Nanoparticles (NPs) elicit sterile inflammation, but the underlying signaling pathways are poorly understood. Here, we report that human monocytes are particularly vulnerable to amorphous silica NPs, as evidenced by single-cell-based analysis of peripheral blood mononuclear cells using cytometry by time-of-flight (CyToF), while silane modification of the NPs mitigated their toxicity. Using human THP-1 cells as a model, we observed cellular internalization of silica NPs by nanoscale secondary ion mass spectrometry (nanoSIMS) and this was confirmed by transmission electron microscopy. Lipid droplet accumulation was also noted in the exposed cells. Furthermore, time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed specific changes in plasma membrane lipids, including phosphatidylcholine (PC) in silica NP-exposed cells, and subsequent studies suggested that lysophosphatidylcholine (LPC) acts as a cell autonomous signal for inflammasome activation in the absence of priming with a microbial ligand. Moreover, we found that silica NPs elicited NLRP3 inflammasome activation in monocytes, whereas cell death transpired through a non-apoptotic, lipid peroxidation-dependent mechanism. Together, these data further our understanding of the mechanism of sterile inflammation.

Antisense oligonucleotide (ASO)-based therapeutics hold great potential for the treatment of a variety of diseases. Therefore, a better understanding of cellular delivery, uptake, and trafficking mechanisms of ASOs is highly important for early-stage drug discovery. In particular, understanding the biodistribution and quantifying the abundance of ASOs at the subcellular level are needed to fully characterize their activity. Here, we used a combination of electron microscopy and NanoSIMS to assess the subcellular concentrations of a 34S-labeled GalNAc-ASO and a naked ASO in the organelles of primary human hepatocytes. We first cross-validated the method by including a 127I-labeled ASO, finding that the absolute concentration of the lysosomal ASO using two independent labeling strategies gave matching results, demonstrating the strength of our approach. This work also describes the preparation of external standards for absolute quantification by NanoSIMS. For both the 34S and 127I approaches used for our quantification methodology, we established the limit of detection (5 and 2 μM, respectively) and the lower limit of quantification (14 and 5 μM, respectively).

For decades, "all-or-none" and "kiss-and-run" were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with 13C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of 13C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope 127I, was introduced during stimulation. A significant drop in the level of 13C-labeled dopamine and a reduction in vesicle size, with an increasing level of 127I-, was observed in vesicles of stimulated cells. Colocalization of 13C and 127I- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically.

The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted endocytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to efficient target knockdown, specifically in pancreatic β-cells. It is presumed that ASOs dissociate from GLP1 intracellularly to enable an ASO interaction with its target RNA. It is unknown where or when this happens following GLP1-ASO binding to GLP1R and endocytosis. Here, we use correlative nanoscale secondary ion mass spectroscopy (NanoSIMS) and transmission electron microscopy to explore GLP1-ASO subcellular trafficking in GLP1R overexpressing HEK293 cells. We isotopically label both eGLP1 and ASO, which do not affect the eGLP1-ASO conjugate function. We found that the eGLP1 peptide and ASO are not detected at the same level in the same endosomes, within 30 min of GLP1R-HEK293 cell exposure to eGLP1-ASO. When we utilized different linker chemistry to stabilize the GLP1-ASO conjugate, we observed more ASO located with GLP1 compared to cell incubation with the less stable conjugate. Overall, our work suggests that the ASO separates from GLP1 relatively early in the endocytic pathway, and that linker chemistry might impact the GLP1-ASO function.

Filamentous, heterocyst-forming cyanobacteria represent a paradigm of multicellularity in the prokaryotic world. Physiological studies at the cellular level in model organisms are crucial to understand metabolic activities and qualify specific aspects related to multicellularity.

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.

Fluid-mediated mineral dissolution and reprecipitation processes are the most common mineral reaction mechanism in the solid Earth and are fundamental for the Earth’s internal dynamics. Element exchange during such mineral reactions is commonly thought to occur via aqueous solutions with the mineral solubility in the coexisting fluid being a rate limiting factor. Here we show in high-pressure/low temperature rocks that element transfer during mineral dissolution and reprecipitation can occur in an alkali-Al–Si-rich amorphous material that forms directly by depolymerization of the crystal lattice and is thermodynamically decoupled from aqueous solutions. Depolymerization starts along grain boundaries and crystal lattice defects that serve as element exchange pathways and are sites of porosity formation. The resulting amorphous material occupies large volumes in an interconnected porosity network. Precipitation of product minerals occurs directly by repolymerization of the amorphous material at the product surface. This mechanism allows for significantly higher element transport and mineral reaction rates than aqueous solutions with major implications for the role of mineral reactions in the dynamic Earth.

Ichthyosaurs are extinct marine reptiles that display a notable external similarity to modern toothed whales. Here we show that this resemblance is more than skin deep. We apply a multidisciplinary experimental approach to characterize the cellular and molecular composition of integumental tissues in an exceptionally preserved specimen of the Early Jurassic ichthyosaur Stenopterygius . Our analyses recovered still-flexible remnants of the original scaleless skin, which comprises morphologically distinct epidermal and dermal layers. These are underlain by insulating blubber that would have augmented streamlining, buoyancy and homeothermy. Additionally, we identify endogenous proteinaceous and lipid constituents, together with keratinocytes and branched melanophores that contain eumelanin pigment. Distributional variation of melanophores across the body suggests countershading, possibly enhanced by physiological adjustments of colour to enable photoprotection, concealment and/or thermoregulation. Convergence of ichthyosaurs with extant marine amniotes thus extends to the ultrastructural and molecular levels, reflecting the omnipresent constraints of their shared adaptation to pelagic life.

Significance Comets are pristine, volatile-rich objects formed beyond the orbits of the gas giants and, thus, thought to preserve a record of the primordial molecular cloud material parental to our Solar System. We use magnesium and chromium isotopes to show that a class of pristine chondrites, the metal-rich carbonaceous chondrites, has a signature distinct from most inner Solar System planets and asteroids. This signature is consistent with that predicted for unprocessed primordial molecular cloud material, suggesting that—similar to comets—metal-rich carbonaceous chondrites are samples of asteroids that accreted in the outer Solar System. Therefore, these objects may provide a direct window into the formation history of the outer Solar System.

We have measured the abundance and isotopic composition of water in apatites from several lunar rocks representing Potassium (K), Rare Earth Elements (REE), and Phosphorus (P) − KREEP − rich lithologies, including felsites, quartz monzodiorites (QMDs), a troctolite, and an alkali anorthosite. The H-isotope data from apatite provide evidence for multiple reservoirs in the lunar interior. Apatite measurements from some KREEP-rich intrusive rocks display moderately elevated δD signatures, while other samples show δD signatures similar to the range known for the terrestrial upper mantle. Apatite grains in Apollo 15 quartz monzodiorites have the lowest δD values measured from the Moon so far (as low as −749‰), and could potentially represent a D-depleted reservoir in the lunar interior that had not been identified until now. Apatite in all of these intrusive rocks contains <267ppm H2O, which is relatively low compared to apatites from the majority of studied mare basalts (200 to >6500ppm H2O). Complexities in partitioning of volatiles into apatite make this comparison uncertain, but measurements of residual glass in KREEP basalt fragments in breccia 15358 independently show that the KREEP basaltic magmas were low in water. The source of 15358 contained ∼10ppm H2O, about an order of magnitude lower than the source of the Apollo 17 pyroclastic glass beads, suggesting potential variations in the distribution of water in the lunar interior.

We describe a procedure to determine precise and accurate elemental abundance by means of quantitative imaging using secondary ion mass spectrometry (on images covering similar to 10 x 10 mu m(2)) applied to natural Insoluble Organic Matter (IOM). Dynamic SIMS conditions are reached for a 16 keV Cs+ fluence of >2.0 x 10(17) Cs+ cm(-2) implanted at the surface of the IOM. Once the sample surface is saturated in cesium, steady-state implantation and sputtering yield are reached: constant secondary ion count rates are then observed. Two calibrations of the nitrogen abundance are presented. The nitrogen abundance is expressed either as [N] or N/C atomic ratio calibrated by means of (CN-)-C-12-N-14/C-12(-) or (CN-)-C-12-N-14/C-12(2)- ionic ratios respectively. The (CN-)-C-12-N-14/C-12(-) uncertainty is always larger than the uncertainty of (CN-)-C-12-N-14/C-12(2)-. At a smaller scale inside an image (similar to 1 x 1 mu m(2)), a little larger than the primary beam size, processes induced by topographic irregularity of the IOM powder result in a variation of the (CN-)-C-12-N-14, C-12(2)- and C-12(-) count rates and increase the variability of the ionic ratios. The chemical contrast produced by a rough sample surface exposed to the Cs+ rastering is evaluated with a scratched epoxy resin for (CN-)-C-12-N-14/C-12(-) and (CN-)-C-12-N-14/C-12(2)- elemental ratios. Owing to similarities in emission parameters, the determination of the N/C ratio in rough organic surfaces is improved by using (CN-)-C-12-N-14/C-12(2)- instead of (CN-)-C-12-N-14/C-12(-).

Quantification of water content is relevant in various topics in geology and planetary sciences. NanoSIMS has capabilities for high spatial resolution imaging and offers opportunities to accurately quantify water contents at fine scale on small surface areas. The main concern using ion microprobe techniques is to estimate and minimize contribution of water contamination, from residual gas in the sample chamber, sticking onto the surface of the sample. Here we tackle a set of sputtering/analytical parameters and we evaluate their relative influence on the OH−/Si− ratio. We demonstrate that a high erosion rate, reached using a primary beam intensity of ~25pA, is sufficient to lower this OH contamination for basaltic glass. This leads us to describe a procedure to correct for OH contamination and thus determine accurate values of OH/Si ratio in order to quantify water contents in silicate materials using NanoSIMS imaging.

The insoluble organic matter (IOM) of an unequilibrated enstatite chondrite Sahara (SAH) 97096 has been investigated using a battery of analytical techniques. As the enstatite chondrites are thought to have formed in a reduced environment at higher temperatures than carbonaceous chondrites, they constitute an interesting comparative material to test the heterogeneities of the IOM in the solar system and to constrain the processes that could affect IOM during solar system evolution. The SAH 97096 IOM is found in situ: as submicrometer grains in the network of fine-grained matrix occurring mostly around chondrules and as inclusions in metallic nodules, where the carbonaceous matter appears to be more graphitized. IOM in these two settings has very similar delta(15)N and delta(13)C; this supports the idea that graphitized inclusions in metal could be formed by metal catalytic graphitization of matrix IOM. A detailed comparison between the IOM extracted from a fresh part and a terrestrially weathered part of SAH 97096 shows the similarity between both IOM samples in spite of the high degree of mineral alteration in the latter. The isolated IOM exhibits a heterogeneous polyaromatic macromolecular structure, sometimes highly graphitized, without any detectable free radicals and deuterium-heterogeneity and having mean H-and N-isotopic compositions in the range of values observed for carbonaceous chondrites. It contains some submicrometer-sized areas highly enriched in (15)N (delta(15)N up to 1600 parts per thousand). These observations reinforce the idea that the IOM found in carbonaceous chondrites is a common component widespread in the solar system. Most of the features of SAH 97096 IOM could be explained by the thermal modification of this main component.