Pulmonary rehabilitation restores limb muscle mitochondria and improves the intramuscular metabolic profile.

BACKGROUND: Exercise, as the cornerstone of pulmonary rehabilitation, is recommended to chronic obstructive pulmonary disease (COPD) patients. The underlying molecular basis and metabolic process were not fully elucidated. METHODS: Sprague-Dawley rats were classified into five groups: non-COPD/rest ( n  = 8), non-COPD/exercise ( n  = 7), COPD/rest ( n  = 7), COPD/medium exercise ( n  = 10), and COPD/intensive exercise ( n  = 10). COPD animals were exposed to cigarette smoke and lipopolysaccharide instillation for 90 days, while the non-COPD control animals were exposed to room air. Non-COPD/exercise and COPD/medium exercise animals were trained on a treadmill at a decline of 5° and a speed of 15 m/min while animals in the COPD/intensive exercise group were trained at a decline of 5° and a speed of 18 m/min. After eight weeks of exercise/rest, we used ultrasonography, immunohistochemistry, transmission electron microscopy, oxidative capacity of mitochondria, airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI), and transcriptomics analyses to assess rectal femoris (RF). RESULTS: At the end of 90 days, COPD rats' weight gain was smaller than control by 59.48 ±â€Š15.33 g ( P  = 0.0005). The oxidative muscle fibers proportion was lower ( P  < 0.0001). At the end of additional eight weeks of exercise/rest, compared to COPD/rest, COPD/medium exercise group showed advantages in weight gain, femoral artery peak flow velocity (Δ58.22 mm/s, 95% CI: 13.85-102.60 mm/s, P  = 0.0104), RF diameters (Δ0.16 mm, 95% CI: 0.04-0.28 mm, P  = 0.0093), myofibrils diameter (Δ0.06 µm, 95% CI: 0.02-0.10 µm, P  = 0.006), oxidative muscle fiber percentage (Δ4.84%, 95% CI: 0.15-9.53%, P  = 0.0434), mitochondria oxidative phosphorylate capacity ( P  < 0.0001). Biomolecules spatial distribution in situ and bioinformatic analyses of transcriptomics suggested COPD-related alteration in metabolites and gene expression, which can be impacted by exercise. CONCLUSION: COPD rat model had multi-level structure and function impairment, which can be mitigated by exercise.

New trends of new psychoactive substances (NPS)-infused chocolate: Identification and quantification of trace level of NPS in complex matrix by GC-MS and NMR.

For the first time, the identification and quantification of trace level of new psychoactive substances (NPS) in a complex chocolate matrix have been reported. Since the beginning of 2022, suspected NPS-infused chocolate samples confiscated in inbound packages have been continuously sent to our laboratory for analysis. The qualitative gas chromatography-mass spectrometry (GC-MS) results were verified by 1H nuclear magnetic resonance (1H NMR) and 19F NMR to distinguish between potential aromatic isomers. A total of 11 NPS including deoxymethoxetamine, 3-OH-PCP, 6-APB, 4-APB, 4-OH-MiPT, 3-FEA, 2-FEA, 3-MMC, bromazolam, 2-FDCK, and ADB-BUTINACA were detected in 65 seized chocolate samples. A general 1H quantitative NMR (1H qNMR) method for quantification of 297 types of NPS in complex chocolate matrixes was devised for the first time after rigorous analysis of various critical features of merit, including suitable deuterated solvent, internal standard, quantitative peaks, and instrument acquisition parameters. Validation of the method using six different types of NPS afforded limits of detection of 0.05-0.1 mg/mL, limits of quantification of 0.01-0.03 mg/mL, repeatability and reproducibility lower than 0.5% and 3.6%, recoveries of 91.7%∼104.4%, and absence of matrix effect. The quantitative analysis of 65 seized chocolate samples by 1H qNMR and 19F qNMR showed that the content of NPS was in the range of 0.5 mg/g∼44.1 mg/g. Generally, the developed qNMR method was simple, fast, precise, and can be performed without reference materials of NPS. Since the type and content of NPS are relatively random, chocolate consumers will face huge health risks. Therefore, this new trend of NPS-infused chocolate deserves and requires more attention from national NPS monitoring departments as well as forensic laboratories.

SEAM is a spatial single nuclear metabolomics method for dissecting tissue microenvironment.

Spatial metabolomics can reveal intercellular heterogeneity and tissue organization. Here we report on the spatial single nuclear metabolomics (SEAM) method, a flexible platform combining high-spatial-resolution imaging mass spectrometry and a set of computational algorithms that can display multiscale and multicolor tissue tomography together with identification and clustering of single nuclei by their in situ metabolic fingerprints. We first applied SEAM to a range of wild-type mouse tissues, then delineated a consistent pattern of metabolic zonation in mouse liver. We further studied the spatial metabolic profile in the human fibrotic liver. We discovered subpopulations of hepatocytes with special metabolic features associated with their proximity to the fibrotic niche, and validated this finding by spatial transcriptomics with Geo-seq. These demonstrations highlighted SEAM's ability to explore the spatial metabolic profile and tissue histology at the single-cell level, leading to a deeper understanding of tissue metabolic organization.

In Situ Identification and Spatial Mapping of Microplastic Standards in Paramecia by Secondary-Ion Mass Spectrometry Imaging.

Microplastics (MPs) are universally present in the ecosystem and pose great threats to the environment and living organisms. Research studies have shown that small MPs (<50 µm in diameter) are especially toxic and account for more than half of all MPs collected in the Atlantic Ocean. Nevertheless, current methods for the detection and analysis of MPs are incapable of achieving rapid and in situ analysis of small MPs in the biota to ultimately enable the study of their biological effects. In this work, we report a method that allows rapid in situ identification and spatial mapping of small MPs directly from paramecia with high accuracy by acquiring chemical composition information using secondary-ion mass spectrometry (SIMS) imaging. Specifically, six types of common MPs (polymethyl methacrylate, polyvinyl chloride, polypropylene, polyethylene terephthalate, polyglycidyl methacrylate, and polyamide 6) with a diameter of 1-50 µm were simultaneously imaged with high chemical specificity at a spatial resolution of 700 nm. In situ spatial mapping of a group of MPs ingested by paramecia was performed using SIMS fragments specific to the plastic composition with no sample pretreatment, revealing the aggregation of MPs in paramecia after ingestion. Compared with existing methods, one additional advantage of the developed method is that the MPs and the organism can be analyzed in the same experimental workflow to record their fingerprint spectra, acquiring biochemical information to evaluate MP fate, toxicity, and the MP-biota interaction.

Metabolic fingerprinting of cell types in mouse skeletal muscle by combining TOF-SIMS with immunofluorescence staining.

Skeletal muscle tissue is composed of various muscle cell types which differ in physiological functions. Changes in cell type composition of skeletal muscle are associated with the development of metabolic diseases. Skeletal muscle cell types are currently distinguished by immunofluorescence (IF) staining based on myosin heavy chain (MHC) isoform difference. However, it remains a challenge to provide metabolic fingerprints of different muscle cell types by IF staining. Therefore, in this study, we proposed a method to examine metabolite distribution within different cell types by time-of-flight secondary ion mass spectrometry (TOF-SIMS) with high spatial resolution. Skeletal muscle samples from C57/BL6 mice were obtained by slicing. Cell types in TOF-SIMS images were labelled corresponding to IF images from the same region of serially cut sections. Mass spectra corresponding to individual muscle cells were extracted to compare metabolic fingerprints among cell types. Skeletal muscle cells were classified into two clusters based on the mass spectra of individual cells. Unsaturated diacylglycerol (DG) and fatty acid (FA) species were found to be distributed in a cell-type dependent manner. Moreover, relative quantification showed that the content of unsaturated DGs, oleic acid and linoleic acid was higher in type I and type IIA cells than in type IIB cells. TOF-SIMS in combination with IF enables us to directly visualize metabolite distribution in different cell types, to find potential biomarkers for cell type classification. TOF-SIMS imaging coupled with IF staining has been proved to be a promising tool for metabolic fingerprinting of different skeletal muscle cell types.

Tuning the pKa of Carboxyfluorescein with Arginine-Rich Cell-Penetrating Peptides for Intracellular pH Imaging.

5-Carboxylfluorescein (FAM) is a conventional pH-responsive fluorophore widely used in fluorescence labeling and imaging. Because of its nonfluorescent structure under acidic conditions, FAM has long been limited to pH determination in a neutral-basic environment. Here, we modified the optical properties of FAM with cationic arginine-rich cell-penetrating peptides (CPPs), tuning the pKa value of FAM to adapt well to pH measurement under diverse pH conditions. With increasing length of polyarginine, the pKa value of FAM was tuned from 6.20 ± 0.06 to 5.17 ± 0.05. The key mechanism for pKa variations was attributed to intramolecular electrostatic attraction and the positive charge of cationic CPPs tend to stabilize the fluorescent dianionic form of FAM. Apart from tunable pKa, arginine-rich CPPs also improved the water solubility, membrane permeability, and organelle-specific localization of FAM. Two conjugated probes FAM-R12 and FAM-(Fxr)3 were selected to monitor intracellular pH fluctuations. Compared to FAM-(Fxr)3, highly positively charged FAM-R12 was more effective in lower pH condition and realized targeted visualization of lysosomal pH changes. The arginine-rich CPP-based strategy offers a promising approach to obtain optimized fluorescent pH probes with adjustable pKa values for organelle-specific pH measurement.

Vacuum Ultraviolet Laser Desorption/Ionization Mass Spectrometry Imaging of Single Cells with Submicron Craters.

Mass spectrometry imaging (MSI) is a crucial label-free method to distinguish the localization patterns in single cells. MALDI-TOF MS and ToF-SIMS are now bearing the responsibility. However, MALDI-TOF MS is limited to micron spatial resolution and ToF-SIMS suffers from severe molecular fragmentation. Here, we proposed a new MSI methodology of vacuum ultraviolet laser desorption/ionization (VUVDI) with high spatial resolution, achieving higher ion yields and less fragmentation compared with ToF-SIMS at submicron level. The fluorescence image and mass spectrum of VUVDI were obtained simultaneously. In addition, the adjustable laser fluence acquired selective detection for different molecular and fragmental ions, thus realizing molecular identification. Furthermore, MSIs of single cells with submicron craters were presented. These results suggest VUVDI is a potential mass spectrometry method that provides a soft ionization source and submicron spatial resolution for molecular analysis in life science.

A cell-penetrating ratiometric probe for simultaneous measurement of lysosomal and cytosolic pH change.

A new ratiometric fluorescent probe based on cell-penetrating peptides (CPPs) was constructed for whole-cell pH mapping and simultaneous measurement of pH changes in the cytoplasm and lysosomes. The arginine-rich CPP, R12K worked as linker, carrier and part of the fluorophore. Benefiting from R12K, the fluorescent probe is completely water soluble, membrane permeable and well biocompatible. It shows high selectivity, sensitivity and reversibility to pH fluctuations. The ratio of fluorescence intensities F519/F582 increased from 0.2 to 9.2 over the pH range from 3.3 to 8.1. Intracellular pH mapping was successfully realized owing to the wide distribution of the probe in live cells (even in nucleus). Moreover, cytosolic and lysosomal pH change caused by the stimuli can be simultaneously detected. Compared to other ratiometric pH probes, RhB-R12K-FITC can provide more precise information about H+ redistribution between different cellular compartments.

Chemical Visualization of Sweat Pores in Fingerprints Using GO-Enhanced TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used in imaging of small molecules (<500 Da) in fingerprints, such as gunshot residues and illicit drugs. However, identifying and mapping relatively high mass molecules are quite difficult owing to insufficient ion yield of their molecular ions. In this report, graphene oxide (GO)-enhanced TOF-SIMS was used to detect and image relatively high mass molecules such as poison, alkaloids (>600 Da) and controlled drugs, and antibiotics (>700 Da) in fingerprints. Detail features of fingerprints such as the number and distribution of sweat pores in a ridge and even the delicate morphology of one pore were clearly revealed in SIMS images of relatively high mass molecules. The detail features combining with identified chemical composition were sufficient to establish a human identity and link the suspect to a crime scene. The wide detectable mass range and high spatial resolution make GO-enhanced TOF-SIMS a promising tool in accurate and fast analysis of fingerprints, especially in fragmental fingerprint analysis.

Imaging specific newly synthesized proteins within cells by fluorescence resonance energy transfer.

Metabolic azide amino acid labelling followed by the use of bioorthogonal chemistry is an efficient technique for imaging newly synthesized proteins. Recently, AHA-labelling together with the proximity-ligation assay was used to identify newly synthesized proteins of interest (POI) (Tom Dieck et al., Nat. Meth. 2015, 12, 411). Here we build on this study replacing the proximity-ligation assay with FRET to improve the spatial resolution. Herein, we develop a FRET-based strategy for imaging the newly synthesized endogenous POI within cells: a FRET acceptor is installed onto the newly synthesized proteins via click chemistry, and a FRET donor onto the POI via immunocytochemistry. We found that a photobleaching based FRET efficiency imaging mode and a fluorescence lifetime imaging mode showed the distribution of newly synthesized proteins more accurately compared to the direct observation of FRET signals. We demonstrated the capability of this FRET-based imaging method by visualizing several newly synthesized proteins including TDP-43, tubulin and CaMKIIα in different cell lines. This novel analytical imaging method could be used to visualize other specific endogenous proteins of interest in situ.

Cell-Penetrating Peptide Spirolactam Derivative as a Reversible Fluorescent pH Probe for Live Cell Imaging.

A colorless and nonfluorescent spirolactam derivative, RhB-R12K, was synthesized by amide condensation between the carboxyl group of rhodamine B (RhB) and the amino group of cell-penetrating peptide (CPP). The fluorescence intensity of RhB-R12K sharply increased as the pH value decreased from 8.0 to 4.9, demonstrating sensitive and reversible response to intracellular pH distribution. This CPP probe was completely water soluble, had low cytotoxicity, was membrane permeable, and was suitable for pH measurement in various organelles by choosing organelle-specific CPP sequences. Interestingly, CPPs acted not only as carriers but also as indispensable parts of fluorophores here. The presence of active groups on the peptides potentially allows for modification with additional dyes to construct multifunctional and ratiometric probes for cell imaging.

Graphene Oxide as a Novel Evenly Continuous Phase Matrix for TOF-SIMS.

Using matrix to enhance the molecular ion signals for biomolecule identification without loss of spatial resolution caused by matrix crystallization is a great challenge for the application of TOF-SIMS in real-world biological research. In this report, graphene oxide (GO) was used as a matrix for TOF-SIMS to improve the secondary ion yields of intact molecular ions ([M + H]+). Identifying and distinguishing the molecular ions of lipids (m/z >700) therefore became straightforward. The spatial resolution of TOF-SIMS imaging could also be improved as GO can form a homogeneous layer of matrix instead of crystalline domain, which prevents high spatial resolution in TOF-SIMS imaging. Lipid mapping in presence of GO revealed the delicate morphology and distribution of single vesicles with a diameter of 800 nm. On GO matrix, the vesicles with similar shape but different chemical composition could be distinguished using molecular ions. This novel matrix holds potentials in such applications as the analysis and imaging of complex biological samples by TOF-SIMS. Graphical Abstract ᅟ.