Proteomic and metabolomic profiling reveals the fungicidal mechanisms of Microsporum canis in response to methylene blue-mediated photodynamic therapy

Transcriptomic analysis reveals the molecular mechanisms of methylene blue-mediated photodynamic therapy against Microsporum canis.

Hemorrhagic mastitis (HM) in dairy cows caused great economic losses in the dairy industry due to decreased milk production and increased costs associated with cattle management and treatment. However, the pathological and molecular mechanisms of HM are not well-understood. The present study aimed to investigate differentially expressed proteins (DEPs) associated with HM according to data-independent acquisition (DIA) proteomics. Compared to the mammary glands of healthylactating Holstein cows (Control, C group), the pathology of the HM group displayed massive alveolar infiltration of hemocytes and neutrophils, and the blood vessels, including arteriole, venules and capillaries were incomplete and damaged, with a loss of endothelial cells. DIA proteomics results showed that a total of 3,739 DEPs and 819 biological process terms were screened in the HM group. We focused on the blood, permeability of blood vessel, vascular and angiogenesis of mammary glands, and a total of 99 candidate DEPs, including 60 up- and 39 down-regulated DEPs, were obtained from the Gene Ontology (GO) and Pathway enrichment analyses. Phenotype prediction and function analysis of the DEPs revealed that three DEPs, particularly Caveolin-1(CAV1), were participated in the regulation of angiogenesis. Immunohistochemical and immunofluorescence staining showed that the CAV1 protein was present mainly in the mammary epithelial cells, vascular endothelial cells and vascular smooth muscle cells. The expression level of CAV1 mRNA and protein in the HM group was significantly down-regulated. The results will be helpful to the further understanding of the pathological and molecular mechanisms of HM in dairy cows.

PurposeAcute pancreatitis can be classified histologically as interstitial edema pancreatitis (IEP) or as acute necrotizing pancreatitis (ANP). ANP has a higher mortality and long-term or short-term sequelae than IEP. Therefore, this work aims to explore the differences in pathogenesis between ANP and IEP and it has great clinical importance for the treatment and prevention of ANP.MethodsIn this work, whole blood samples from IEP and ANP patients were analyzed by whole gene sequencing (WGS). Serum samples from IEP and ANP patients were evaluated via enzyme-linked immunosorbent assay (ELISA). Meanwhile, pancreatic tissues of IEP and ANP rat models were subjected to data independent acquisition (DIA) proteomics assays. Then, the WGS analysis and DIA proteomics assay data were analyzed comprehensively.ResultsSix pathways were found to be significantly different in the ANP/IEP groups through WGS analysis. DIA proteomics found eleven different pathways. In both assays, the complement and coagulation cascades pathway was the most significantly different (p < 0.01) pathway between the two groups. WGS analysis showed base mutations in ten genes in the complement and coagulation cascades pathway. These results were consistent with the ten proteins detected by DIA proteomics analysis, which were significantly upregulated in the ANP/IEP groups. In addition, five of these proteins, complement C3, complement Factor I, alpha-2-macroglobulin, complement C9, and serpin family C member 1, were successfully verified by parallel reaction monitoring analysis and ELISA.ConclusionC3, CFI, A2m, C9, and Serpinc1, which belong to complement and coagulation cascades pathway, may promote pancreatic necrosis and aggravate the severity of ANP.

During estrus, the poll glands of male Bactrian Camels (Camelus Bactrianus) become slightly raised, exuding a large amount of pale yellow watery secretion with a characteristic odor that may contain hydrogen sulfide (H2S). However, whether H2S can be synthesized in the poll glands of male Bactrian Camels and its role in inducing camel estrus remains unclear. This study aimed to identify differentially expressed proteins (DEPs) and signaling pathways in the poll gland tissues of male Bactrian Camels using data independent acquisition (DIA) proteomics. Additionally, gas chromatography-mass spectrometry (GC-MS) was performed to identify differentially expressed metabolites (DEMs) in the neck hair containing secretions during estrus in male Bactrian Camels, to explore the specific expression patterns and mechanisms in the poll glands of camels during estrus. The results showed that cystathionine-γ-lyase (CTH) and cystathionine-β-synthase (CBS), which are closely related to H2S synthesis in camel poll glands during estrus, were mainly enriched in glycine, serine, and threonine metabolism, amino acid biosynthesis, and metabolic pathways. In addition, both enzymes were widely distributed and highly expressed in the acinar cells of poll gland tissues in camels during estrus. Meanwhile, the neck hair secretion contains high levels of amino acids, especially glycine, serine, threonine, and cystathionine, which are precursors for H2S biosynthesis. These results demonstrate that the poll glands of male Bactrian Camels can synthesize and secrete H2S during estrus. This study provides a basis for exploring the function and mechanism of H2S in the estrus of Bactrian Camels.

Anthracyclines, as a type of chemotherapy drugs, are widely used in the first-line treatment of cancers. However, anthracycline-induced cardiotoxicity (AIC) in the cumulative and dose-dependent manner greatly limits the clinical application. Insufficient energy supply caused by myocardial metabolic reconstruction is an established factor for AIC. At present, the left ventricular maximum standardized uptake value (LV SUVmax) and left ventricular mean standardized uptake value (LV SUVmean) detected via 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) are effective metrics reflecting alterations of myocardial metabolism after anthracycline medications, serving as promising targets for predicting the risk of AIC. To investigate AIC-induced myocardial metabolism changes by an integration of 18F-FDG PET/CT and data-independent acquisition (DIA) proteomics, thus providing novel targets for predicting AIC. A total of 30 patients with diffuse large B-cell lymphomas and treated with anthracycline-based chemotherapy in the Hematology Department of Jiangsu Province Hospital of Chinese Medicine from December 2023 to December 2024 were enrolled. Finally, 17 participants were included as the Diffuse Large B-cell Lymphoma (DLBCL) group. Additionally, 13 non-oncologic participants without organic heart disease, who required 18F-FDG PET/CT for disease screening, were recruited as the control group. General data, dosage of anthracyclines, physical and chemical examination findings, and cardiac function indicators were collected. 18F-FDG PET/CT was performed before and after 6 cycles of chemotherapy in the DLBCL group, whereas the control group underwent a single scan during the same period. Serum samples were harvested for analyzing alterations of myocardial metabolism before and after chemotherapy via DIA proteomics. Among 17 patients in the DLBCL group, 6 received epirubicin chemotherapy with an average dose of 59.28 ± 12.54 mg/m2 per cycle, and 11 received liposomal doxorubicin chemotherapy with an average dose of 25.21 ± 3.14 mg/m2 per cycle. Compared to the control group, no significant differences were observed in pre-chemotherapy LV SUVmax (median: 2.40, 95% CI: 2.04-2.74) and LV SUVmean (median: 1.34, 95% CI: 1.15-1.55) in the DLBCL group (P > 0.05). After anthracycline-based chemotherapy, both LV SUVmax (median: 4.82, 95% CI: 3.50-6.05) and LV SUVmean (median: 1.93, 95% CI: 1.57-2.56) significantly increased compared to pre-chemotherapy values (P < 0.05). The proportion of patients without 18F-FDG uptake in the left ventricular myocardium significantly decreased relative to both pre-chemotherapy levels and the control group (P < 0.001). Conversely, the proportions of patients exhibiting diffuse uptake and focal-on-diffuse uptake significantly increased compared to pre-chemotherapy measurements (P < 0.01). Moreover, the incidence of abnormal 18F-FDG uptake in the left ventricular myocardium was significantly higher after chemotherapy than both before treatment and in the control group (P < 0.001). DIA proteomics showed that CPT1A, ACOX1, ECH1, and ACAT1 were significantly downregulated after anthracycline-based chemotherapy, which were mainly enriched in fatty acid metabolism. Their protein levels were negatively associated with left ventricular standardized uptake value (LV SUV). The Mantel test consistently proved a significant association between fatty acid metabolism and LV SUV. An integration of 18F-FDG PET/CT and DIA proteomics reveals a decreased fatty acid oxidation (FAO) and an increased myocardial glucose uptake after anthracycline-based chemotherapy, serving as potential mechanisms of AIC. Alterations of myocardial metabolism monitored by 18F-FDG PET/CT may represent early indicators of metabolic remodeling, potentially identifying patients at risk for AIC. https://www.chictr.org.cn/, identifier ChiCTR2400088740.

PurposeThis study is aimed to delve into the crucial proteins associated with hormonal osteonecrosis of the femoral head (ONFH) and its intra-articular lesions through data-independent acquisition (DIA) proteomics and bioinformatics analysis.MethodsWe randomly selected samples from eligible ONFH patients and collected samples from the necrotic area of the femoral head and load-bearing cartilage. The control group comprised specimens from the same location in patients with femoral neck fractures. With DIA proteomics, we quantitatively and qualitatively tested both groups and analyzed the differentially expressed proteins (DEPs) between groups. Additionally, we enriched the analysis of DEP functions using gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways and verified the key proteins in ONFH through Western blot.ResultsProteomics experiment uncovered 937 common DEPs (422 upregulated and 515 downregulated) between the two groups. These DEPs mainly participate in biological processes such as hidden attributes, catalytic activity, molecular function regulators, and structural molecule activity, and in pathways such as starch and sucrose metabolism, ECM–receptor interaction, PI3K-Akt signaling, complement and coagulation cascades, IL-17 signaling, phagosome, transcriptional misregulation in cancers, and focal adhesion. Through protein–protein interaction network target gene analysis and Western blot validation, we identified C3, MMP9, APOE, MPO, LCN2, ELANE, HPX, LTF, and THBS1 as key proteins in ONFH.ConclusionsWith DIA proteomics and bioinformatics analysis, this study reveals the molecular mechanisms of intra-articular lesions in ONFH. A correlation in the necrotic area and load-bearing cartilage of ONFH at ARCO stages IIIB-IV as well as potential key regulatory proteins was identified. These findings will help more deeply understand the pathogenesis of ONFH and may provide important clues for seeking more effective treatment strategies.

Early diagnosis and intervention are essential for improving the prognosis and survival of gastric cancer (GC) patients. However, specific biomarkers for early GC diagnosis are still unavailable. Data-independent acquisition (DIA) proteomics was employed to identify differentially expressed proteins (DEPs) between GC and adjacent non-tumor tissues. Functional and pathway enrichment analyses were conducted, with subsequent genomic-level validation. Methyltransferase-like 7A (METTL7A) expression in GC versus adjacent tissues was confirmed via tissue microarray analysis. Correlations between METTL7A expression, clinical characteristics, and immune infiltration were also explored. Additionally, co-expressed genes related to METTL7A were analyzed, and gene set variation analysis (GSVA) was performed. DIA proteomics identified 84 DEPs, mainly involved in protein binding and enriched in complement and coagulation pathways. Eight DEPs overlapped with results from the gene expression omnibus (GEO) dataset. METTL7A expression was significantly lower in GC tissues compared to adjacent tissues, confirmed at the genomic level. The cancer genome atlas (TCGA) analysis revealed an area under the receiver operating characteristic (ROC) curve (AUC) of 0.81, with METTL7A expression inversely correlated with age (p = 7.307e-05). Tissue microarray analysis further confirmed reduced METTL7A expression in GC tissues (p = 0.000). METTL7A expression was positively correlated with activated B cells and negatively correlated with activated CD4 T cells. METTL7A is a promising biomarker for early GC diagnosis.

Based on data-independent acquisition (DIA) proteomics technology, to analyze the proteomic characteristics of colorectal tubular adenoma and explore the expression changes of SLC30A10 and their potential association with the cGAS-STING pathway. A self-controlled design was adopted, collecting colorectal tubular adenoma (TA) and paired normal mucosa (NM) from 15 patients with TA. Differentially expressed proteins were screened by DIA proteomics, followed by GO and KEGG enrichment analyses. Immunohistochemistry was performed to detect the expression of SLC30A10, cGAS, STING, p-IRF3, ISG15, and β-catenin; immunofluorescence double staining was used to observe the co-localization of p-IRF3 and β-catenin; inductively coupled plasma mass spectrometry (ICP-MS) was employed to determine tissue manganese content. DIA analysis showed that SLC30A10 protein expression was significantly downregulated in TA tissues. Functional enrichment analysis indicated abnormalities in signal transduction, metabolic reprogramming, and nitrogen metabolism in TA tissues. IHC results demonstrated that, compared with NM, TA tissues exhibited reduced expression of SLC30A10, while the expression of cGAS, STING, p-IRF3, ISG15, and β-catenin was upregulated. Manganese content in TA tissues was also significantly increased. Immunofluorescence revealed enhanced nuclear signals of p-IRF3 in TA cells, with co-localization of p-IRF3 and β-catenin observed in the nucleus. Downregulation of SLC30A10 in colorectal tubular adenoma is associated with manganese accumulation and alterations in the cGAS-STING pathway, suggesting its potential role in the development and progression of adenoma, a finding with promising research implications.

Dental pulp stem cells hold significant prospects for tooth regeneration and repair. However, a comprehensive understanding of the molecular differences between dental pulp stem cells (DPSC, from permanent teeth) and stem cells from human exfoliated deciduous teeth (SHED, from deciduous teeth) remains elusive, which is crucial for optimizing their therapeutic potential. To address this gap, we employed a novel data-independent acquisition (DIA) proteomics approach to compare the protein expression profiles of DPSC and SHED. Based on nano-LC-MS/MS DIA proteomics, we identified over 7,000 proteins in both cell types. By comparing their expression levels, 209 differentially expressed proteins were identified. Subsequent Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, along with protein-protein interaction network construction, revealed significant metabolic differences and key regulatory nodes. DPSC exhibited significantly higher expression of proteins belonging to the NDUFB family, SMARC family, RPTOR and TLR3. These proteins are known to be involved in critical cellular processes such as mitochondrial energy metabolism, mTOR-related autophagy pathway, and innate immune response. Conversely, SHED displayed elevated expression of AKR1B family, which participated in glycerolipid metabolism and adipogenic differentiation, PRKG1, MGLL and UQCRB proteins associated with thermogenesis. These findings highlight the specific proteomic landscape of DPSC and SHED, suggesting their distinct biological roles and potential applications.

Data-independent acquisition (DIA) proteomics enables reproducible and systematic peptide detection and quantification, and trapped ion mobility spectrometry (TIMS) on the timsTOF platform further improves DIA by synchronizing ion mobility separation with quadrupole precursor sampling. Analyzing the highly multiplexed spectra generated by DIA typically relies on spectral libraries, and fully leveraging the additional ion mobility dimension requires these libraries to include accurate retention time, fragment ion intensity, and ion mobility annotations. Existing in silico spectral library generation tools either lack ion mobility support entirely or rely on models trained on data-dependent acquisition (DDA) data, that can introduce a mismatch that may not capture unique experiment-specific biases when applied to each respective timsTOF dataset. Carafe is a software tool that uses deep learning models to generate high-quality, experiment-specific in silico libraries by training directly on DIA data. In this study, we extend Carafe to generate libraries for timsTOF DIA data, which involves fine-tuning retention time (RT), fragment ion intensity, and ion mobility prediction models using timsTOF DIA data. Carafe2 operates directly on native timsTOF raw data (Bruker .d directories) without the need for data conversion. We demonstrate the performance of Carafe2 across a wide range of DIA applications, including global proteome, phosphoproteome, and plasma proteome datasets. Comparing Carafe2 fine-tuned RT, fragment ion intensity, and ion mobility prediction models with pretrained DDA models, we find that Carafe2 models outperform pretrained models on a variety of DIA datasets. We then demonstrate the utility of in silico libraries generated by Carafe2 for peptide detection on several different types of timsTOF DIA datasets by comparing with the libraries generated with DDA-trained AlphaPeptDeep models, DIA-NN built-in models, and empirical spectral libraries generated from DDA experiments.

Cigars are a type of tobacco product made entirely from dried tobacco, primarily consisting of the filler, binder, and wrapper. Fermentation is a key step in improving the quality of cigar tobacco leaves (CTLs). To investigate how fermentation affects quality, this study employed non-targeted metabolomics and data-independent acquisition (DIA) proteomics to examine the metabolic changes and protein expression levels in tobacco leaves. The results reveal that a total of 112 differential metabolites were identified through untargeted metabolomics, with 87 compounds demonstrating a decrease in relative abundance post-fermentation, including 20 amino acids and their derivatives. Utilizing DIA proteomics, 341 differentially expressed proteins were identified. Functional analysis of these proteins revealed variations in biological functions at different fermentation stages. A total of 21 driver proteins exhibited significant correlations with the metabolic regulation of eight amino acids. This study revealed the transformation of amino acid metabolism significantly affects the quality of CTLs. It enhanced the understanding of amino acids among the differential metabolites before and after fermentation. This research provides a theoretical basis for the control of amino acids during the artificial fermentation process of CTLs, aiming to further improve their quality.

Cigars are a type of tobacco product made entirely from dried tobacco, primarily consisting of the filler, binder, and wrapper. Fermentation is a key step in improving the quality of cigar tobacco leaves (CTLs). To investigate how fermentation affects quality, this study employed non-targeted metabolomics and data-independent acquisition (DIA) proteomics to examine the metabolic changes and protein expression levels in tobacco leaves. The results reveal that a total of 112 differential metabolites were identified through untargeted metabolomics, with 87 compounds demonstrating a decrease in relative abundance post-fermentation, including 20 amino acids and their derivatives. Utilizing DIA proteomics, 341 differentially expressed proteins were identified. Functional analysis of these proteins revealed variations in biological functions at different fermentation stages. A total of 21 driver proteins exhibited significant correlations with the metabolic regulation of eight amino acids. This study revealed that the transformation of amino acid metabolism significantly affects the quality of CTLs. It enhanced the understanding of amino acids among the differential metabolites before and after fermentation. This research provides a theoretical basis for the control of amino acids during the artificial fermentation process of CTLs, aiming to further improve their quality.

This study aims to explore the anti-gastric cancer mechanism of combined prescription of modified Liangfu and Jianpi Yiqi(CPMLJY) using a zebrafish xenograft model with gastric cancer, ultra-performance liquid chromatography(UPLC), and data-independent acquisition(DIA) proteomics. The active ingredients of CPMLJY were identified by UPLC. A zebrafish xenograft model was established by microinjecting human gastric cancer HGC-27 cells into the yolk sac of wild-type AB strain zebrafish. The maximum tolerated concentration(MTC) of CPMLJY was measured by a gradient dilution method. Zebrafish were randomly divided into normal control group, model control group, capecitabine group, and low-(1/4 MTC), medium-(1/2 MTC), and high-dose(MTC) groups of CPMLJY, with 30 zebrafish in each group. After 48 h at 35 ℃, the effect of CPMLJY on tumor proliferation and apoptosis was assessed via fluorescence labeling. Transgenic zebrafish(T-cell-red and vascular-green Fil-1 strains) were used for modelling to evaluate immune response and angiogenesis. DIA proteomics was used to reveal potential anti-gastric cancer mechanisms. The results showed that the main active ingredients of CPMLJY were flavonoids, steroidal saponins, and triterpenoid saponins. The medium-and high-dose of CPMLJY significantly inhibited tumor growth(P&lt;0.05). High-dose of CPMLJY significantly increased the number of T cells, while medium-dose of CPMLJY significantly suppressed angiogenesis and induced apoptosis(P&lt;0.05). Proteomics identified 143 upregulated and 233 downregulated proteins(P&lt;0.05). CPMLJY exerted anti-tumor effects by modulating adipocyte lipolysis, forkhead box O(FoxO), wingless-related integration site(Wnt), and peroxisome proliferator-activated receptor(PPAR) signaling pathways, and amino acid metabolism(P&lt;0.05). These findings support further clinical translation of CPMLJY for gastric cancer therapy.

microRNAs (miRs) function in cancer progression as post-transcriptional regulators. We previously reported that endogenous circular RNAs (circRNAs) function as efficient miR sponges and could act as novel gene regulators in oral squamous cell carcinoma (OSCC). In this study, we carried out cellular and luciferase reporter assays to examine competitive inhibition of miR-1269a, which is upregulated expression in several cancers, by circRNA-1269a, a synthetic circRNA that contains miR-1269a binding sequences. We also used data-independent acquisition (DIA) proteomics and in silico analyses to determine how circRNA-1269a treatment affects molecules downstream of miR-1269a. First, we confirmed the circularization of the linear miR-1269a binding site sequence using RT-PCR with divergent/convergent primers and direct sequencing of the head-to-tail circRNA junction point. In luciferase reporter and cellular functional assays, circRNA-1269a significantly reduced miR-1269a function, leading to a significant decrease in cell proliferation and migration. DIA proteomics and gene set enrichment analysis of OSCC cells treated with circRNA-1269a indicated high differential expression for 284 proteins that were mainly enriched in apoptosis pathways. In particular, phospholipase C gamma 2 (PLCG2), which is related to OSCC clinical stage and overall survival, was affected by the circRNA-1269a/miR-1269a axis. Taken together, synthetic circRNA-1269a inhibits tumor progression via miR-1269a and its downstream targets, indicating that artificial circRNAs could represent an effective OSCC therapeutic.

Multi-omics analysis reveals the protective effects of Tibetan Medicine dalijuzhou powder on high-altitude polycythemia.