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Abstract Background Long‐term overgrazing has led to severe degradation of grasslands, posing a significant threat to the sustainable use of grassland resources. Methods Based on the investigation of changes in functional traits and photosynthetic physiology of Stipa breviflora under no grazing, moderate grazing, and heavy grazing treatments, the changes in expression patterns of genes and proteins associated with different grazing intensities were assessed through integrative transcriptomic and proteomic analyses. Results Differentially expressed genes and proteins were identified under different grazing intensities. They were mainly related to RNA processing, carbon metabolism, and secondary metabolite biosynthesis. These findings suggest that long‐term grazing leads to molecular phenotypic plasticity, affecting various biological processes and metabolic pathways in S. breviflora. Correlation analysis revealed low correlation between the transcriptome and the proteome, indicating a large‐scale regulation of gene expression at the posttranscriptional and translational levels during the response of S. breviflora to grazing. The expression profiles of key genes and proteins involved in photosynthesis and phenylpropanoid metabolism pathways suggested their synergistic response to grazing in S. breviflora. Conclusions Our study provides insight into the adaptation mechanisms of S. breviflora to grazing and provides a scientific basis for the development of more efficient grassland protection and utilization practices.

Abstract Prevotella intermedia, a Gram‐negative bacterium from the Bacteroidota phylum, is associated with periodontitis. Other species within this phylum are known to possess the general O‐glycosylation system. The O‐glycoproteome has been characterized in several species, including Tannerella forsythia, Porphyromonas gingivalis, and Flavobacterium johnsoniae. In our study, we used electron cryotomography (cryoET) and glycoproteomics to reveal the ultrastructure of P. intermedia and characterize its O‐glycoproteome. Our cryoET analysis unveiled the ultrastructural details of the cell envelope and outer membrane vesicles (OMVs) of P. intermedia. We observed an electron‐dense surface layer surrounding both cells and OMVs. The OMVs were often large (>200 nm) and presented two types, with lumens being either electron‐dense or translucent. LC‐MS/MS analyses of P. intermedia fractions led to the identification of 1655 proteins, which included 62 predicted T9SS cargo proteins. Within the glycoproteome, we identified 443 unique O‐glycosylation sites within 224 glycoproteins. Interestingly, the O‐glycosylation motif exhibited a broader range than reported in other species, with O‐glycosylation found at D(S/T)(A/I/L/M/T/V/S/C/G/F/N/E/Q/D/P). We identified a single O‐glycan with a delta mass of 1531.48 Da. Its sequence was determined by MS2 and MS3 analyses using both collision‐induced dissociation and high‐energy collisional dissociation fragmentation modes. After partial deglycosylation with trifluoromethanesulfonic acid, the O‐glycan sequence was confirmed to be dHex‐dHex‐HexNAc (HPO3‐C6H12O5)‐dHex‐Hex‐HexA‐Hex(dHex). Bioinformatic analyses predicted the localization of O‐glycoproteins, with 73 periplasmic proteins, 53 inner membrane proteins, 52 lipoproteins, 26 outer membrane proteins, and 14 proteins secreted by the T9SS.

Abstract Background This study aimed to investigate dynamic urinary proteome changes of electroacupuncture (EP) on cerebral ischemia‑reperfusion (CI/R) injured rats and to explore the therapeutic biological mechanisms of EP. Methods First, changed urinary proteins were found in EP stimulation in healthy rats. Then, we used a CI/R injury rat model induced by Pulsinelli's four‐vessel occlusion (4‐VO) method to explore the function of EP on urinary proteome in CI/R injury. Urine samples were collected for proteome analysis by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) and bioinformatics analysis. Results In total, 384 proteins were identified, among which 47 proteins (23 upregulated, 24 downregulated) were differentially expressed with 0.6‐log FC and p

Abstract Periodontitis is a chronic inflammatory disease caused by periodontal pathogens in subgingival plaque and is associated with systemic inflammatory diseases. Extracellular vesicles (EVs) released from host cells and pathogens carry a variety of biological molecules and are of interest for their role in disease progression and as diagnostic markers. In the present study, we analysed the proteome and inflammatory response of EVs derived from macrophages infected with Tannerella forsythia, a periodontal pathogen. The EVs isolated from the cell conditioned medium of T. forsythia‐infected macrophages were divided into two distinct vesicles, macrophage‐derived EVs and T. forsythia‐derived OMVs, by size exclusion chromatography combined with density gradient ultracentrifugation. Proteome analysis showed that in T. forsythia infection, macrophage‐derived EVs were enriched with pro‐inflammatory cytokines and inflammatory mediators associated with periodontitis progression. T. forsythia‐derived OMVs harboured several known virulence factors, including BspA, sialidase, GroEL and various bacterial lipoproteins. T. forsythia‐derived OMVs induced pro‐inflammatory responses via TLR2 activation. In addition, we demonstrated that T. forsythia actively released OMVs when T. forsythia encountered macrophage‐derived soluble molecules. Taken together, our results provide insight into the characterisation of EVs derived from cells infected with a periodontal pathogen.

Abstract Background The pathogenesis of moyamoya disease (MMD) is unclear. Inflammation and immune imbalance have been identified as potential factors contributing to the occurrence and progression of MMD. However, the specific proteins and metabolites responsible for triggering this process are yet to be established. The purpose of this study is to identify differentially expressed proteins and metabolites in patients with MMD and perform Kyoto Encyclopedia of Genes and Genomes pathway integration analysis to pinpoint crucial proteins and metabolites involved in the disease. Methods We performed untargeted metabolomic and data‐independent acquisition proteomic analyses on the serum samples of individuals with MMD and healthy controls (HC). Results In patients with MMD versus HC, 24 proteins and 60 metabolites, including 21 anionic metabolites and 39 cationic metabolites, which were significantly different, were identified. In patients with MMD, several proteins involved in inflammation and immune metabolism, such as tubulin beta‐6 and complement C4, were found to have significantly altered levels. Similarly, many metabolites involved in inflammation and immune metabolisms, such as dimethyl 4‐hydroxyisophthalate, beta‐nicotinamide mononucleotide, 2‐(3‐(4‐pyridyl)‐1H‐1,2,4‐triazol‐5‐yl)pyridine, and PC (17:1/18:2), were significantly altered. Intriguingly, these proteins and metabolites are involved in the progression of atherosclerosis through immune and inflammatory pathways, although some have never been reported in MMD. Moreover, integrated proteomics and metabolomics studies were conducted to determine shared pathways involving cholesterol metabolism, vitamin digestion, fat digestion, and absorption pathways of proteins and metabolites, which warrant further investigation. Conclusions Significant increases in pro‐inflammatory and immunosuppressive abilities have been observed in patients with MMD, accompanied by significant reductions in anti‐inflammatory and immune regulation. Various metabolites and proteins implicated in these processes have been identified for the first time. These findings hold immense significance for comprehending the pathogenesis of MMD and for the development of future drug therapies.

Abstract Cells are basic building blocks of life with vast heterogeneity. Nowadays, the rapid development of single‐cell multiomics (scMulti‐Omics) has facilitated comprehensive understanding of gene regulatory networks, cellular characteristics, and temporal dynamics. However, simultaneous analysis of transcriptome and proteome at single‐cell level still faces huge challenges due to their differences in molecular modalities. Recent technological advances in single‐cell manipulations, barcoding, and ultrasensitive instrument recently offer unprecedented opportunities for the co‐profiling of genes and proteins. In this review, multiple types of single‐cell isolation, lysis, and molecular separation technologies are first introduced. Second, various approaches for co‐measurement of transcriptome and proteome in single‐cells are summarized, with their advantages, limitations, and capacity for targeted or unbiased deep analysis. Then we highlight the cutting‐edge spatial multiomics methodologies that operate at the single‐cell or subcellular resolution level, providing a comprehensive understanding of cell function and heterogeneity within the tissue spatial environment. The emerging biomedical applications of multiomics are also discussed. Finally, the challenges and prospects of this field are proposed.

Abstract Alterations of the glycosylation machinery are common events in cancer, leading to the synthesis of aberrant glycan structures by tumor cells. Extracellular vesicles (EVs) play a modulatory role in cancer communication and progression, and interestingly, several tumor‐associated glycans have already been identified in cancer EVs. Nevertheless, the impact of 3D tumor architecture in the selective packaging of cellular glycans into EVs has never been addressed. In this work, the capacity of gastric cancer cell lines with differential glycosylation is evaluated in producing and releasing EVs when cultured under conventional 2D monolayer or in 3D culture conditions. Furthermore, the proteomic content is identified and specific glycans are studied in the EVs produced by these cells, upon differential spatial organization. Here, it is observed that although the proteome of the analyzed EVs is mostly conserved, an EV differential packaging of specific proteins and glycans is found. In addition, protein–protein interaction and pathway analysis reveal individual signatures on the EVs released by 2D‐ and 3D‐cultured cells, suggesting distinct biological functions. These protein signatures also show a correlation with clinical data. Overall, this data highlight the importance of tumor cellular architecture when assessing the cancer‐EV cargo and its biological role.

Societal Impact Statement Genetic engineering is used to introduce new genes into plants to obtain crops with novel traits. However, the unintended effects of genetically engineered (GE) crops on nontarget organisms—such as beneficial insects—are a topic of much concern. We evaluate the potential unintended effects of pollen from three GE maize and seven commercialized conventional maize lines on the ladybird beetle Propylea japonica, by combining omics approaches with feeding assays. Our results suggest that unintended changes caused by genetic engineering in maize pollen may not lead to biologically relevant effects on P. japonica. Meanwhile our study provides a useful strategy to assess the biological impacts of genetic engineering on nontarget organisms. Summary The potential effects caused by the inserted traits in genetically engineered (GE) plants on nontarget organisms (NTOs) have been well assessed. However, whether the process of genetic engineering itself causes unintended changes that go beyond the natural variation of the crop and further poses any biological effects to NTOs is still under debate. Here, we evaluated the potential unintended effects of pollen from three GE maize and seven commercialized conventional maize lines on the NTO Propylea japonica by combining omics approaches with feeding assays. The results showed that genetic breeding indeed brought somewhat differences at both proteome and metabolome levels in maize pollen, although such differences were far more common in conventionally crossbred plants. Feeding experiments indicated that the changes in proteins and metabolites caused by genetic breeding did not lead to unintended effects on the NTOs that go beyond those measured for the conventional crossbred lines. Together, our results suggest that the differences detected by omics experiments may not cause any biological relevant effects on NTOs and the combination of omics approaches and NTOs feeding assays provide a valid approach to assess the biological relevance of compositional effects caused by genetic breeding.

Abstract The model pennate diatom Phaeodactylum tricornutum is able to assimilate a range of iron sources. It therefore provides a platform to study different mechanisms of iron processing concomitantly in the same cell. In this study, we follow the localization of three iron starvation induced proteins (ISIPs) in vivo, driven by their native promoters and tagged by fluorophores in an engineered line of P. tricornutum. We find that the localization patterns of ISIPs are dynamic and variable depending on the overall iron status of the cell and the source of iron it is exposed to. Notwithstanding, a shared destination of the three ISIPs both under ferric iron and siderophore‐bound iron supplementation is a globular compartment in the vicinity of the chloroplast. In a proteomic analysis, we identify that the cell engages endocytosis machinery involved in the vesicular trafficking as a response to siderophore molecules, even when these are not bound to iron. Our results suggest that there may be a direct vesicle traffic connection between the diatom cell membrane and the periplastidial compartment (PPC) that co‐opts clathrin‐mediated endocytosis and the “cytoplasm to vacuole” (Cvt) pathway, for proteins involved in iron assimilation. Proteomics data are available via ProteomeXchange with identifier PXD021172. Highlight The marine diatom P. tricornutum engages a vesicular network to traffic siderophores and phytotransferrin from the cell membrane directly to a putative iron processing site in the vicinity of the chloroplast.

The domestic silkworm is a type of lepidopteran insect that feeds on mulberry leaves and has high economic value because of its ability to spin cocoons. Sericin 1 is an important component of silkworm cocoons, accounting for approximately 25% of the material. In this study, CRISPR/Cas9‐mediated gene editing was successfully used to destroy the sericin 1 gene, and homozygous mutants were obtained after continuous screening. Homozygous mutation resulted in premature termination of the translation of sericin 1 protein at 323 amino acids. Comparative transcriptomic and proteomic analyses of middle silk gland cells from wild‐type individuals and mutants were performed on the fourth day of the fifth instar, and the results suggest that sericin 1 plays an important role in the cellular immune system. In addition, the results suggest that sericin 1 has a synergistic effect with some protease inhibitors and that the secretion of these proteins is strictly regulated. These results will provide new insights into the function and expression pattern of sericin 1 and the mechanism of silk secretion.

Abstract Rapeseed (Brassica napus L.) is the second most important oilseed crop in edible vegetable oil and bioenergy; however, drought stress generally causes a decrease in rapeseed yield and oil content, especially during the reproductive stage. In our study, we measured the oil and protein contents and gibberellic acid (GA) and abscisic acid (ABA) levels in seeds that were acquired on the 30th, 40th, and 50th days after flowering under control and drought treatments. RNA and protein libraries were constructed from the stressed seeds to perform transcriptome and proteome analyses, respectively. Our results demonstrated that the oil content decreased due to four primary mechanisms: downregulation of fatty acid biosynthesis‐associated genes and proteins; upregulation of fatty acid degradation‐associated genes and proteins; enhancement of protein storage due to changes in the abundances of relevant genes and proteins; and upregulation of Gly‐Asp‐Ser‐Leu (GDSL) gene expression, potentially as the result of upregulating the GA biosynthesis gene GA20ox3 and downregulating the GA inactivating gene GA2ox3 and thus an increase in GA content. During seed maturation, oil storage change may also relate to increasing ABA content as the upregulation of two members of NCED6 (9‐cis‐epoxycarotenoid dioxygenase) gene family involved in ABA biosynthesis, and the upregulation of genes involved in ABA signal transduction. These results will help to establish a foundation for breeding excellent varieties of rapeseed with high oil content for areas with frequent droughts to promote the supply of edible vegetable oil and biofuel.

Despite significant advancements in breast cancer (BC) research, clinicians lack robust serological protein markers for accurate diagnostics and tumor stratification. Tumor interstitial fluid (TIF) accumulates aberrantly externalized proteins within the local tumor space, which can potentially gain access to the circulatory system. As such, TIF may represent a valuable starting point for identifying relevant tumor‐specific serological biomarkers. The aim of the study was to perform comprehensive proteomic profiling of TIF to identify proteins associated with BC tumor status and subtype. A liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis of 35 TIFs of three main subtypes: luminal (19), Her2 (4), and triple‐negative (TNBC) (12) resulted in the identification of > 8800 proteins. Unsupervised hierarchical clustering segregated the TIF proteome into two major clusters, luminal and TNBC/Her2 subgroups. High‐grade tumors enriched with tumor infiltrating lymphocytes (TILs) were also stratified from low‐grade tumors. A consensus analysis approach, including differential abundance analysis, selection operator regression, and random forest returned a minimal set of 24 proteins associated with BC subtypes, receptor status, and TIL scoring. Among them, a panel of 10 proteins, AGR3, BCAM, CELSR1, MIEN1, NAT1, PIP4K2B, SEC23B, THTPA, TMEM51, and ULBP2, was found to stratify the tumor subtype‐specific TIFs. In particular, upregulation of BCAM and CELSR1 differentiates luminal subtypes, while upregulation of MIEN1 differentiates Her2 subtypes. Immunohistochemistry analysis showed a direct correlation between protein abundance in TIFs and intratumor expression levels for all 10 proteins. Sensitivity and specificity were estimated for this protein panel by using an independent, comprehensive breast tumor proteome dataset. The results of this analysis strongly support our data, with eight of the proteins potentially representing biomarkers for stratification of BC subtypes. Five of the most representative proteomics databases currently available were also used to estimate the potential for these selected proteins to serve as putative serological markers.

Abstract Background Lymphoma (LSA) is a common malignancy in dogs. Epigenetic changes are linked to LSA pathogenesis and poor prognosis in humans, and LSA pathogenesis in dogs. Sulforaphane (SFN), an epigenetic‐targeting compound, has recently gained interest in relation to cancer prevention and therapy. Objective Examine the impact of oral supplementation with SFN on the lymph node proteome of dogs with multicentric LSA. Animals Seven client‐owned dogs with multicentric LSA. Methods Prospective, nonrandomized, noncontrolled study in treatment‐naïve dogs with intermediate or large cell multicentric LSA. Lymph node cell aspirates were obtained before and after 7 days of oral supplementation with SFN, and analyzed via label‐free mass spectrometry, immunoblots, and Gene Set Enrichment Analysis. Results There was no clinical response and no adverse events attributed to SFN. For individual dogs, the expression of up to 650 proteins changed by at least 2‐fold (range, 2‐100) after supplementation with SFN. When all dogs where analyzed together, 14 proteins were significantly downregulated, and 10 proteins were significantly upregulated after supplementation with SFN (P

Objectives To determine the mechanisms of ubiquitination in postmenopausal osteoporosis and investigate the ubiquitinated spectrum of novel targets between healthy postmenopausal women and postmenopausal osteoporosis patients, we performed ubiquitylome analysis of the whole blood of postmenopausal women and postmenopausal osteoporosis patients. Methods To obtain a more comprehensive understanding of the postmenopausal osteoporosis mechanism, we performed a quantitative assessment of the ubiquitylome in whole blood from seven healthy postmenopausal women and seven postmenopausal osteoporosis patients using high‐performance liquid chromatography fractionation, affinity enrichment, and liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS). To examine the ubiquitylome data, we performed enrichment analysis using an ubiquitylated amino acid motif, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Results Altogether, 133 ubiquitinated sites and 102 proteins were quantified. A difference of more than 1.2 times is considered significant upregulation and less than 0.83 significant downregulation; 32 ubiquitinated sites on 25 proteins were upregulated and 101 ubiquitinated sites on 77 proteins were downregulated. These quantified proteins, both with differently ubiquitinated sites, participated in various cellular processes, such as cellular processes, biological regulation processes, response to stimulus processes, single‐organism and metabolic processes. Ubiquitin conjugating enzyme activity and ubiquitin‐like protein conjugating enzyme activity were the most highly enriched in molecular function of upregulated sites with corresponding proteins, but they were not enriched in downregulated in sites with corresponding proteins. The KEGG pathways analysis of quantified proteins with differentiated ubiquitinated sites found 13 kinds of molecular interactions and functional pathways, such as glyoxylate and decarboxylate metabolism, dopaminergic synapse, ubiquitin‐mediated proteolysis, salivary secretion, coagulation and complement cascades, Parkinson's disease, and hippo signaling pathway. In addition, hsa04120 ubiquitin‐mediated proteolysis was the most highly enriched in proteins with upregulated sites, hsa04610 complement and coagulation cascades was the most highly enriched in proteins with downregulated ubiquitinated sites, and hsa04114 Oocyte meiosis was the most highly enriched among all differential proteins. Conclusion Our study expands the understanding of the spectrum of novel targets that are differentially ubiquitinated in whole blood from healthy postmenopausal women and postmenopausal osteoporosis patients. The findings will contribute toward our understanding of the underlying proteostasis pathways in postmenopausal osteoporosis and the potential identification of diagnostic biomarkers in whole blood.

Multiple species within the order Hemiptera cause severe agricultural losses on a global scale. Aphids and whiteflies are of particular importance due to their role as vectors for hundreds of plant viruses, many of which enter the insect via the gut. To facilitate the identification of novel targets for disruption of plant virus transmission, we compared the relative abundance and composition of the gut plasma membrane proteomes of adult Bemisia tabaci (Hemiptera: Aleyrodidae) and Myzus persicae (Hemiptera: Aphididae), representing the first study comparing the gut plasma membrane proteomes of two different insect species. Brush border membrane vesicles were prepared from dissected guts, and proteins extracted, identified and quantified from triplicate samples via timsTOF mass spectrometry. A total of 1699 B. tabaci and 1175 M. persicae proteins were identified. Following bioinformatics analysis and manual curation, 151 B. tabaci and 115 M. persicae proteins were predicted to localize to the plasma membrane of the gut microvilli. These proteins were further categorized based on molecular function and biological process according to Gene Ontology terms. The most abundant gut plasma membrane proteins were identified. The ten plasma membrane proteins that differed in abundance between the two insect species were associated with the terms "protein binding" and "viral processes." In addition to providing insight into the gut physiology of hemipteran insects, these gut plasma membrane proteomes provide context for appropriate identification of plant virus receptors based on a combination of bioinformatic prediction and protein localization on the surface of the insect gut., (© 2024 The Author(s). Archives of Insect Biochemistry and Physiology published by Wiley Periodicals LLC.)

Formalin‐fixed, paraffin‐embedded (FFPE), biobanked tissue samples offer an invaluable resource for clinical and biomarker research. Here, we developed a pressure cycling technology (PCT)‐SWATH mass spectrometry workflow to analyze FFPE tissue proteomes and applied it to the stratification of prostate cancer (PCa) and diffuse large B‐cell lymphoma (DLBCL) samples. We show that the proteome patterns of FFPE PCa tissue samples and their analogous fresh‐frozen (FF) counterparts have a high degree of similarity and we confirmed multiple proteins consistently regulated in PCa tissues in an independent sample cohort. We further demonstrate temporal stability of proteome patterns from FFPE samples that were stored between 1 and 15 years in a biobank and show a high degree of the proteome pattern similarity between two types of histological regions in small FFPE samples, that is, punched tissue biopsies and thin tissue sections of micrometer thickness, despite the existence of a certain degree of biological variations. Applying the method to two independent DLBCL cohorts, we identified myeloperoxidase, a peroxidase enzyme, as a novel prognostic marker. In summary, this study presents a robust proteomic method to analyze bulk and biopsy FFPE tissues and reports the first systematic comparison of proteome maps generated from FFPE and FF samples. Our data demonstrate the practicality and superiority of FFPE over FF samples for proteome in biomarker discovery. Promising biomarker candidates for PCa and DLBCL have been discovered.

Background We recently showed that, in patients with heart failure, lower high‐density lipoprotein (HDL) cholesterol concentration was a strong predictor of death or hospitalization for heart failure. In a follow‐up study, we suggested that this association could be partly explained by HDL proteome composition. However, whether the emerging concept of HDL function contributes to the prognosis of patients with heart failure has not been addressed. Methods and Results We measured 3 key protective HDL function metrics, namely, cholesterol efflux, antioxidative capacity, and anti‐inflammatory capacity, at baseline and after 9 months in 446 randomly selected patients with heart failure from BIOSTAT‐CHF (A Systems Biology Study to Tailored Treatment in Chronic Heart Failure). Additionally, the relationship between HDL functionality and HDL proteome composition was determined in 86 patients with heart failure. From baseline to 9 months, HDL cholesterol concentrations were unchanged, but HDL cholesterol efflux and anti‐inflammatory capacity declined (both P

Abstract High temperature (HT) is an important environmental factor affecting crops growth, development, and yield. Transcriptome and proteome technologies developed in recent years can provide deeper and more reliable data to decipher the mechanisms of HT response. In the present study, the transcriptome analysis with RNA sequencing (RNA‐Seq) and integrative analysis on obtained proteomic data were performed in tomato (Solanum lycopersicum) leaves under HT stress (42°C for 4 hr). In total, 3,531 differentially expressed genes (DEGs) and 268 differentially expressed proteins (DEPs) were explored. The quantitative data were validated by qRT‐PCR and parallel reaction monitoring (PRM). Upon mapping of the DEGs/DEPs to the KEGG pathway database, “protein processing in the endoplasmic reticulum” was found to be the most significant enrichment pathway both at the transcriptional and protein levels, suggesting that endoplasmic reticulum stress may play a central role in HT tolerance. Furthermore, transcriptome‐proteome integrative analysis revealed 91 genes shared both in DEGs and DEPs, of which 79 correlations with the same changing trends were most associated with stress response and protein folding. Among these, a HT response gene designated SlBAG5 of unknown function received attention. The full‐length of SlBAG5 was cloned and ectopically overexpressed in Arabidopsis, which displayed thermosensitive phenotype. Taken together, this work provides deep insight into the molecular mechanisms of plant thermotolerance and also facilitates the identification of the key potential genes/proteins for HT response.

Abstract Recently emerged mass cytometry (cytometry by time‐of‐flight [CyTOF]) technology permits the identification and quantification of inherently diverse cellular systems, and the simultaneous measurement of functional attributes at the single‐cell resolution. By virtue of its multiplex ability with limited need for compensation, CyTOF has led a critical role in immunological research fields. Here, we present an overview of CyTOF, including the introduction of CyTOF principle and advantages that make it a standalone tool in deciphering immune mysteries. We then discuss the functional assays, introduce the bioinformatics to interpret the data yield via CyTOF, and depict the emerging clinical and research applications of CyTOF technology in sketching immune landscape in a wide variety of diseases.

Background: Early screening for colorectal cancer (CRC) is essential to improve its prognosis. Liquid biopsies are increasingly being considered for diagnosing cancer due to low invasiveness and high reproducibility. In addition, circulating extracellular vesicles (crEVs, extracellular vesicles isolated from plasma) expressing tumour-specific proteins are potential biomarkers for various cancers. Here, we present a data-independent acquisition (DIA)-mass spectrometry (MS)-based diagnostic method for liquid biopsies. Methods: Extracellular vesicles (EVs) were isolated from culture supernatants of human CRC cell lines, and plasma of patients with CRC at different tumour stages, by overnight ultracentrifugation coupled with sucrose density gradient centrifugation. Tumour-specific EV proteins were prioritized using Tandem Mass Tag (TMT)-based shotgun proteomics and phosphoproteomics. The results were verified in a second independent cohort and a mouse tumour-bearing model using Western blotting (WB). The candidate biomarkers were further validated in a third cohort by DIA-MS. Finally, the DIA-MS methodology was accelerated to permit high-throughput detection of EV biomarkers in another independent cohort of patients with CRC and healthy controls. Results: High levels of total and phosphorylated fibronectin 1 (FN1) in crEVs, haptoglobin (HP), S100A9 and fibrinogen α chain (FGA) were significantly associated with cancer progression. FGA was the most dominant biomarker candidate. Analysis of the human CRC cell lines and the mouse model indicated that FGA+ crEVs were likely released by CRC cells. Furthermore, fast DIA-MS and parallel reaction monitoring (PRM)-MS both confirmed that FGA+ crEVs could distinguish colon adenoma with an area of curve (AUC) in the receiver operating characteristic (ROC) curve of 0.949 and patients with CRC (AUC of ROC is 1.000) from healthy individuals. The performance outperformed conventional tumour biomarkers. The DIA-MS quantification of FGA+ crEVs among three groups agreed with that from PRM-MS. Conclusion: DIA-MS detection of FGA+ crEVs is a potential rapid and non-invasive screening tool to identify early stage CRC. Abbreviations: FGA: fibrinogen α chain; CRC: colorectal cancer; crEVs: circulating extracellular vesicles; EV: extracellular vesicles;MS: mass spectrometry; WB: Western blotting; ROC: receiver operating characteristic; PRM: Parallel Reaction Monitoring; GPC1: Glypican-1; GO: Gene ontology; TEM: transmission electron microscopy; FN1: Fibronectin 1; HP: haptoglobin; TMT: Tandem Mass Tag; LC-MS/MS: liquid chromatography coupled to tandem mass spectrometry; DIA: data-independent acquisition; DDA: data-dependent acquisition; CiRT: Common internal Retention Time standards;AGC: Automatic gain control; AUC: area under curve.

Abstract China launched the Tiangong‐2 space laboratory in 2016 and will eventually build a basic space station by the early 2020s. These spaceflight missions require astronauts to stay on the space station for more than 6 months, and they inevitably carry microbes into the space environment. It is known that the space environment affects microbial behavior, including growth rate, biofilm formation, virulence, drug resistance, and metabolism. However, the mechanisms of these alternations have not been fully elucidated. Therefore, it is beneficial to monitor microorganisms for preventing infections among astronauts in a space environment. Salmonella enteritidis is a Gram‐negative bacterial pathogen that commonly causes acute gastroenteritis in humans. In this study, to better understand the effects of the space environment on S. enteritidis, a S. enteritidis strain was taken into space by the Shenzhou‐11 spacecraft from 17 October 2016 to 18 November 2016, and a ground simulation with similar temperature conditions was simultaneously performed as a control. It was found that the flight strain displayed an increased growth rate, enhanced amikacin resistance, and some metabolism alterations compared with the ground strain. Enrichment analysis of proteome revealed that the increased growth rate might be associated with differentially expressed proteins involved in transmembrane transport and energy production and conversion assembly. A combined transcriptome and proteome analysis showed that the amikacin resistance was due to the downregulation of the oppA gene and oligopeptide transporter protein OppA. In conclusion, this study is the first systematic analysis of the phenotypic, genomic, transcriptomic, and proteomic variations in S. enteritidis during spaceflight and will provide beneficial insights for future studies on space microbiology.

Abstract D‐Alanyl‐D‐alanine ligase A (DdlA) catalyses the dimerization of two D‐alanines yielding D‐alanyl‐D‐alanine required for mycobacterial peptidoglycan biosynthesis, and is a promising antimycobacterial drug target. To better understand the roles of DdlA in mycobacteria in vivo, we established a cell model in which DdlA expression was specifically downregulated by ddlA antisense RNA by introducing a 380 bp ddlA fragment into pMind followed by transforming the construct into nonpathogenic Mycobacterium smegmatis. The M. smegmatis cell model was verified by plotting the growth inhibition curves and quantifying endogenous DdlA expression using a polyclonal anti‐DdlA antibody produced from the expressed DdlA. Scanning electron microscopy and transmission electron microscopy were used to investigate mycobacterial morphology. Bidimensional gel electrophoresis and mass spectrometry were used to analyze differentially expressed proteins. Consequently, the successful construction of the M. smegmatis cell model was verified. The morphological investigation of the model indicated that DdlA deficiency led to an increased number of Z rings and a rearrangement of intracellular content, including a clear nucleoid and visible filamentous DNA. Proteomic techniques identified six upregulated and 14 downregulated proteins that interacted with each other to permit cell survival by forming a regulatory network under DdlA deficiency. Finally, our data revealed that DdlA deficiency inhibited cell division in mycobacteria and attenuated the process of carbohydrate catabolism and the pathway of fatty acid anabolism, while maintaining active protein degradation and synthesis. N‐Nitrosodimethylamine (NDMA)‐dependent methanol dehydrogenase (MSMEG_6242) and fumonisin (MSMEG_1419) were identified as potential antimycobacterial drug targets.

Brain-derived extracellular vesicles (EVs) play an active role in Alzheimer's disease (AD), relaying important physiological information about their host tissues. The internal cargo of EVs is protected from degradation, making EVs attractive AD biomarkers. However, it is unclear how circulating EVs relate to EVs isolated from disease-vulnerable brain regions. We developed a novel method for collecting EVs from the hippocampal interstitial fluid (ISF) of live mice. EVs (EV ISF ) were isolated via ultracentrifugation and characterized by nanoparticle tracking analysis, immunogold labelling, and flow cytometry. Mass spectrometry and proteomic analyses were performed on EV ISF cargo. EV ISF were 40-150 nm in size and expressed CD63, CD9, and CD81. Using a model of cerebral amyloidosis (e.g., APPswe, PSEN1dE9 mice), we found protein concentration increased but protein diversity decreased with Aβ deposition. Genotype, age, and Aβ deposition modulated proteostasis- and immunometabolic-related pathways. Changes in the microglial EV ISF proteome were sexually dimorphic and associated with a differential response of plaque associated microglia. We found that female APP/PS1 mice have more amyloid plaques, less plaque associated microglia, and a less robust- and diverse- EV ISF microglial proteome. Thus, in vivo microdialysis is a novel technique for collecting EV ISF and offers a unique opportunity to explore the role of EVs in AD., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

The release of extracellular vesicles (EVs) in cell cultures as well as their molecular cargo can be influenced by cell culture conditions such as the presence of foetal bovine serum (FBS). Although several studies have evaluated the effect of removing FBS-derived EVs by ultracentrifugation (UC), less is known about the influence of FBS heat inactivation (HI) on the cell-derived EVs. To assess this, three protocols based on different combinations of EV depletion by UC and HI were evaluated, including FBS ultracentrifuged but not heat inactivated (no-HI FBS), FBS heat inactivated before EV depletion (HI-before EV-depl FBS), and FBS heat inactivated after EV depletion (HI-after EV-depl FBS). We isolated large (L-EVs) and small EVs (S-EVs) from FBS treated in the three different ways, and we found that the S-EV pellet from HI-after EV-depl FBS was larger than the S-EV pellet from no-HI FBS and HI-before EV-depl FBS. Transmission electron microscopy, protein quantification, and particle number evaluation showed that HI-after EV-depl significantly increased the protein amount of S-EVs but had no significant effect on L-EVs. Consequently, the protein quantity of S-EVs isolated from three cell lines cultured in media supplemented with HI-after EV-depl FBS was significantly increased. Quantitative mass spectrometry analysis of FBS-derived S-EVs showed that the EV protein content was different when FBS was HI after EV depletion compared to EVs isolated from no-HI FBS and HI-before EV-depl FBS. Moreover, we show that several quantified proteins could be ascribed to human origin, thus demonstrating that FBS bovine proteins can mistakenly be attributed to human cell-derived EVs. We conclude that HI of FBS performed after EV depletion results in changes in the proteome, with molecules that co-isolate with EVs and can contaminate EVs when used in subsequent cell cultures. Our recommendation is, therefore, to always perform HI of FBS prior to EV depletion., (© 2024 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Periodontitis is a chronic inflammatory disease caused by periodontal pathogens in subgingival plaque and is associated with systemic inflammatory diseases. Extracellular vesicles (EVs) released from host cells and pathogens carry a variety of biological molecules and are of interest for their role in disease progression and as diagnostic markers. In the present study, we analysed the proteome and inflammatory response of EVs derived from macrophages infected with Tannerella forsythia, a periodontal pathogen. The EVs isolated from the cell conditioned medium of T. forsythia-infected macrophages were divided into two distinct vesicles, macrophage-derived EVs and T. forsythia-derived OMVs, by size exclusion chromatography combined with density gradient ultracentrifugation. Proteome analysis showed that in T. forsythia infection, macrophage-derived EVs were enriched with pro-inflammatory cytokines and inflammatory mediators associated with periodontitis progression. T. forsythia-derived OMVs harboured several known virulence factors, including BspA, sialidase, GroEL and various bacterial lipoproteins. T. forsythia-derived OMVs induced pro-inflammatory responses via TLR2 activation. In addition, we demonstrated that T. forsythia actively released OMVs when T. forsythia encountered macrophage-derived soluble molecules. Taken together, our results provide insight into the characterisation of EVs derived from cells infected with a periodontal pathogen., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

We have previously uncovered the impact of oncogenic and differentiation processes on extracellular vesicles (EVs) in cancer. This is of interested in the context of glioma stem cells (GSC) that are responsible for recurrent nature of glioblastoma multiforme (GBM), while retaining the potential to undergo differentiation and self renewal. GSCs reside in vascular niches where they interact with endothelial cells through a number of mediators including bioactive cargo of EVs. GSCs can be classified as proneural (PN) or mesenchymal (MES) subtypes on the basis of their gene expression profiles and distinct biological characteristics. In the present study we investigated how GSC diversity and differentiation programmes influence their EV-mediated communication potentials. Indeed, molecular subtypes of GBMs and GSCs differ with respect to their expression of EV-related genes (vesiculome) and GSCs with PN or MES phenotypes produce EVs with markedly different characteristics, marker profiles, proteomes and endothelial stimulating activities. For example, while EVs of PN GSC are largely devoid of exosomal markers their counterparts from MES GSCs express ample CD9, CD63 and CD81 tetraspanins. In both GSC subtypes serum-induced differentiation results in profound, but distinct changes of cellular phenotypes including the enhanced EV production, reconfiguration of their proteomes and the related functional pathways. Notably, the EV uptake was a function of both subtype and differentiation state of donor cells. Thus, while, EVs produced by differentiated MES GSCs were internalized less efficiently than those from undifferentiated cells they exhibited an increased stimulatory potential for human brain endothelial cells. Such stimulating activity was also observed for EVs derived from differentiated PN GSCs, despite their even weaker uptake by endothelial cells. These findings suggest that the role of EVs as biological mediators and biomarkers in GBM may depend on the molecular subtype and functional state of donor cancer cells, including cancer stem cells. Abbreviations: CryoTEM: cryo-transmission electron microscopy; DIFF: differentiated GSCs; EGF: epidermal growth factor; DUC: differential ultracentrifugation; EV: extracellular vesicle; FGF: fibroblast growth factor; GBM: glioblastoma multiforme; GFAP: glial fibrillary acidic protein; GO: gene ontology; GSC: glioma stem cells; HBEC-5i: human brain endothelial cells; MES: mesenchymal cells; MTS - [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMT1: proneural-to-mesenchyman transition cell line 1; PN: proneural cells; TEM: transmission electron microscopy; WB: western blotting