sri Lanka. It has been hypothesised that exposure to nephrotoxic arsenic, cadmium and lead could play a role in the development of CKDu, and these metals have previously been found in unsafe concentrations in Sri Lankan rice. Traditional varieties of Sri Lankan rice remain popular due to their perceived health benefits, but their uptake of trace and toxic heavy metals remained unexplored. Here, we report a one-time, cross-sectional dataset on the concentrations of essential and toxic elements present in eleven samples of polished and unpolished traditional rice varieties, all regularly grown and sold in the Anuradhapura district, a CKDu hotspot. All rice was sourced from the same farm, with the exception of one store bought sample grown on another, unidentified farm. Cadmium concentrations varied significantly between varieties, and potentially unsafe concentrations of cadmium were detected in the store-bought sample (Suwadel, 113±13 µg kg-1). Elemental imaging of the grains revealed lead to be stored mainly in the rice bran, which is removed during polishing, while cadmium was distributed in the edible portion of the grain. Essential elements were generally higher in the traditional rice varieties than those reported for non-traditional varieties and are a potential source of trace elements for nutrient-deficient communities. The concentration of selenium, an element that plays a protective role in the kidneys, was too low to provide the minimum recommended intake. The methods developed in this study could be applied to a more comprehensive study of elemental uptake of rice under controlled growing conditions.
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The analysis of natural and anthropogenic nanomaterials (NMs) in the environment is challenging and requires methods capable to identify and characterise structures on the nanoscale regarding particle number concentrations (PNCs), elemental composition, size, and mass distributions. In this study, we employed single particle inductively coupled plasma-mass spectrometry (SP ICP-MS) to investigate the occurrence of NMs in the Melbourne area (Australia) across 63 locations. Poisson statistics were used to discriminate between signals from nanoparticulate matter and ionic background. TiO2-based NMs were frequently detected and corresponding NM signals were calibated with an automated data processing platform. Additionally, a method utilising a larger mass bandpass was developed to screen for particulate high-mass elements. This procedure identified Pb-based NMs in various samples. The effects of different environmental matrices consisting of fresh, brackish, or seawater were mitigated with an aerosol dilution method reducing the introduction of salt into the plasma and avoiding signal drift. Signals from TiO2- and Pb-based NMs were counted, integrated, and subsequently calibrated to determine PNCs as well as mass and size distributions. PNCs, mean sizes, particulate masses, and ionic background levels were compared across different locations and environments.
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Nanoestructuras , Titanio , Plomo , Tamaño de la Partícula , Análisis Espectral , Titanio/análisis , AguaRESUMEN
Open-sourced software is a key component of the mass spectrometry imaging field, where transparency in data processing is vital. Imaging of trace elements and immunohistochemically labeled biomolecules in tissue sections is typically performed using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). However, efficient and facile processing of images is hampered by a lack of verifiable and user-friendly software that supports multiple LA-ICP-MS platforms. In this technical note, we introduce Pew2, a LA-ICP-MS specific and feature-rich open-source image processing software that is compatible with common ICP-MS vendors. Pew2 is designed to be fast and easy to use and adheres to modern visualization philosophies to maximize productivity and to minimize data interpretation errors and image anomalies.
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Terapia por Láser , Oligoelementos , Espectrometría de Masas , Programas Informáticos , Análisis Espectral , Oligoelementos/análisisRESUMEN
The discharge of plastic waste and subsequent formation and global distribution of microplastics (MPs) has caused great concern and highlighted the need for dedicated methods to characterise MPs in complex environmental matrices like seawater. Single particle inductively coupled plasma - mass spectrometry (SP ICP-MS) is an elegant method for the rapid analysis of nano- and microparticles and to characterise number concentrations, mass, and size distributions. However, the analysis of carbon (C)-based microstructures such as MPs by SP ICP-MS is at an early stage. This paper investigates various strategies to improve figures of merit to detect and characterise MPs in complex matrices, such as seawater. Ten methods operating distinct acquisition modes with various collision/reaction gases, tandem MS (ICP-MS/MS) and targeting 12C or 13C were developed and compared for the analysis of polystyrene-based MPs standards in ultra-pure water and seawater. The robust analysis of MPs in seawater was accomplished by on-line aerosol dilution enabling repeatable size calibration while minimising drift effects. However, the direct analysis of seawater decreased ion transmission and required matrix-matching for accurate size calibration. Analysis of the 12C isotope instead of 13C improved the size detection limits (sDL) to 0.62 µm in ultra-pure water and to 0.96 µm in seawater. ICP-MS/MS methods decreased ion transmission but also reduced background signal and increased selectivity, particularly in the presence of spectral interferences. In the second part of this study, it was demonstrated that the developed methods were applicable for the analysis of C in unicellular organisms and allowed calibration of physical dimensions. This is relevant for the investigation and understanding of phenotypical traits associated, for example, with climate change resilience as well as oceanic C storage. SP/SC ICP-MS was employed to target five different intact Symbiodiniaceae algae strains with diverse life-histories in seawater and polystyrene-based MPs were used to calibrate cellular C masses, which were between 51 and 83 pg. The C mass distribution across the analysed unicellular cells was used for modelling cell sizes, which were in the range of 7.6 and 10.1 µm. Determined values were in line with values obtained with complementary techniques (Coulter-counting, total organic C analysis and microscopic analysis).
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Microplásticos , Plásticos , Carbono , Agua de Mar , Espectrometría de Masas en TándemRESUMEN
Standard preparation for elemental bio-imaging by laser ablation-inductively coupled plasma-mass spectrometry is confounded by the chemical and physical differences between standard and sample matrices. These differences lead to variable ablation, aerosol generation and transportation characteristics and must be considered when designing matrix-matched standards for reliable calibration and quantification. The ability to precisely mimic sample matrices is hampered due to the complexity and heterogeneity of biological tissue and small variabilities in standard matrices and sample composition often negatively impact accuracy, precision and robustness. Furthermore, cumbersome preparation protocols may limit reproducibility and traceability. This work presents novel facile methods for the preparation of gelatine standards using both commercial and laboratory-made moulds. Surface roughness, thickness and robustness of the mould-prepared standards were compared against cryo-sectioned gelatine and homogenised brain tissue standards. The mould-prepared standards had excellent thickness accuracy and signal precision which allowed robust quantification, were easier to prepare and therefore easier to reproduce. We also compared gelatine standards prepared from a variety of animal sources and discuss their suitability to calibrate low level elemental concentrations. Finally, we present a simple method to remove background metals in gelatine using various chelating resins to increase the dynamic calibration range and to improve limits of analysis.
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Gelatina/normas , Animales , Química Encefálica , Calibración , Bovinos , Quelantes/química , Peces , Gelatina/química , Terapia por Láser/métodos , Pulmón/química , Masculino , Espectrometría de Masas/métodos , Metales/análisis , Metales/química , Ratones Endogámicos C57BL , Músculo Cuádriceps/química , Estándares de Referencia , Reproducibilidad de los Resultados , Extracción en Fase Sólida/métodos , PorcinosRESUMEN
Growing concern over the environmental and health impacts of per- and polyfluoroalkyl substances (PFASs) has led to the development of increasingly stringent regulatory guidelines. To meet these guidelines for the determination of PFASs in surface-water, solid-phase extraction (SPE) is commonly implemented for clean-up and pre-concentration of samples. In this paper a micro-SPE method for the clean-up and pre-concentration of PFASs from surface-water was developed. A micro-SPE packing phase was created to retain 13 long and short chain PFAS after examining combinations of four 3⯵m particle size sorbents, with the optimal phase consisting of a 50:50 mixture of C18 and aminopropyl silica. Micro-SPE achieved similar results to conventional SPE methods while reducing sample preparation time to 5 min and using only 2â¯mL of sample. The method was validated using spiked recoveries (100â¯ng L-1) from PFAS contaminated surface-water samples with recoveries ranging from 86% to 111% and relative standard deviations below 18%. Concentrations of the PFASs in the samples ranged from below the limit of quantification to 898 ± 15â¯ng L-1. Automation of sample preparation, including the micro-SPE extraction, was also demonstrated. These results show the potential for automated micro-SPE to replace conventional SPE, with the decreases in sample preparation time, sample and solvent volumes crucial for incorporation into routine analyses in commercial laboratories.