Metal-rich organic matter and hot continental passive margin: drivers for Devonian copper-cobalt-germanium mineralization in dolomitized reef-bearing carbonate platform.

The abundance and types of reef-bearing carbonate platforms reflect the evolution of Devonian climate, with conspicuous microbial-algal reefs in the warm Early and Late Devonian and sponge-coral reefs in the cooler Middle Devonian. A dolomitized Wenlock-Lower Devonian microbial-algal reef-bearing carbonate platform hosts epigenetic copper-cobalt-germanium (Cu-Co-Ge) sulfide mineralization at Ruby Creek-Bornite in the Brooks Range, Alaska. Here, we present rhenium-osmium (Re-Os) radiometric ages and molybdenum and sulfur (δ98/95Mo = +2.04 to +5.48‰ and δ34S = -28.5 to -1.8‰) isotope variations for individual Cu-Co-Fe sulfide phases along the paragenetic sequence carrollite-bornite-pyrite. In the context of a hot, extensional passive margin, greenhouse conditions in the Early Devonian favored restriction of platform-top seawater circulation and episodic reflux of oxidized brines during growth of the carbonaceous carbonate platform. Molybdenum and sulfur isotope data signal the stepwise reduction of hot brines carrying Cu during latent reflux and geothermal circulation for at least ca. 15 million years from the Early Devonian until Cu-Co sulfide mineralization ca. 379-378 million years ago (Ma) in the Frasnian, Late Devonian (weighted mean of Re-Os model ages of carrollite at 379 ± 15 Ma [n = 4]; Re-Os isochron age of bornite at 378 ± 15 Ma [n = 6]). On the basis of petrographic relationships between sulfides and solid bitumen, and the Mo and S isotope data for sulfides, we imply that the endowment in critical metals (e.g., Co, Ge, Re) in the Ruby Creek-Bornite deposit is linked to the activity of primary producers that removed trace metals from the warm Early Devonian seawater and concentrated Co, Ge, and Re in algal-bacterial organic matter in carbonate sediments. Supplementary Information: The online version contains supplementary material available at 10.1007/s00126-022-01123-1.

Subcellular antibiotic visualization reveals a dynamic drug reservoir in infected macrophages.

Tuberculosis, caused by the intracellular pathogen Mycobacterium tuberculosis, remains the world's deadliest infectious disease. Sterilizing chemotherapy requires at least 6 months of multidrug therapy. Difficulty visualizing the subcellular localization of antibiotics in infected host cells means that it is unclear whether antibiotics penetrate all mycobacteria-containing compartments in the cell. Here, we combined correlated light, electron, and ion microscopy to image the distribution of bedaquiline in infected human macrophages at submicrometer resolution. Bedaquiline accumulated primarily in host cell lipid droplets, but heterogeneously in mycobacteria within a variety of intracellular compartments. Furthermore, lipid droplets did not sequester antibiotic but constituted a transferable reservoir that enhanced antibacterial efficacy. Thus, strong lipid binding facilitated drug trafficking by host organelles to an intracellular target during antimicrobial treatment.

Hair analysis to monitor adherence to prescribed chronic inhaler drug therapy in patients with asthma or COPD.

BACKGROUND: Poor adherence to inhaled drug therapy in individuals with asthma and/or chronic obstructive pulmonary disease (COPD) may be associated with suboptimal therapeutic outcomes. Measurement of drug residues in hair samples has been employed to assess oral medication use over time. Here, we test the feasibility of analyzing hair samples from patients with asthma and/or COPD for assessing adherence to prescribed inhaled medication. METHODS: In total, 200 male and female subjects, ≥ 18 years of age, with stable asthma and/or COPD who were receiving an acceptable standard of care daily inhaled product consistently, were recruited. Head hair samples were taken during a single visit to the clinical site and grouped by hair color according to the Fischer-Saller scale. Drug residues were extracted from milled hair samples using solid-phase extraction and analyzed using liquid chromatography-tandem mass spectrometry. RESULTS: Inhaled drugs were detected in hair for 72% of subjects from whom it was possible to analyze hair samples (n = 157/200). Most hair samples obtained from subjects receiving formoterol or vilanterol had amounts of drug present that allowed determination of a quantifiable concentration, and demonstrated a dose response. Drugs were detected in all hair colors, with higher concentrations of formoterol observed in dark-haired versus light-haired individuals. CONCLUSIONS: This is the first study to demonstrate that inhaled medication can be measured in hair samples from subjects with asthma and/or COPD. The results show that hair drug concentration data could potentially provide a record of historical adherence to inhaled therapeutics.

The 3D OrbiSIMS-label-free metabolic imaging with subcellular lateral resolution and high mass-resolving power.

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 µm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters-gamma-aminobutyric acid, dopamine and serotonin-with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.

Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS.

ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery.

Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (Fic) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined Fic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. Fic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery.

Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability.

One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.

Expert consensus document: Defining the major health modifiers causing atrial fibrillation: a roadmap to underpin personalized prevention and treatment.

Despite remarkable advances in antiarrhythmic drugs, ablation procedures, and stroke-prevention strategies, atrial fibrillation (AF) remains an important cause of death and disability in middle-aged and elderly individuals. Unstructured management of patients with AF sharply contrasts with our detailed, although incomplete, knowledge of the mechanisms that cause AF and its complications. Altered calcium homeostasis, atrial fibrosis and ageing, ion-channel dysfunction, autonomic imbalance, fat-cell infiltration, and oxidative stress, in addition to a susceptible genetic background, contribute to the promotion, maintenance, and progression of AF. However, clinical management of patients with AF is currently guided by stroke risk parameters, AF pattern, and symptoms. In response to this apparent disconnect between the known pathophysiology of AF and clinical management, we propose a roadmap to develop a set of clinical markers that reflect the major causes of AF in patients. Thereby, the insights into the mechanisms causing AF will be transformed into a format that can underpin future personalized strategies to prevent and treat AF, ultimately informing better patient care.

Alternative surfactants for improved efficiency of in situ tryptic proteolysis of fingermarks.

Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual's fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis.

Demonstrating enhanced throughput of RapidFire mass spectrometry through multiplexing using the JmjD2d demethylase as a model system.

Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true high-throughput screens (HTS) of large compound collections (>1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using "mass-tagged" substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.

Benefits to shorebirds from invasion of a non-native shellfish.

Introductions of non-native species are seen as major threats to ecosystem function and biodiversity. However, invasions of aquatic habitats by non-native species are known to benefit generalist consumers that exhibit dietary switches and prey upon the exotic species in addition to or in preference to native ones. There is, however, little knowledge concerning the population-level implications of such dietary changes. Here, we show that the introduction of the Manila clam Tapes philippinarum into European coastal waters has presented the Eurasian oystercatcher Haematopus ostralegus ostralegus with a new food resource and resulted in a previously unknown predator-prey interaction between these species. We demonstrate, with an individuals-based simulation model, that the presence of this non-native shellfish, even at the current low density, has reduced the predicted over-winter mortality of oystercatchers at one recently invaded site. Further increases in clam population density are predicted to have even more pronounced effects on the density dependence of oystercatcher over-winter mortality. These results suggest that if the Manila clam were to spread around European coastal waters, a process which is likely to be facilitated by global warming, this could have considerable benefits for many shellfish-eating shorebird populations.