Differential Protein Expression Research Articles

Autophagy is involved in the toxicity of the biocontrol agent GC16 against Tetranychus pueraricola (Acari: Tetranychidae) based on transcriptomic and proteomic analyses

BackgroundGC16 is a novel pesticide with acaricidal properties against the spider mite Tetranychus pueraricola (Ehara & Gotoh). Its physiological mechanisms have been described previously, but its molecular mechanisms of action remain unclear. Thus, we aimed to explore the acaricidal mechanisms of GC16 through transcriptomic and proteomic analyses. The results were verified using transmission electron microscopy (TEM), immunofluorescence assay, and western blotting.ResultsTranscriptomic and proteomic analyses revealed 2717 differentially expressed genes (DEGs) and 374 differentially expressed proteins (DEPs) between the GC16-treated and control mites. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the DEGs and DEPs were enriched in the autophagy pathway. TEM showed that the number of autophagosomes and autolysosomes was higher in the GC16-treated mites than in the control mites. Immunofluorescence assay and western blot results consistently indicated that GC16 treatment significantly enhanced the relative expression of the autophagy protein LC3 in insect Sf9 cells. The intracellular calcium concentration in the GC16-treated Sf9 cells was 2.30 times higher than that in the control cells, suggesting that GC16 disrupted calcium homeostasis and potentially acted as a calcium-driven nerve agent.ConclusionsAutophagy is involved in the toxicity of GC16 against T. pueraricola and may be activated by elevated Ca2+ levels. This study reveals the molecular insecticidal mechanisms of GC16 and provides rationale for the field application of GC16 to control pest mites.

Allicin Binds to Cysteine Residues of Fungal Dihydrolipoamide Dehydrogenase to Control Gray Mold (Botrytis cinerea) of Tomato.

Allicin exhibits strong inhibitory activity against phytopathogenic fungi; however, its antifungal mechanism remains unclear. This study assessed allicin's inhibitory effects on several phytopathogenic fungi, revealing a half-maximal effective concentration of 125.47 μg/ml against the hyphal growth of Botrytis cinerea. Micromorphological analysis showed that allicin caused abnormalities in the hyphae, including unclear organelle boundaries and organelle dissolution. Integrated transcriptomic, and proteomic, and metabolomic assays indicated that allicin induced differential gene and protein expression, particularly in the plasma membrane, oxidative stress processes, and energy metabolism pathways. Additionally, differentially expressed metabolites were involved in the inhibition of hyphal growth. Biochemical assays demonstrated that allicin inhibited ATP production and damaged hyphal cell membranes. Molecular docking revealed that allicin could bind to dihydrolipoamide dehydrogenase (DLDH), an enzyme rich in cysteine residues, with a binding free energy of -6.322 kcal/mol. Through antimicrobial activity testing of allicin analogues and molecular docking analysis, the active groups of allicin and its interaction with DLDH were identified. This study shows that allicin interferes with energy metabolism, impacts cell membrane and wall integrity by targeting cysteine-containing proteins, and inhibits the proliferation of plant pathogenic fungi. These insights into the antifungal mechanism will provide valuable data for the development and field application of allicin analogues.

Integrating proteomics and metabolomics to elucidate the regulatory mechanisms of pimpled egg production in chickens: Multi-omics analysis of the mechanism of pimpled egg formation.

Eggshells not only protect the contents of the egg from external damage but are also a key factor influencing consumer choice, second only to price. In the later stages of egg production, the incidence of pimpled eggs significantly increases, severely affecting the hatchability and food safety of the eggs. This study compares the differences in the uterine proteomes and metabolomes of hens producing pimpled eggs and those producing normal eggs, aiming to identify the proteins and metabolites that may play a crucial role in the formation of pimpled eggs. A total of 242 differentially expressed proteins (DEPs) were identified in uterine tissue, of which 116 were upregulated and 126 were downregulated. Enrichment analysis revealed that the DEPs were enriched in pathways related to ion transport, energy metabolism, and immune responses. The study found that in the normal eggs (NE) group, HCO₃⁻ was predominantly transported via SLC4A1, although other transport pathways may also play a role. In contrast, in the pimpled eggs (PE) group, bicarbonate ions (HCO₃⁻) was primarily transported through SLC4A4. Additionally, a total of 44 differentially metabolites (DMs) were identified in the uterus, with 5'-Adenylic acid (ATP) being significantly downregulated in the PE group. The ions and matrix proteins required for eggshell formation are transported from uterine cells to the uterine fluid against a concentration gradient, a process that consumes a substantial amount of energy. The decrease in ATP concentration in the PE group may be a significant factor influencing the formation of pimpled eggs. Subsequently, we found that the DEPs and DMs were jointly enriched in several signaling pathways, including the FoxO signaling pathway related to energy metabolism, nicotinate and nicotinamide metabolism, and tryptophan metabolism associated with immune response. Notably, the DMs involved in these signaling pathways were all downregulated in the PE group. Our research findings indicate that SLC4A1, SLC4A2, and ATP2B4 (DEPs), along with 5'-adenylic acid and trigonelline (DMs), influence the formation of eggshells through mechanisms related to energy metabolism, ion transport, and immune response. These DEPs and DMs may serve as potential biomarkers for the genetic improvement of eggshell quality.

Comprehensive proteomic analysis and multidimensional model construction of peritoneal metastasis in gastric cancer.

Peritoneal metastasis following gastric cancer surgery is often associated with a poor prognosis. This study aimed to investigate the mechanisms underlying peritoneal metastasis and to develop a predictive model for the risk of postoperative peritoneal metastases in gastric cancer. We performed a comprehensive analysis of the protein mass spectra and tumor microenvironment in paraffin-embedded primary tumor sections from gastric cancer patients, both with and without postoperative peritoneal metastases. Using proteomic profiling, we identified 9,595 proteins and stratified patients into three distinct proteomic subgroups (Pro1, Pro2, Pro3) based on differential protein expression. Simultaneously, immune cell profiling allowed us to classify patients into four immune subgroups (IG-I, IG-II, IG-III, IG-IV). The relationships between these proteomic, immune, and metastasis classifications were further explored to uncover potential associations and mechanisms driving metastasis. Building on these insights, we developed an integrative model combining proteomics, immunological, and radiomics data for predicting postoperative peritoneal metastases. This model demonstrated high predictive efficacy, offering a robust tool for identifying high-risk patients. Our findings provide a deeper understanding of the biological processes underlying peritoneal metastasis in gastric cancer, highlighting the interplay between proteomic and immune factors. By establishing novel patient subgroups and an effective prediction model, this study lays the groundwork for early diagnosis and tailored therapeutic strategies to improve outcomes for gastric cancer patients.

Identification of TRAF2, CAMK2G, and TIMM17A as biomarkers distinguishing mechanical asphyxia from sudden cardiac death base on 4D-DIA Proteomics: A pilot study.

In the context of forensic medicine, the differential diagnosis between mechanical asphyxia and sudden cardiac death is very important regarding the establishment of the cause of death. Traditional autopsy findings have generally been very nonspecific; accordingly, highlighting the need for more specific molecular biomarkers. This study employed four-dimensional data-independent acquisition (4D-DIA) proteomics technology, in combination with both animal models and human samples, to conduct a comprehensive protein expression analysis of cardiac tissues, identifying 7557 proteins, among which 142 shared differentially expressed proteins (DEPS) were screened out. Based on the protein interaction network and through rigorous screening, this study identified three proteins, namely TNF receptor-associated factor 2 (TRAF2), Calcium/calmodulin-dependent protein kinase II gamma (CAMK2G), and translocase of inner mitochondrial membrane 17 homolog A (TIMM17A), as biomarkers for differentiating mechanical asphyxia from sudden cardiac death. Further verification using Western Blot (WB) and immunohistochemistry (IHC) proved the differential expression of these biomarkers in both animal and human samples. These findings, besides deepening the molecular understanding of the pathophysiological differences between sudden cardiac death and mechanical asphyxia, also provided new biomarkers for forensic applications that could enable the improvement of accuracy and reliability in the determination of the cause of death.

Establishment and evaluation of rabbit model for corneal ectasia by photorefractive keratectomy.

The study aimed to compare the effects of different types of excimer laser keratectomy on rabbit corneas and to identify the optimal disease model for corneal ectasia. Additionally, investigating the structural and molecular alterations in the novel disease model helped explore the mechanisms underlying biomechanical cues in corneal ectasia. 2.0-2.5kg New Zealand white rabbits were treated with different types of excimer laser keratectomy, including comparisons between photorefractive keratectomy (PRK) and phototherapeutic keratectomy (PTK) surgeries, as well as comparisons of different ablation depths of PRK. Detailed tests on post-surgery corneas included pentacam analyzer, H&E staining and optical coherence tomography (OCT), transmission electron microscopy (TEM), raman spectroscopy and uniaxial tensile tests. Later, tandem mass tag-labeled proteomics and multiply statistic analysis were performed on post-PRK75 corneas. Western blot was used to validate protein expression. Herein, we found that tapered corneal thinning in post-PRK corneas predisposed to corneal ectasia. Greater ablation depth increased ectasia risk. PRK75 (ablation of 75% of corneal thickness using PRK mode) emerged as the optimal modeling approach, evidenced by significant and sustained corneal ectasia for 4 weeks. The 4-week post-PRK75 corneas were evaluated by changes in stromal cell microstructure, basement membrane, collagen lamellae, collagen covalent bonds and decreased corneal biomechanical strength. Additionally, PRK75 surgery induced 109 differentially expressed proteins (DEPs), with 51 previously linked to human corneal ectasia. The statistic analysis demonstrated the dysregulation of immue response was involved in the post-PRK75 corneas, and identified nine core proteins involved in corneal ectasia, including SERPINH1, ALDH1A1, MMP10, A2M, GSTM3, CD44, CLU, C3, and ITGB2. Therefore, we concluded that PRK75 was a novel and reliable modeling method for corneal ectasia, resemble human corneal ectasia. The intrinsic structural remodeling and molecular alteration in post-PRK75 corneas could shed lights on understanding the mechanism of biomechanical cues in corneal ectasia in the future.

The anti-neuroinflammatory effects of cepharanthine in uric acid-induced neuroinflammation.

Cepharanthine (CEP) is an alkaloid extracted from Stephania cephalantha Hayata, a traditional Chinese medicine (TCM) renowned for its heatclearing and dehumidifying properties. For centuries, Stephania cephalantha Hayata has been employed in the treatment of a wide range of diseases, including pain, edema, inflammation, and fever. Our research aims to investigate the role and mechanism of Cepharanthine in ameliorating uric acid (UA) induced neuroinflammatory responses. The Connectivity Map (CMap) was utilized to identify the therapeutic drug Cepharanthine, based on the proteomic disturbances associated with uric acid (UA). Limited proteolysis small molecule mapping (LiP-SMap) and thermal proteome profiling (TPP) technologies were used to identify the direct target proteins for UA and Cepharanthine. Additionally, we used the induced-fit docking algorithm integrated within the Schrodinger suite to explore the interactions between Cepharanthine and uric acid targets. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology was employed to determine the concentration of Cepharanthine in the mice cerebral cortex. The pro-inflammatory cytokine genes were also quantified by qPCR in U251cells and in hyperuricemic mice. The findings indicated that uric acid increased the transcription of pro-inflammatory cytokines and the expression levels of proteins linked to inflammation in U251cells. PPP2R1A was identified as a potential candidate for direct interaction with uric acid, potentially initiating inflammation. Based on the CMap prediction, Cepharanthine was identified as a candidate drug for interaction with PPP2R1A. TPP analysis indicated that Cepharanthine could reduce the thermal stability of PPP2R1A. Molecular docking confirmed that Cepharanthine could directly bind to PPP2R1A. Furthermore, the detection of Cepharanthine in the cerebral cortex suggested its ability to cross the blood-brain barrier. Proteomic analysis of Cepharanthine-treated mice revealed significant enrichments of differentially expressed proteins (DEPs) in inflammation-related pathways and biological processes. Additionally, Cepharanthine was effective in decreasing the expression of pro-inflammatory cytokine genes induced by uric acid in U251cells and in hyperuricemic mice. Cepharanthine could effectively alleviate hyperuricemia-induced neuroinflammation via binding to PPP2R1A. This study offers a novel approach for prevention and treatment of neurological diseases caused by hyperuricemia.

Differential Expression of Hard Tissue Proteins in Hypomineralized Second Primary Molars in Comparison to Normal Teeth.

This study aims to identify the proteins in hypomineralized second primary molars (HSPMs) and correlate their function in Amelogenesis. HSPM is a qualitative defect of the enamel of the second primary molars with no clear etiology. Total protein quantification was performed using the Bradford Protein Assay, followed by the electrophoretic separation of samples using 2D-Gel electrophoresis to identify the proteins. The results from the Bradford Protein Assay unveiled a five-fold increase in the protein content in HSPM. Proteins such as Dentin sialo-phosphoprotein (DSPP), Keratin, type I, Serum Albumin, Anti-thrombin III, Alpha-1-Antitrypsin, Histone H3.2, Actin, Heat shock Protein, Vimentin, Desmoglein-3, Glyceraldehyde-3-phosphate dehydrogenase, Inosine-5'-monophosphate dehydrogenase 2, Zinc Alpha 2 glycoprotein, Lysozyme C, Prothrombin, Vit-D binding Protein, Apolipoprotein A-1, Defensin 1, Immunoglobulin Gamma, Immunoglobulin Kappa, and Alpha-Amylase were all upregulated (p < 0.05) in HSPM. This investigation conclusively demonstrates that HSPM-affected teeth have higher protein content than healthy teeth. The study also supports the theory of proteolytic inhibition attributed to reduced protease activity and heightened protease inhibitor activity.

Epimedin C promotes mitochondrial transfer and delays thymus atrophy in 4-VCD induced mimetic-menopausal mice.

Prevention of thymus atrophy during menopause is of great significance for improving the immune function and overall health of menopausal women. Epimedin C is one of the major bioactive compounds in Herba Epimedii, a traditional herbal medicine for the treatment of menopausal syndrome in China, but the action and mechanism of Epimedin C in the treatment of menopausal thymus atrophy remains unclear. To study the effect of Epimedin C on thymus atrophy in 4-vinylcyclohexene diepoxide (4-VCD) induced mimetic-menopausal mice and explore its mechanism from new perspectives of tunneling nanotubes (TNTs) formation and mitochondrial transfer (MitoT). The effects of Epimedin C in the 4-VCD induced menopause-like phenotype in mice were observed, and the thymic output function was evaluated by the quantitative detection of T cell receptor excision circles (TRECs). The structure of the thymus was observed by H&E. The arrangement and quantity of different cell subpopulations of thymic epithelial cells (TECs) and thymocytes were detected by multiple fluorescent staining and flow cytometry. Mitochondrial morphology was observed with transmission electron microscopy. LC-MS/MS was used to analyze and identify the differential protein expression in thymus before and after Epimedin C treatment. Actin polymerization inhibitor was used to verify the possible mechanism of Epimedin C. The treadmilling-balance of actin, TNTs formation, and MitoT processes were observed by specific fluorescent probe labeling. The interaction between G-actin, Thymosin β4 (Tβ4), and Epimedin C were studied by protein cross-linking assay. Epimedin C significantly increased the thymus weight and the area of the thymus medulla, increased the grip strength and bone strength in 4-VCD induced mimetic-menopausal mice, and enhanced ovarian secretion function. It could affect the thymus output, increase CK5 and CK8 expression, maintain the reticular structure of TECs, inhibit the differentiation of thymocytes into double positive cells (CD4+CD8+) and CD4SP (CD3+TCR β+CD4+CD8-) cells. Epimedin C promoted the conversion of G-actin to F-actin and accelerated MitoT via stimulating the TNTs formation, which related with the downregulation of Tβ4 and obstruction to the formation of Tβ4-G-actin complex. Epimedin C can promote TEC activity in 4-VCD induced mimetic-menopausal mice by decreasing the expression of Tβ4, inhibiting the binding of Tβ4 to G-actin, promoting the F-actin polymerization and the TNTs-depended MitoT.

Proteome Analysis Reveals Paraspinal Muscle Fiber Type Changes in Patients with Degenerative Lumbar Scoliosis.

Degenerative lumbar scoliosis (DLS) is a common aging-related spinal deformity. Paraspinal muscle degeneration is highly correlated with the rapid progression of DLS. However, understanding of the role of the degeneration is limited because of a lack of histologic and molecular evidence. Our study profiled the proteomic alteration of paraspinal muscles and investigated the muscle fiber type transition that occurs in DLS, along with its correlation with clinical parameters. Cross-sectional basic science study using clinical data and biological samples. Paraspinal muscle samples were collected intraoperatively from the concave and convex sides of the apex vertrebrae in patients with DLS (n = 10) and either side of L3 level from age- and sex-matched participants without DLS (n = 10). Analysis was perfomed using isobaric tagging for relative and absolute quantitation (iTRAQ) and liquid chromatography with tandem mass spectrometry on muscle tissue from the convex side of spines in patients with DLS and in participants without DLS to identify differentially expressed proteins (DEPs). Western blotting was used to validate the DEPs. The measurement of acidity/basicity of ATPase (pH = 9.4), succinic acid dehydrogenase staining, and real-time quantitative polymerase chain reaction were performed to assess the muscle fiber type change in DLS. The Pearson correlation coefficient was used to analyze the correlation between the myofiber transition and the Cobb angle of the main curve. This study was supported by the National Natural Science Foundation of China (NSFC) (No. 82272545), $ 8,000-10,000 and the Jiangsu Provincial Key Medical Center, and the China Postdoctoral Science Foundation (2021M701677, $ 5,000-7,000). We identified 62 DEPs, of which 16 were downregulated and 46 were upregulated. Gene ontology indicated significant changes in biological processes including muscle contraction. Protein-protein interaction network analysis showed that structural muscle proteins such as MYH1 (myosin heavy chain 1) and TNNT3 (troponin T) were the key nodes. Western blotting further validated the downregulation of MYH1. Histologically, ATPase staining showed a significant reduction of type II muscle fibers in DLS, consistent with the functional changes of the DEPs. Furthermore, we found that the reduction of type II muscle fibers percentage was correlated with the severity of DLS. This study is the first to elucidate the underlying molecular basis and pathways that implicate the paraspinal muscle fiber type transition in DLS. Type II myofiber percentage was diminished both on the concave side and the convex side of the paraspinal muscles in DLS, especially on the convex side, which may play an important role in the onset and/or progression of the disease. This study shows a potential molecular basis for histopathologic change in the paraspinal muscles of DLS and provides a potential tool for assessing paraspinal muscle quality and predicting the poor prognosis of DLS.

A window into intracellular events in myositis through subcellular proteomics

Objective and designIdiopathic inflammatory myopathies (IIM) are a heterogeneous group of inflammatory muscle disorders of unknown etiology. It is postulated that mitochondrial dysfunction and protein aggregation in skeletal muscle contribute to myofiber degeneration. However, molecular pathways that lead to protein aggregation in skeletal muscle are not well defined.SubjectsHere we have isolated membrane-bound organelles (e.g., nuclei, mitochondria, sarcoplasmic/endoplasmic reticulum, Golgi apparatus, and plasma membrane) from muscle biopsies of normal (n = 3) and muscle disease patients (n = 11). Of the myopathy group, 10 patients displayed mitochondrial abnormalities (IIM (n = 9); mitochondrial myopathy (n = 1)), and one IIM patient did not show mitochondrial abnormalities (polymyositis).MethodsGlobal proteomic analysis was performed using an Orbitrap Fusion mass spectrometer. Upon unsupervised clustering, normal and mitochondrial myopathy muscle samples clustered separately from IIM samples.ResultsWe have confirmed previously known protein alterations in IIM and identified several new ones. For example, we found differential expression of (i) nuclear proteins that control cell division, transcription, RNA regulation, and stability, (ii) ER and Golgi proteins involved in protein folding, degradation, and protein trafficking in the cytosol, and (iii) mitochondrial proteins involved in energy production/metabolism and alterations in cytoskeletal and contractile machinery of the muscle.ConclusionsOur data demonstrates that molecular alterations are not limited to protein aggregations in the cytosol (inclusions) and occur in nuclear, mitochondrial, and membrane compartments of IIM skeletal muscle.

Effect of potato glycoside alkaloids on mitochondria energy metabolism of Fusarium solani, the root rot pathogen of Lycium barbarum

Fusarium solani is a widely distributed pathogenic fungus that can cause soil borne diseases in various plants and is also one of the main pathogenic bacteria of Lycium barbarum root rot. This study employed tandem mass labeling (TMT) quantitative proteomics technology to investigate the antifungal mechanism of potato glycoside alkaloids (PGA) against Fusarium solani. We elucidated the antifungal mechanism of PGA from the perspective of mitochondrial proteome molecular biology. Furthermore, we identified and annotated the differentially expressed proteins (DEP) of F. solani under PGA stress. A total of 2,412 DEPs were identified, among which 1,083 were significantly up-regulated and 1,329 significantly down-regulated. Subsequent analysis focused on five DEPs related to energy metabolism for verification at both protein and gene levels. Gene Ontology (GO) function analysis revealed that the DEPs were primarily involved in the integral component of the membrane, intrinsic component of the membrane, pyridine-containing compound metabolic processes, carbon-oxygen lyase activity, and the endoplasmic reticulum, with a notable enrichment in membrane components. Furthermore, a total of 195 pathways were identified through KEGG analysis, with significant enrichment in critical pathways including pentose and glucuronate interconversions, propanoate metabolism, various types of N-glycan biosynthesis, the pentose phosphate pathway, and carbon fixation in photosynthetic organisms. The results from both parallel reaction monitoring (PRM) and real-time RT-qPCR were consistent with the overall trends observed in TMT proteomics, thereby confirming the validity of the TMT proteomics analysis. These findings indicate that PGA inhibits the growth of F. solani by impacting mitochondrial energy metabolism. This study reveals the antifungal mechanism of PGA from the perspective of energy metabolism, providing a theoretical basis for the development and application of PGA as a biopesticide.

Proteomic Approach to Study the Effect of Pneumocystis jirovecii Colonization in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and interstitial disease with an unclear cause, believed to involve genetic, environmental, and molecular factors. Recent research suggested that Pneumocystis jirovecii (PJ) could contribute to disease exacerbations and severity. This article explores how PJ colonization might influence the pathogenesis of IPF. We performed a proteomic analysis to study the profile of control and IPF patients, with/without PJ. We recruited nine participants from the Virgen del Rocio University Hospital (Seville, Spain). iTRAQ and bioinformatics analyses were performed to identify differentially expressed proteins (DEPs), including a functional analysis of DEPs and of the protein–protein interaction networks built using the STRING database. We identified a total of 92 DEPs highlighting the protein vimentin when comparing groups. Functional differences were observed, with the glycolysis pathway highlighted in PJ-colonized IPF patients; as well as the pentose phosphate pathway and miR-133A in non-colonized IPF patients. We found 11 protein complexes, notably the JAK-STAT signaling complex in non-colonized IPF patients. To our knowledge, this is the first study that analyzed PJ colonization’s effect on IPF patients. However, further research is needed, especially on the complex interactions with the AKT/GSK-3β/snail pathway that could explain some of our results.

P-2232. Tracheal aspirate and plasma proteomics reveals the local and systemic host immune response to severe pediatric lower respiratory viral infections

Abstract Background Viral lower respiratory tract infections (LRTI) are a leading cause of child mortality worldwide. Improved understanding of local and systemic host immune responses to severe viral LRTI could reveal insights into pathophysiology, lead to novel host-based diagnostic tests, and inform personalized treatment. To date, no studies have yet employed proteomics to simultaneously characterize the lower airway and systemic proteome in pediatric viral LRTI. Figure 1 Methods We used SomaScan® to assess relative expression of 1,305 proteins from tracheal aspirate (TA) and plasma in 63 patients with acute respiratory failure, a subset of a larger prospective multicenter cohort. We performed differential protein expression and pathway enrichment analyses between viral LRTI (n=41; n=24 with bacterial superinfection) and non-infectious respiratory failure (n=22), developed a diagnostic classifier using LASSO regression, and comparatively analyzed TA and plasma samples. Subanalyses were performed to investigate the impact of bacterial superinfection and viral load. Figure 2 Comparison of host protein expression between Viral LRTI and No LRTI cohorts in tracheal aspirate. (A) Volcano plot of the differentially expressed proteins, with proteins significantly upregulated in LRTI shown in red, and proteins significantly downregulated in LRTI shown in blue. The top ten proteins based on adjusted P-value are labeled. (B) Heat map showing differential expression of the top 20 proteins based on adjusted P-value (rows) across all patients (columns). Dendrogram clustering (top) highlights the proteomic differences between the two groups. (C) Pathway enrichment analysis showing the top ten pathways (all upregulated) ordered by Normalized Enrichment Score. Dot color indicates the false discovery rate (FDR) adjusted P-value, and size indicates the number of proteins included in the pathway. (D) Receiver operator characteristic (ROC) curve of the proteomic classifier to distinguish Viral LRTI from No LRTI. Results We identified a distinct proteomic signature of severe viral LRTI in TA with 200 differentially expressed proteins (adjusted P-value&amp;lt; 0.05), with upregulation of proteins key for type I interferon response, NK cells, and cytotoxic T cells and downregulation of inflammation-modulating proteins. A parsimonious diagnostic classifier achieved an AUC of 0.96 (95% CI 0.90-1.00) for diagnosing LRTI. In contrast, the systemic host response to viral LRTI in plasma was more subtle with 56 differentially expressed proteins. Correlation between TA and plasma proteomics was limited, although the interferon-stimulated protein ISG15 demonstrated increased expression across both compartments. Bacterial superinfection showed upregulation of TNF-stimulated protein TSG-6, C-reactive protein, and interferon signaling compared to viral infection alone. Viral load exhibited positive correlation with interferon proteins and negative correlation with neutrophil aggregation proteins. Conclusion We characterized the lower airway and systemic proteomic host response of severe pediatric viral LRTI and identified proteins with potential mechanistic role in disease severity that could be targeted with novel interventions. Disclosures Jack Kamm, PhD, Genentech: Employee (started there after leaving this study group) Nadir Yehya, MD, AstraZeneca: Advisor/Consultant Lilliam Ambroggio, PhD, Pfizer Inc.: Grant/Research Support

Targeting Hepatocellular Carcinoma Growth: Haprolid’s Inhibition of AKT Signaling Through DExH-Box Helicase 9 Downregulation

Objective: Haprolid, a novel compound extracted from Myxobacterium, has been proven to possess selective toxicity towards various tumor cells, effectively inhibiting the growth of hepatocellular carcinoma (HCC). However, the underlying molecular mechanism remains unclear. Methods: To identify differentially expressed proteins (DEPs), isobaric tags for relative and absolute quantitation (iTRAQ) were employed. The clinical significance of DExH-Box Helicase 9 (DHX9) was determined using tissue microarrays in HCC patients. Changes in protein expression were detected using Western blotting, qPCR, and immunohistochemistry. Cell proliferation was evaluated using CCK-8 and crystal violet staining. Cell apoptosis was assessed using Alexa Fluor 647 Annexin V. Xenograft tumor experiments were conducted in animals. Results: iTRAQ screening identified DHX9 as a DEP. DHX9 was discovered to be highly expressed in HCC tissues, correlating with poor prognosis in patients. Haprolid downregulated DHX9 expression, while knockdown of DHX9 suppressed HCC cell proliferation and migration and promoted apoptosis. Meanwhile, overexpression of DHX9 mitigated the inhibitory effect of Haprolid on HCC cells. Knockdown of DHX9 inhibited the AKT signaling pathway, and SC79 reversed the inhibitory effect of DHX9 knockdown on HCC cells. Xenograft experiments confirmed that the knockdown of DHX9 inhibited HCC growth, while the overexpression of DHX9 attenuated the inhibitory effect of Haprolid on HCC growth. Conclusions: Haprolid inhibits the AKT signaling pathway by downregulating DHX9, ultimately suppressing HCC growth. This finding opens up new avenues for targeted HCC therapy.

Proteomic Characterization of NEDD4 Unveils Its Potential Novel Downstream Effectors in Gastric Cancer.

The E3 ubiquitin ligase neural precursor cell-expressed developmentally down-regulated 4 (NEDD4) is involved in various cancer signaling pathways, including PTEN/AKT. However, its role in promoting gastric cancer (GC) progression is unclear. This study was conducted to elucidate the role of NEDD4 in GC progression. We found that the inhibition of NEDD4 expression significantly reduced the migratory and proliferative abilities of GC cells, with minimal impact on the PTEN expression or p-AKT activation, suggesting that NEDD4 may exert its GC-promoting effects through alternative pathways. To gain novel insights into the role of NEDD4 in GC, we performed a comprehensive proteomic analysis to search for proteins with altered expression levels following NEDD4 gene knockdown, identifying a total of 3916 proteins. Pathway analysis of differentially expressed proteins (DEPs) indicated the potential involvement of NEDD4 in cancer-related metabolic pathways. Furthermore, the protein-protein interaction network of the DEPs revealed enriched core modules, highlighting key cellular processes and signaling pathways regulated by NEDD4 in GC. Additionally, we identified proteins whose expression was altered by NEDD4 inhibition, some of which were associated with poor prognosis in GC. These findings suggest that these proteins may act as downstream effectors that contribute to NEDD4-mediated GC progression.

Proteomic analysis of whole blood to investigate the therapeutic effects of nervonic acid on cerebral ischemia-reperfusion injury in rats

IntroductionBlood proteomics offers a powerful approach for identifying disease-specific biomarkers. However, no reliable blood markers are currently available for the diagnosis stroke. Nervonic acid (NA), a vital long-chain monounsaturated fatty acid found in mammalian nervous tissue, shows promising therapeutic potential in neurological disorders. This study aimed to develop a reliable methodology for whole blood proteomics to identify early warning biomarkers and evaluate drug treatment efficacy.MethodsAfter modeling via the classic thread embolization method, whole blood samples were collected from the rats. Morphological assessments of brain tissue indicated that NA significantly mitigated brain and neuronal damage in rats. The differential protein expression profile was analyzed using Liquid Chromatography—Tandem Mass Spectrometry (LC-MS/MS) whole blood proteomics.ResultsZZZGene Ontology (GO) analysis revealed that, compared to ginkgo biloba extract (EGb), the proteins differentially expressed under NA intervention were predominantly involved in oxidative stress response and calcium-dependent adhesion processes. Key targets of NA in the treatment of middle cerebral artery occlusion (MCAO) models included ENO1, STAT3, NME2, VCL, and CCT3.DiscussionThis whole blood proteomic approach provides a comprehensive understanding of protein profiles associated with disease states, offering valuable insights into potential therapeutic targets and enabling the evaluation of NA and EGb intervention efficacy. Our findings underscore the protective effects of NA against cerebral ischemia-reperfusion injury and highlight its potential as a treatment for stroke.

Proteomics profiling of inflammatory responses to elexacaftor/tezacaftor/ivacaftor in cystic fibrosis

BackgroundCFTR modulator therapies have positive clinical outcomes, yet chronic inflammation and bacterial infections persist in people with CF (pwCF). How elexacaftor–tezacaftor–ivacaftor (ETI) fails to improve innate immune signaling responsible for bacterial clearance and inflammation resolution remains unknown.MethodsWe used an unbiased proteomics approach to measure the effect of ETI on inflammatory proteins. Plasma from 20 pediatric pwCF and 20 non-CF (NCF) was collected during routine examination and 3 months after ETI initiation. Protein screening was performed with an inflammation panel (Target 96, Olink®). Bioinformatics analysis was used to determine changes in protein expression.ResultsThere were significantly fewer pulmonary exacerbations after ETI initiation, along with sustained improvement in lung function and reduced bacterial colonization. Unpaired analysis of CF pre-ETI and NCF resulted in 34 significantly different proteins. Of these, CCL20, MMP-10, EN-RAGE, and AXIN1 had a log2 fold change of 1.2 or more. There was a modest shift in overall CF protein profiles post-ETI toward the NCF cluster. Unpaired analysis of protein differential expression between NCF and CF post-ETI identified a total of 24 proteins significantly impacted by ETI therapy (p-value ≤ 0.05), with only CCL20 having a log2 fold change higher than 1.2. Paired analysis (CF pre- and CF post-ETI) of differential protein expression demonstrated significant expression changes of MMP-10, EN-RAGE, and IL-17A. Pathway analysis identified significantly impacted pathways such as the NF-κB pathway.ConclusionThis study showed that ETI in a pediatric cohort had a modest effect on several inflammatory proteins with potential as biomarkers. Pathways significantly impacted by ETI can be further studied for future therapies to combat persistent inflammation and dysregulated immunity.

Multi-Omics Sequencing Dissects the Atlas of Seminal Plasma Exosomes from Semen Containing Low or High Rates of Sperm with Cytoplasmic Droplets

Sperm cytoplasmic droplets (CDs) are remnants of cytoplasm that can cause a number of problems if it not shed from the sperm after ejaculation. Exosomes can rapidly bind to sperm, but it is not clear whether exosomes can affect the migration and shedding of CDs. We first extracted and characterized seminal plasma exosomes from boar semen containing sperm with low or high rates of CDs. Then, the transcriptomic and proteomic detection of these exosomes were performed to analyze the differences between the two groups of seminal plasma exosomes. The results revealed that 486 differentially expressed genes (DEGs), 40 differentially expressed proteins (DEPs), and 503 differentially expressed lncRNAs (DElncRNAs) were identified between the low CD rate group and high CD rate group. Integrative multi-omics analysis showed that exosome components may affect migration and shedding of cytoplasmic droplets by influencing cytoskeletal regulation and insulin signaling, including regulation of the actin cytoskeleton, ECM–receptor interaction, axon guidance, insulin secretion, and the insulin signaling pathway. Overall, our study systematically revealed the DEGs, DEPs, and DElncRNAs in seminal plasma exosomes between low CD rate semen and high CD rate semen, which will help broaden our understanding of the complex molecular mechanisms involved in the shedding of CDs.

Cross-sectional study of proteomic differences between moderate and severe psoriasis

Although an ongoing understanding of psoriasis vulgaris (PV) pathogenesis, little is known about the proteomic differences between moderate and severe psoriasis. In this cross-sectional study, we evaluated the proteomic differences between moderate and severe psoriasis using data-independent acquisition mass spectrometry (DIA-MS). 173 differentially expressed proteins (DEPs) were significantly differentially expressed between the two groups. Among them, 85 proteins were upregulated, while 88 were downregulated (FC ≥ ± 1.5, P < 0.05). Eighteen DEPs were mainly enriched in the IL − 17 signalling pathway, Neutrophil extracellular trap formation, Neutrophil degranulation and NF − kappa B signalling pathway, which were associated with psoriasis pathogenesis. Ingenuity pathway Analysis (IPA) identified TNF and TDP53 as the top upstream up-regulators, while Lipopolysaccharide and YAP1 were the top potential down-regulators. The main active pathways were antimicrobial peptides and PTEN signalling, while the inhibitory pathways were the neutrophil extracellular trap pathway, neutrophil degranulation, and IL-8 signalling. 4D-parallel reaction monitoring (4D-PRM) suggested that KRT6A were downregulated in severe psoriasis. Our data identify Eighteen DEPs as biomarkers of disease severity, and are associated with IL − 17 signalling pathway, Neutrophil extracellular trap formation, NF − kappa B signalling pathway, and defence response to the bacterium. Targeting these molecules and measures to manage infection may improve psoriasis’s severity and therapeutic efficacy.