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Molecular & Cellular Proteomics : MCP Jun 2024Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a...
Microglia are resident immune cells of the brain and regulate its inflammatory state. In neurodegenerative diseases, microglia transition from a homeostatic state to a state referred to as disease associated microglia (DAM). DAM express higher levels of proinflammatory signaling molecules, like STAT1 and TLR2, and show transitions in mitochondrial activity toward a more glycolytic response. Inhibition of Kv1.3 decreases the proinflammatory signature of DAM, though how Kv1.3 influences the response is unknown. Our goal was to identify the potential proteins interacting with Kv1.3 during transition to DAM. We utilized TurboID, a biotin ligase, fused to Kv1.3 to evaluate potential interacting proteins with Kv1.3 via mass spectrometry in BV-2 microglia following TLR4-mediated activation. Electrophysiology, western blotting, and flow cytometry were used to evaluate Kv1.3 channel presence and TurboID biotinylation activity. We hypothesized that Kv1.3 contains domain-specific interactors that vary during a TLR4-induced inflammatory response, some of which are dependent on the PDZ-binding domain on the C-terminus. We determined that the N-terminus of Kv1.3 is responsible for trafficking Kv1.3 to the cell surface and mitochondria (e.g. NUDC, TIMM50). Whereas, the C-terminus interacts with immune signaling proteins in an LPS-induced inflammatory response (e.g. STAT1, TLR2, and C3). There are 70 proteins that rely on the C-terminal PDZ-binding domain to interact with Kv1.3 (e.g. ND3, Snx3, and Sun1). Furthermore, we used Kv1.3 blockade to verify functional coupling between Kv1.3 and interferon-mediated STAT1 activation. Overall, we highlight that the Kv1.3 potassium channel functions beyond conducting the outward flux of potassium ions in an inflammatory context and that Kv1.3 modulates the activity of key immune signaling proteins, such as STAT1 and C3.
PubMed: 38936775
DOI: 10.1016/j.mcpro.2024.100809 -
MSystems Jun 2024Airway microbiota are known to contribute to lung diseases, such as cystic fibrosis (CF), but their contributions to pathogenesis are still unclear. To improve our...
Airway microbiota are known to contribute to lung diseases, such as cystic fibrosis (CF), but their contributions to pathogenesis are still unclear. To improve our understanding of host-microbe interactions, we have developed an integrated analytical and bioinformatic mass spectrometry (MS)-based metaproteomics workflow to analyze clinical bronchoalveolar lavage (BAL) samples from people with airway disease. Proteins from BAL cellular pellets were processed and pooled together in groups categorized by disease status (CF vs. non-CF) and bacterial diversity, based on previously performed small subunit rRNA sequencing data. Proteins from each pooled sample group were digested and subjected to liquid chromatography tandem mass spectrometry (MS/MS). MS/MS spectra were matched to human and bacterial peptide sequences leveraging a bioinformatic workflow using a metagenomics-guided protein sequence database and rigorous evaluation. Label-free quantification revealed differentially abundant human peptides from proteins with known roles in CF, like neutrophil elastase and collagenase, and proteins with lesser-known roles in CF, including apolipoproteins. Differentially abundant bacterial peptides were identified from known CF pathogens (e.g., ), as well as other taxa with potentially novel roles in CF. We used this host-microbe peptide panel for targeted parallel-reaction monitoring validation, demonstrating for the first time an MS-based assay effective for quantifying host-microbe protein dynamics within BAL cells from individual CF patients. Our integrated bioinformatic and analytical workflow combining discovery, verification, and validation should prove useful for diverse studies to characterize microbial contributors in airway diseases. Furthermore, we describe a promising preliminary panel of differentially abundant microbe and host peptide sequences for further study as potential markers of host-microbe relationships in CF disease pathogenesis.IMPORTANCEIdentifying microbial pathogenic contributors and dysregulated human responses in airway disease, such as CF, is critical to understanding disease progression and developing more effective treatments. To this end, characterizing the proteins expressed from bacterial microbes and human host cells during disease progression can provide valuable new insights. We describe here a new method to confidently detect and monitor abundance changes of both microbe and host proteins from challenging BAL samples commonly collected from CF patients. Our method uses both state-of-the art mass spectrometry-based instrumentation to detect proteins present in these samples and customized bioinformatic software tools to analyze the data and characterize detected proteins and their association with CF. We demonstrate the use of this method to characterize microbe and host proteins from individual BAL samples, paving the way for a new approach to understand molecular contributors to CF and other diseases of the airway.
PubMed: 38934598
DOI: 10.1128/msystems.00929-23 -
MSystems Jun 2024The application of fecal metaproteomics to large-scale studies of the gut microbiota requires high-throughput analysis and standardized experimental protocols. Although...
The application of fecal metaproteomics to large-scale studies of the gut microbiota requires high-throughput analysis and standardized experimental protocols. Although high-throughput protein cleanup and digestion methods are increasingly used in shotgun proteomics, no studies have yet critically compared such protocols using human fecal samples. In this study, human fecal protein extracts were processed using several different protocols based on three main approaches: filter-aided sample preparation (FASP), solid-phase-enhanced sample preparation (SP3), and suspension trapping (S-Trap). These protocols were applied in both low-throughput (i.e., microtube-based) and high-throughput (i.e., microplate-based) formats, and the final peptide mixtures were analyzed by liquid chromatography coupled to high-resolution tandem mass spectrometry. The FASP-based methods and the combination of SP3 with in-StageTips (iST) yielded the best results in terms of the number of peptides identified through a database search against gut microbiome and human sequences. The efficiency of protein digestion, the ability to preserve hydrophobic peptides and high molecular weight proteins, and the reproducibility of the methods were also evaluated for the different protocols. Other relevant variables, including interindividual variability of stool, duration of protocols, and total costs, were considered and discussed. In conclusion, the data presented here can significantly contribute to the optimization and standardization of sample preparation protocols in human fecal metaproteomics. Furthermore, the promising results obtained with the high-throughput methods are expected to encourage the development of automated workflows and their application to large-scale gut microbiome studies.IMPORTANCEFecal metaproteomics is an experimental approach that allows the investigation of gut microbial functions, which are involved in many different physiological and pathological processes. Standardization and automation of sample preparation protocols in fecal metaproteomics are essential for its application in large-scale studies. Here, we comparatively evaluated different methods, available also in a high-throughput format, enabling two key steps of the metaproteomics analytical workflow (namely, protein cleanup and digestion). The results of our study provide critical information that may be useful for the optimization of metaproteomics experimental pipelines and their implementation in laboratory automation systems.
PubMed: 38934547
DOI: 10.1128/msystems.00661-24 -
Heliyon Jun 2024(PC) is a traditional Chinese medicine (TCM) and food as well as an important essential oil plant in China. PC essential oil exerts pharmacological effects such as...
GC-MS method for simultaneous determination and pharmacokinetic investigation of five volatile components in rat plasma after oral administration of the essential oil extract of .
(PC) is a traditional Chinese medicine (TCM) and food as well as an important essential oil plant in China. PC essential oil exerts pharmacological effects such as anti-inflammatory, anti-oxidant, anti-platelet, anti-thrombotic, and anti-depressant. This study established a reliable and sensitive gas chromatography-mass spectrometry (GC-MS) method for the simultaneous determination of the pharmacokinetics of patchouli alcohol, β-elemene, β-caryophyllene, caryophyllene oxide, and farnesol in the plasma of rats after oral administration of PC essential oil extract. Using ethyl acetate to prepare the plasma samples, and p-menthone was used as the internal standard (IS). An HP5-MS column (0.25 μm × 0.25 mm × 30 m) was used for chromatographic separation, and detection was performed in selected ion monitoring (SIM) mode. The accuracies of intra-day and inter-day for all analytes displayed a range of -6.7 %-9.2 %, with precision below 12.5 %. Extraction recoveries for analytes ranged from 74.0 to 106.4 % and matrix effects ranged from 92.4 to 106.9 %. Stability results have demonstrated that the relative standard deviations (RSD) of analytes were below 12.1 %. Therefore, the developed GC-MS method successfully evaluated the pharmacokinetics of five volatile components in PC essential oil extract administered orally to rats.
PubMed: 38933986
DOI: 10.1016/j.heliyon.2024.e32444 -
Heliyon Jun 2024Colon cancer is a common gastrointestinal malignancy that ranks third in incidence among gastrointestinal cancers. Therefore, screening bioactive compounds for treatment...
Colon cancer is a common gastrointestinal malignancy that ranks third in incidence among gastrointestinal cancers. Therefore, screening bioactive compounds for treatment of colon cancer is urgently needed. L. (SO) has been demonstrated that the extractions or monomers possess potential anti-tumor effect. In this study, we firstly used cell membrane chromatography (CMC) and ultra-performance liquid chromatography coupled with (quadrupole) time-of-flight mass spectrometry (UHPLC-(Q) TOF-MS/MS) to identify a novel active ingredient, octyl gallate (OG), from SO methanol extract (SO-MtOH). HCT116 and SW620 cells lines were used for research, which showed OG presents great anti-colon cancer effect by inhibiting proliferation, inducing apoptosis, and repressing the migration and invasion. Furthermore, SW620 bearing athymic nude mice was used to investigate the potential antitumor activity , which exhibited OG treatment remarkably lessened the tumor volume. Mechanism studies showed that OG downregulated the PI3K/AKT/mTOR signaling axis and induced apoptosis by upregulating the Bax/Bcl-2 protein and the cleaved caspase-3, caspase-9. In conclusion, our research innovatively applied the method of CMC to intriguingly unearth the potential anti-colon cancer ingredient OG and demonstrated its the great antineoplastic activity, which provide a new insight for researchers efficiently developing the novel apoptosis-inducing compound for colon cancer therapy.
PubMed: 38933948
DOI: 10.1016/j.heliyon.2024.e32230 -
Frontiers in Nutrition 2024Lignans are phytoestrogens found in various forms such as glycosides, ester-linked oligomers, and aglycones in a variety of foods, including soy products, legumes,...
Lignans are phytoestrogens found in various forms such as glycosides, ester-linked oligomers, and aglycones in a variety of foods, including soy products, legumes, grains, nuts, vegetables, and fruits. This study aimed to optimize the extraction of lignans from cereal grains using response surface methodology (RSM). Lignans, including secoisolariciresinol (Seco), matairesinol (Mat), pinoresinol (Pin), lariciresinol (Lar), and syringaresinol (Syr), were quantified using high-performance liquid chromatography-tandem mass spectrometry. A Box-Behnken design was employed to determine the optimal values for three extraction parameters: temperature (X1: 20°C-60°C), methanol concentration (X2: 60%-100%), and extraction time (X3: 30-90 min). The highest lignan contents were obtained at X1 = 44.24°C, X2 = 84.64%, and X3 = 53.63 min. To apply these experimental conditions to the actual experiment, the optimal conditions were slightly adjusted to X1 = 40°C, X2 = 80%, and X3 = 60 min. The predicted results closely matched the experimental results obtained using the modified optimal extraction conditions. The highest lignan content found in barley sprouts (85.930 μg/100 g), however, most grains exhibited relatively low concentrations of lignans. These findings provide valuable insights into the lignan content of grains and contribute to the generation of reliable data in this field.
PubMed: 38933882
DOI: 10.3389/fnut.2024.1409309 -
Frontiers in Veterinary Science 2024Feline chronic enteropathies (FCE) are common causes of chronic gastrointestinal signs in cats and include different diseases such as food-responsive enteropathy (FRE),...
Feline chronic enteropathies (FCE) are common causes of chronic gastrointestinal signs in cats and include different diseases such as food-responsive enteropathy (FRE), inflammatory bowel diseases (IBD), and low-grade intestinal T-cell lymphoma (LGITL). Although changes in intestinal microbiota and fecal metabolites have been reported in dogs and humans with chronic enteropathy, research in cats has been limited. Therefore, this study aimed to evaluate the fecal microbiota and lipid-related fecal metabolites in cats with FCE to a clinically healthy comparison group (CG). A total of 34 cats with FCE (13 FRE, 15 IBD, and 6 LGITL) and 27 cats in the CG were enrolled in this study. The fecal microbiota was evaluated by the qPCR-based feline Dysbiosis Index (DI). The feline DI in cats with CE (median: 1.3, range: -2.4 to 3.8) was significantly higher ( < 0.0001) compared to CG (median: - 2.3, Range: -4.3 to 2.3), with no difference found among the FCE subgroups. The fecal abundances of Faecalibacterium ( < 0.0001), ( < 0.0001), ( = 0.0398), Bifidobacterium ( = 0.0004), and total bacteria ( = 0.0337) significantly decreased in cats with FCE. Twenty-seven targeted metabolites were measured by gas chromatography-mass spectrometry, including long-chain fatty acids (LCFAs), sterols, and bile acids (BAs). Fecal concentrations of 5 of 12 LCFAs were significantly increased in cats with FCE compared to CG. Fecal concentrations of zoosterol ( = 0.0109), such as cholesterol ( < 0.001) were also significantly increased in cats with FCE, but those of phytosterols were significantly decreased in this group. No differences in fecal BAs were found between the groups. Although no differences were found between the four groups, the fecal metabolomic pattern of cats with FRE was more similar to that of the CG than to those with IBD or LGITL. This could be explained by the mild changes associated with FRE compared to IBD and LGITL. The study showed changes in intestinal microbiota and alteration of fecal metabolites in FCE cats compared to the CG. Changes in fecal lipids metabolites suggest a dysmetabolism of lipids, including LCFAs, sterols, and unconjugated BAs in cats with CE.
PubMed: 38933703
DOI: 10.3389/fvets.2024.1401592 -
Frontiers in Cellular and Infection... 2024This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of...
BACKGROUND
This study unveils the intricate functional association between cyclic di-3',5'-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in , a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive.
METHODS
We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLAT.
RESULTS
We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within LPS. Notably, the predicted regulator gene , serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile.
CONCLUSION
The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.
Topics: Porphyromonas gingivalis; Lipopolysaccharides; Signal Transduction; Virulence Factors; Gene Expression Regulation, Bacterial; Energy Metabolism; Dinucleoside Phosphates; Fatty Acids; Humans; Bacterial Proteins
PubMed: 38933693
DOI: 10.3389/fcimb.2024.1418651 -
Frontiers in Plant Science 2024, widely used in clinical practice as a medicinal herb, belongs to the genus in the family Ephedraceae. However, the presence of numerous varieties and variants...
OBJECTIVE
, widely used in clinical practice as a medicinal herb, belongs to the genus in the family Ephedraceae. However, the presence of numerous varieties and variants requires differentiation for accurate identification.
METHODS
In this study, we employed headspace gas chromatography mass spectrometry (HS-GC-MS), ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), and global natural products social molecular networking (GNPS) for chemical component identification. Chemometric analysis was used to analyze the differential components. Metabolic analysis and Kyoto encyclopedia of genes and genomes (KEGG) enrichment were utilized to explore the synthesis pathways of different components.
RESULT
A total of 83 volatile and 79 non-volatile components were identified in species. Differential analysis revealed that among the eight stems, 18 volatile and 19 non-volatile differential compounds were discovered, whereas roots exhibited 21 volatile and 17 non-volatile markers. Volatile compounds were enriched in four synthetic pathways, while non-volatile components were enriched in five pathways among the differentiated components.
CONCLUSION
This study is the first to conduct a comparative analysis of chemical components in different species and parts. It provides a foundational reference for authenticating herbs, evaluating medicinal resources, and comparing quality in future studies.
PubMed: 38933459
DOI: 10.3389/fpls.2024.1421008 -
Fundamental Research May 2024The aerosol transmission of coronavirus disease in 2019, along with the spread of other respiratory diseases, caused significant loss of life and property; it impressed... (Review)
Review
The aerosol transmission of coronavirus disease in 2019, along with the spread of other respiratory diseases, caused significant loss of life and property; it impressed upon us the importance of real-time bioaerosol detection. The complexity, diversity, and large spatiotemporal variability of bioaerosols and their external/internal mixing with abiotic components pose challenges for effective online bioaerosol monitoring. Traditional methods focus on directly capturing bioaerosols before subsequent time-consuming laboratory analysis such as culture-based methods, preventing the high-resolution time-based characteristics necessary for an online approach. Through a comprehensive literature assessment, this review highlights and discusses the most commonly used real-time bioaerosol monitoring techniques and the associated commercially available monitors. Methods applied in online bioaerosol monitoring, including adenosine triphosphate bioluminescence, laser/light-induced fluorescence spectroscopy, Raman spectroscopy, and bioaerosol mass spectrometry are summarized. The working principles, characteristics, sensitivities, and efficiencies of these real-time detection methods are compared to understand their responses to known particle types and to contrast their differences. Approaches developed to analyze the substantial data sets obtained by these instruments and to overcome the limitations of current real-time bioaerosol monitoring technologies are also introduced. Finally, an outlook is proposed for future instrumentation indicating a need for highly revolutionized bioaerosol detection technologies.
PubMed: 38933213
DOI: 10.1016/j.fmre.2023.05.012