Differential Protein Profiles Research Articles

COL8A2 activation enhances function of corneal endothelial cells through HIPPO signaling/mitochondria pathway

Corneal endothelial cells (CECs) are essential for maintaining corneal transparency and hydration through their barrier and pump functions. The COL8A2 gene encodes a component of the extracellular matrix of the cornea, which is crucial for the normal functioning of these cells. Mutations in COL8A2 are linked to corneal dystrophies, emphasizing the gene's importance in corneal health. The purpose of this research is to explore the effects of COL8A2 activation within CECs, to understand its contribution to cellular behavior and health. COL8A2 CRISPR/dCas9 activation system (aCOL8A2) was used to activate the COL8A2. In rats, wound healing and mitochondrial function were assessed after COL8A2 activation. As a result, aCOL8A2 promoted wound healing of rat corneal endothelium by increasing mitochondrial membrane potential. In cultured human CECs, proteomic analysis was performed to screen and identify the differential protein profiles between control and aCOL8A2 cells. Western blot was used to validate the differential proteins from both cells. Mitochondrial function and intracellular distribution were assessed by measuring ATP production and mitochondrial membrane potential. In cultured human CECs, aCOL8A2 increased COL8A2 and phospho-YAP levels. Transendothelial electrical resistance (TEER) was increased and actin cytoskeleton was attenuated by aCOL8A2. Gene ontology analysis revealed that the proteins were mainly involved in the regulation of folate biosynthesis, ECM-receptor interaction, cell differentiation, NADP activity and cytoskeleton. ATP production was increased, mitochondrial membrane potential was polarized and mitochondrial distribution was widespread in the aCOL8A2 group. In conclusion, aCOL8A2 induces a regulatory cascade affecting mitochondrial positioning and efficiency, mediated by alterations in the cytoskeletal architecture and the YAP signaling pathway. This sequence of events serves to bolster the functional capacities of corneal endothelial cells, including their pump and barrier functions, essential for corneal health and transparency.

Potential protein biomarkers in saliva for detection of frailty syndrome by targeted proteomics

Frailty is a physiological geriatric syndrome, caused by immunosenescence, inflammation and alterations at the protein level leading to metabolic and microbiota changes. Currently, this syndrome is evaluated clinically with the Frailty-VIG index. The aim of the study was therefore to investigate the potential suitability of saliva as a non-invasive proximal biological fluid for the characterisation and identification of possible protein-level biomarkers in frailty syndrome. This cross-sectional study was conducted in a rural population of older Spanish adults using the SMR proteomics technique. A differential protein profile of eight potential and surrogate proteins (CYTC, CYTD, CYTS, CYTB, MIF, ALBU, CD44 and B2MG) was detected in saliva, all of which correlated with factors characterising frailty syndrome, such as vascular ageing (arterial stiffness and cardiovascular disease), obesity, mood problems, global cognitive impairment, changes in gait and hand pressure strength. The proteins CYTD (r = 0.415, p = 0.013) and CYTC (r = 0.280, p = 0.026), which were detected differentially in the protein profile, were associated with the Frailty-VIG index. All analysed proteins are associated not only with the clinical symptoms of frailty syndrome, but also with an acute inflammatory response, endothelial cell proliferation and the complement system, among others.

Proteomic profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures

Leptospirosis is a global zoonotic disease affecting humans, domestic, and wild animals. Leptospira are typically shed in the urine of reservoir hosts which persist in suitable environments where incidental host transmission occurs after direct contact with infected urine or contaminated environments. Interestingly, serologically identical L. borgpetersenii serovar Hardjo strains JB197 and HB203 show divergent disease severity in the hamster model; JB197 causes severe acute infection while HB203 causes persistent chronic infection. Historically, serovar Hardjo was limited to culture at 29 °C, but utilization of HAN media allows propagation from host tissues at 37 °C. Here, the proteome of strains JB197 and HB203 were characterized after culture from experimentally challenged hamsters at 29 °C and 37 °C. Comparative analyses of JB197 and HB203 samples cultured at 29 °C yielded 425 significantly differentially expressed (DE) proteins, while strains at 37 °C yielded 613 DE proteins including prominent outer membrane proteins and known virulence factors. In agreement, membrane protein GO terms were identified by STRING network analyses along with numerous metabolic KEGG pathways consistent with condition differences. Within strain, JB197 cultured at 29 °C vs 37 °C identified 529 DE proteins, while HB203 identified 524 DE proteins. Investigating differential protein profiles provide insights into strain specific behaviors with implications for better understanding host-pathogen interactions, disease transmission, and response to environmental conditions which can contribute to vaccine development, diagnostic improvement, and ultimately leptospirosis control. SignificanceLeptospirosis is a devastating zoonotic disease affecting humans, wild and domestic animals around the globe. Different species and serovars of Leptospira can affect various animal host species differently; for instance, a serovar that is asymptomatic in the rat may cause severe disease in a dog or human. These differences in host response are not only found at the species and serovar level for Leptospira, but also at the strain level. A prime example comes from strains JB197 and HB203, both species L. borgpetersenii, both serovar Hardjo. Interestingly, JB197 causes a severe acute infection in the hamster while HB203 causes an asymptomatic chronic infection. Understanding these unique relationships between pathogen and host species is important, especially in the context of prevention technologies such as vaccine design, where the strain of Leptospira used as a bacterin might have different efficiencies in different hosts. In this study, proteomic profiles of strains JB197 and HB203 were analyzed, and results revealed diverse protein expression profiles of outer membrane proteins, as well as proteins functioning in motility and growth.

Co-Analysis of Serum and Urine Differentially Expressed Proteins in Mucopolysaccharidosis Type I.

Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disease caused by the deficiency of the enzyme α-l-iduronidase (IDUA), typically leading to devastating secondary pathophysiological cascades. Due to the irreversible nature of the disease's progression, early diagnosis and interventional treatment has become particularly crucial. Considering the fact that serum and urine are the most commonly used specimens in clinical practice for detection, we conducted an analysis to identify the differential protein profile in the serum and urine of MPS I patients using the tandem mass tag (TMT) technique. A total of 182 differentially expressed proteins (DEPs) were detected in serum, among which 9 showed significant differences as confirmed by parallel reaction monitoring (PRM) analysis. The proteins APOA1 and LGFBP3 were downregulated in serum, while the expression levels of ALDOB, CD163, CRTAC1, DPP4, LAMP2, SHBG, and SPP2 exhibited an increase. In further exploratory studies of urinary proteomics, 32 identified DEPs were consistent with the discovered findings in serum tests, specifically displaying a high diagnostic area under the curve (AUC) value. Thus, our study demonstrates the value of serum-urine integrated proteomic analysis in evaluating the clinical course of MPS I and other potential metabolic disorders, shedding light on the importance of early detection and intervention in these conditions.

Proteomics identifies apoptotic markers as predictors of histological transformation in patients with follicular lymphoma

AbstractFollicular lymphoma (FL) is an indolent lymphoma with a generally favorable prognosis. However, histological transformation (HT) to a more aggressive disease leads to markedly inferior outcomes. This study aims to identify biological differences predictive of HT at the time of initial FL diagnosis. We show differential protein expression between diagnostic lymphoma samples from patients with subsequent HT (subsequently-transforming FL [st-FL]; n = 20) and patients without HT (nontransforming FL [nt-FL]; n = 34) by label-free quantification nano liquid chromatography-tandem mass spectrometry analysis. Protein profiles identified patients with high risk of HT. This was accompanied by disturbances in cellular pathways influencing apoptosis, the cytoskeleton, cell cycle, and immune processes. Comparisons between diagnostic st-FL samples and paired transformed FL (n = 20) samples demonstrated differential protein profiles and disrupted cellular pathways, indicating striking biological differences from the time of diagnosis up to HT. Immunohistochemical analysis of apoptotic proteins, CASP3, MCL1, BAX, BCL-xL, and BCL-rambo, confirmed higher expression levels in st-FL than in nt-FL samples (P < .001, P = .015, P = .003, P = .025, and P = .057, respectively). Moreover, all 5 markers were associated with shorter transformation-free survival (TFS; P < .001, P = .002, P < .001, P = .069, and P = .010, respectively). Notably, combining the expression of these proteins in a risk score revealed increasingly inferior TFS with an increasing number of positive markers. In conclusion, proteomics identified altered protein expression profiles (particularly apoptotic proteins) at the time of FL diagnosis, which predicted later transformation.

Human platelet lysate stimulates neurotrophic properties of human adipose-derived stem cells better than Schwann cell-like cells

BackgroundTrauma-associated peripheral nerve injury is a widespread clinical problem causing sensory and motor disabilities. Schwann cells (SCs) contribute to nerve regeneration, mainly by secreting nerve growth factor (NGF) and brain-derived neurotrophic factor. In the last years, adipose-derived stem cells (ASCs) differentiated into SCs (SC-ASCs) were considered as promising cell therapy. However, the cell trans-differentiation process has not been effectively showed and presents several drawbacks, thus an alternative approach for increasing ASCs neurotrophic properties is highly demanded. In the context of human cell-based therapies, Good Manufacturing Practice directions indicate that FBS should be substituted with a xenogeneic-free supplement, such as Human Platelet Lysate (HPL). Previously, we demonstrated that neurotrophic properties of HPL-cultured ASCs were superior compared to undifferentiated FBS-cultured ASCs. Therefore, as following step, here we compared the neurotrophic properties of differentiated SC-like ASCs and HPL-cultured ASCs.MethodsBoth cell groups were investigated for gene expression level of neurotrophic factors, their receptors and neuronal markers. Moreover, the expression of nestin was quantitatively evaluated by flow cytometry. The commitment toward the SC phenotype was assessed with immunofluorescence pictures. Proteomics analysis was performed on both cells and their conditioned media to compare the differential protein profile. Finally, neurotrophic abilities of both groups were evaluated with a functional co-culture assay, assessing dorsal root ganglia survival and neurite outgrowth.ResultsHPL-cultured ASCs demonstrated higher gene expression of NGF and lower expression of S100B. Moreover, nestin was present in almost all HPL-cultured ASCs and only in one quarter of SC-ASCs. Immunofluorescence confirmed that S100B was not present in HPL-cultured ASCs. Proteomics analysis validated the higher expression of nestin and the increase in cytoskeletal and ECM proteins involved in neural regeneration processes. The co-culture assay highlighted that neurite outgrowth was higher in the presence of HPL-ASCs or their conditioned medium compared to SC-ASCs.ConclusionsAll together, our results show that HPL-ASCs were more neurotrophic than SC-ASCs. We highlighted that the HPL triggers an immature neuro-induction state of ASCs, while keeping their stem properties, paving the way for innovative therapies for nerve regeneration.Graphical

Network pharmacological analysis of corosolic acid reveals P4HA2 inhibits hepatocellular carcinoma progression

BackgroundCorosolic acid is a pentacyclic triterpene acid with hypoglycemic, anti-inflammatory, and anti-cancer effects. However, its potential targets in hepatocellular carcinoma (HCC) are unknown, hindering clinical utilization.MethodsDifferentially expressed proteins of the Bel-7404 cell line were identified with tandem mass tag analysis and differentially expressed genes (DEGs) of an HCC TCGA dataset using bioinformatics. Gene functions and pathways were inferred using the DAVID database. Online databases were used to establish P4HA2 expression in HCC (GEPIA2) and its relationship with patient survival (UALCAN and The Human Protein Atlas), the association between P4HA2 expression and immune cell infiltration (TIMER2), and DNA methylation of the P4HA2 gene (MethSurv). Cell proliferation, cell cycle, and cell death were assessed with PI and SYTOX-Green staining, CCK-8, and colony formation assays. Protein expression levels were detected by Western blotting.ResultsA total of 44 differentially expressed proteins and 4498 DEGs were identified. Four genes whose proteins were also found in the differential protein profile but with opposing expressions were selected as candidate targets. The candidate gene prolyl 4-hydroxylase subunit alpha 2 (P4HA2) was recognized as the only potential target due to its high expression in public datasets, association with poor patient survival, and relation to immune cell infiltration in HCC tissues. Moreover, the DNA methylation status in 4 CpG islands of the P4HA2 gene correlated with a poor prognosis. Furthermore, corosolic acid treatment inhibited the proliferation of HCC cell lines Bel-7404 and HepG2 in a dose-dependent manner, caused G2/M phase cell cycle arrest, and promoted cell death. In addition, the treatment reduced P4HA2 protein levels.ConclusionOur results indicate that P4HA2 is a potential target of corosolic acid. Thus, they contribute to understanding molecular changes in HCC after corosolic acid treatment and facilitate finding new treatment regimens.

Proteomic profiling and functional characterization of serum-derived extracellular vesicles in the mucinous and non-mucinous colon adenocarcinoma.

Mucinous adenocarcinoma (MC) is a distinct pathological subtype of colon adenocarcinoma, which is associated with a worse prognosis compared with non-mucinous adenocarcinoma (AC). However, clear distinctions between MC and AC remain unknown. Extracellular vesicles (EVs) are a class of enclosed vesicles containing proteins, lipids, and nucleic acids that are secreted by cells into surrounding tissues or into serum. The EVs could facilitate tumorigenesis by regulating tumor cell proliferation, invasiveness, metastasis, angiogenesis, and evasion of immune surveillance. Quantitative proteomics analysis was performed to determine the characterization and biological differences of serum-derived EVs in two subtypes of colon adenocarcinoma (MC and AC). Serum-derived EVs from patients with MC, AC, and healthy volunteers were included in this study. The role of PLA2G2A in cell migration and invasion were evaluate with transwell assay, and its prognostic predictive value was further assessed based on TCGA database. Quantitative proteomics analysis revealed 846 differentially expressed proteins (DEPs) in EVs from MC patients compared with those from AC patients. Bioinformatics analysis revealed that the most prominent protein cluster included those involved in cell migration and the tumor microenvironment. Overexpression of PLA2G2A, one of the key EV proteins upregulated in patients with MC, in colon cancer cell line SW480 promoted the cell invasion and migration ability. In addition, the high level of PLA2G2A is associated with poor prognosis of colon cancer patients harboring BRAF mutations. Further, after EV stimulation, proteomic analysis of recipient SW480 cells showed that MC-derived EVs activated multiple cancer-related pathways, including the Wnt/β-Catenin signaling pathway, and might promote the malignancy of mucinous adenocarcinoma through these pathways. The identification of differential protein profiles between MC and AC helps to elucidate the underlying molecular mechanisms of MC pathogenesis. The PLA2G2A in EVs is a potential prognostic predictive marker for those patients harboring with BRAF mutations.

Discovery and Mechanism of Azatryptanthrin Derivatives as Novel Anti-Phytopathogenic Bacterial Agents for Potent Bactericide Candidates.

The natural alkaloids of tryptanthrin and their derivatives have a wide range of biological activities. In this research, four series of azatryptanthrin derivatives containing 4-aza/3-aza/2-aza/1-aza tryptanthrin were prepared by condensation cyclization reaction against plant pathogens to develop a new natural product-based bacterial pesticide. Compound 4Aza-8 displayed a remarkable growth inhibitory effect on pathogenic bacteria of Xanthomonas axonopodis pv. citri (Xac), Xanthomonas oryzae pv. Oryzae (Xoo), and Pseudomonas syringae pv. actinidiae (Psa) with the final corrected EC50 values of 0.312, 1.91, and 18.0 μg/mL, respectively, which were greatly superior than that of tryptanthrin (Tryp). Moreover, 4Aza-8 also showed effective therapeutic and protective activities in vivo on citrus canker. Further mechanism studies on Xac elucidated that compound 4Aza-8 was able to affect the growth curve of Xac and the formation of biofilm, cause severe shrinkage in bacterial morphology, increase reactive oxygen species levels, and induce apoptosis in bacterial cells. Quantitative analysis of differential protein profiles found that the major differences were mainly concentrated on the endometrial protein in the bacterial secretion system pathway, which blocked the membrane transport and affected the transfer of DNA to the host cell. In summary, these research results suggest that 4Aza-8 represents a promising anti-phytopathogenic-bacteria agent, which is worth being further investigated as a bactericide candidate.

Differential Serum Proteomic Signatures between Acute Aortic Dissection and Acute Myocardial Infarction.

Acute aortic dissection (AAD) and acute myocardial infarction (AMI) are both severe cardiovascular diseases that may cause sudden death. However, whether serum proteins are differentially expressed between AAD and AMI remains unclear. Here, we aimed to explore serum protein profiles between AAD and AMI patients. A total of 75 serum samples were collected, including AAD patients without AMI (n = 25), AMI patients without AAD (n = 25), and normal subjects (n = 25). Protein identities and expression levels were assessed by LC-MS/MS analysis and a label-free quantitation method, respectively. After depletion of albumin and IgG, a total of 117 proteins with differential expression (fold change ≥2 or ≤-2.0, p &lt; 0.05) were identified, of which 60 were upregulated and 57 were downregulated in AAD sera as compared to AMI sera. Bioinformatic analysis revealed that the differentially expressed serum proteins were mainly derived from exosomes and the extracellular space, and their molecular functions and biological processes were primarily involved in the activity of transporters and complements and the immune response. In addition, the serum level of Cadherin-5, an identified protein with significant regulation in AAD, was further evaluated by ELISA and the results showed that Cadherin-5 in AAD sera was higher that in AMI and healthy sera. Collectively, these findings reveal the differential serum protein profiles between AAD and AMI, which may reflect the divergent pathophysiological progression between the two cardiovascular diseases.

Serum Proteomic Analysis for New Types of Long-Term Persistent COVID-19 Patients in Wuhan.

The emergence of a new type of COVID-19 patients, who were retested positive after hospital discharge with long-term persistent SARS-CoV-2 infection but without COVID-19 clinical symptoms (hereinafter, LTPPs), poses novel challenges to COVID-19 treatment and prevention. Why was there such a contradictory phenomenon in LTPPs? To explore the mechanism underlying this phenomenon, we performed quantitative proteomic analyses using the sera of 12 LTPPs (Wuhan Pulmonary Hospital), with the longest carrying history of 132 days, and mainly focused on 7 LTPPs without hypertension (LTPPs-NH). The results showed differential serum protein profiles between LTPPs/LTPPs-NH and health controls. Further analysis identified 174 differentially-expressed-proteins (DEPs) for LTPPs, and 165 DEPs for LTPPs-NH, most of which were shared. GO and KEGG analyses for these DEPs revealed significant enrichment of "coagulation" and "immune response" in both LTPPs and LTPPs-NH. A unity of contradictory genotypes in the 2 aspects were then observed: some DEPs showed the same dysregulated expressed trend as that previously reported for patients in the acute phase of COVID-19, which might be caused by long-term stimulation of persistent SARS-CoV-2 infection in LTPPs, further preventing them from complete elimination; in contrast, some DEPs showed the opposite expression trend in expression, so as to retain control of COVID-19 clinical symptoms in LTPPs. Overall, the contrary effects of these DEPs worked together to maintain the balance of LTPPs, further endowing their contradictory steady-state with long-term persistent SARS-CoV-2 infection but without symptoms. Additionally, our study revealed some potential therapeutic targets of COVID-19. Further studies on these are warranted. IMPORTANCE This study reported a new type of COVID-19 patients and explored the underlying molecular mechanism by quantitative proteomic analyses. DEPs were significantly enriched in "coagulation" and "immune response". Importantly, we identified 7 "coagulation system"- and 9 "immune response"-related DEPs, the expression levels of which were consistent with those previously reported for patients in the acute phase of COVID-19, which appeared to play a role in avoiding the complete elimination of SARS-CoV-2 in LTPPs. On the contrary, 6 "coagulation system"- and 5 "immune response"-related DEPs showed the opposite trend in expression. The 11 inconsistent serum proteins seem to play a key role in the fight against long-term persistent SARS-CoV-2 infection, further retaining control of COVID-19 clinical symptom of LTPPs. The 26 proteins can serve as potential therapeutic targets and are thus valuable for the treatment of LTPPs; further studies on them are warranted.

Differential proteomic of plasma provides a new perspective on scientific diagnosis and drug screening for dampness heat diarrhea in calves.

Dampness heat diarrhea (DHD) is one of the most common syndromes of calf diarrhea. Its complex etiology and lack of objective diagnostic criteria bring great challenges to the diagnosis and treatment of this disease. This study aims to screen some prospective diagnostic biomarkers or therapeutic targets for calves with DHD by investigating the differential protein profiles of plasma between DHD calves and clinically healthy calves by mass spectrometry-based proteomic. A total of 120 DHD calves and 90 clinically healthy calves were divided into two groups randomly, 30 DHD calves and 30 clinically healthy calves in the test group, and 90 DHD calves and 60 clinically healthy calves in the validation group. In the test group, a total of 52 proteins were differentially expressed between calves with DHD and clinically healthy calves, 13 proteins were significantly increased and 39 proteins were significantly decreased. The differentially expressed proteins were associated with the intestinal immune network of IgA production, caffeine metabolism, purine metabolism, and PI3K signaling pathway. In the validation group, 13 proteins were selected from 52 differential expression proteins for parallel reaction monitoring validation to verify their associations with DHD calves. The targeted proteomic results showed that fibronectin precursor (FN1) and apolipoprotein C-IV precursor (APOC4) were significantly associated with DHD in calves, and they were downregulated in sick calves. In conclusion, the differential expression of plasma proteins was associated with DHD pathogenesis in calves, and the FN1 and APOC4 might be the potential clinical biomarkers for diagnosis of DHD in calves, and the intestinal immune network of IgA production, caffeine metabolism, purine metabolism, and PI3K signaling pathway are the candidate targets to treat DHD in calves. Our finding provides a reference for further investigating the pathogenesis, developing techniques of diagnosis, and screening treatment drugs for DHD in calves.

Typical response of CD14++CD16– monocyte to knee synovial derived mediators as a key target to overcome the onset and progression of osteoarthritis

ObjectiveSynovitis with increased infiltration of immune cells is observed in osteoarthritis (OA). Given the inflammatory condition of synovitis, we explored the protein profile of OA synovium (OAS) and its effect on circulating monocytes activation, migration, and functional commitments.MethodsKnee-synovium was acquired from end-stage OA (N = 8) and trauma patients (Trauma baseline control: TBC; N = 8) for characterization using H&E histology, IHC (iNOS), LCMS-QTOF, and MALDI-imaging. Response of peripheral blood monocytes to OAS conditioned-media (OACM) was observed using transwell (n = 6). The migrated cells were captured in SEM, quantified using phase-contrast microphotographs, and their activation receptors (CCR2, CXCR2, CX3CR1, and CD11b), pro-inflammatory genes, and phagocytic potential were studied using flow cytometry, gene expression array/qPCR, and latex beads (LB) phagocytosis assay, respectively.ResultsThe Venn diagram displayed 119 typical proteins in OAS, while 55 proteins in TBCS. The STRING protein network analysis indicated distinctive links between proteins and gene ontology (GO) and revealed proteins associated with leukocyte-mediated immunity in OAS as compared to TBC. The MALDI-imaging showed typical localized proteins at 2234.97, 2522.61, 2627.21, 3329.50, and 3539.69 m/z and IHC confirmed pro-inflammatory iNOS expression in OA synovium. CD14++CD16– classical monocytes significantly migrated in OACM and expressed CCR2, CXCR2, and CD11b receptors, TNFRSF11A, MAPK1, S100A8, HSPB1, ITGAL, NFATC1, IL13RA1, CD93, IL-1β, TNF-α, and MYD88 genes and increased LB uptake as compared to SFM.ConclusionOur findings suggest that the differential protein profile of OA synovium and the classical monocytes migrated, activated, and functionally committed in response to these mediators could be of therapeutic advantage.

Identification of nine signature proteins involved in periodontitis by integrated analysis of TMT proteomics and transcriptomics.

Recently, there are many researches on signature molecules of periodontitis derived from different periodontal tissues to determine the disease occurrence and development, and deepen the understanding of this complex disease. Among them, a variety of omics techniques have been utilized to analyze periodontitis pathology and progression. However, few accurate signature molecules are known and available. Herein, we aimed to screened and identified signature molecules suitable for distinguishing periodontitis patients using machine learning models by integrated analysis of TMT proteomics and transcriptomics with the purpose of finding novel prediction or diagnosis targets. Differential protein profiles, functional enrichment analysis, and protein–protein interaction network analysis were conducted based on TMT proteomics of 15 gingival tissues from healthy and periodontitis patients. DEPs correlating with periodontitis were screened using LASSO regression. We constructed a new diagnostic model using an artificial neural network (ANN) and verified its efficacy based on periodontitis transcriptomics datasets (GSE10334 and GSE16134). Western blotting validated expression levels of hub DEPs. TMT proteomics revealed 5658 proteins and 115 DEPs, and the 115 DEPs are closely related to inflammation and immune activity. Nine hub DEPs were screened by LASSO, and the ANN model distinguished healthy from periodontitis patients. The model showed satisfactory classification ability for both training (AUC=0.972) and validation (AUC=0.881) cohorts by ROC analysis. Expression levels of the 9 hub DEPs were validated and consistent with TMT proteomics quantitation. Our work reveals that nine hub DEPs in gingival tissues are closely related to the occurrence and progression of periodontitis and are potential signature molecules involved in periodontitis.

Rapid Multivariate Analysis Approach to Explore Differential Spatial Protein Profiles in Tissue.

Spatially targeted proteomics analyzes the proteome of specific cell types and functional regions within tissue. While spatial context is often essential to understanding biological processes, interpreting sub-region-specific protein profiles can pose a challenge due to the high-dimensional nature of the data. Here, we develop a multivariate approach for rapid exploration of differential protein profiles acquired from distinct tissue regions and apply it to analyze a published spatially targeted proteomics data set collected from Staphylococcus aureus-infected murine kidney, 4 and 10 days postinfection. The data analysis process rapidly filters high-dimensional proteomic data to reveal relevant differentiating species among hundreds to thousands of measured molecules. We employ principal component analysis (PCA) for dimensionality reduction of protein profiles measured by microliquid extraction surface analysis mass spectrometry. Subsequently, k-means clustering of the PCA-processed data groups samples by chemical similarity. Cluster center interpretation revealed a subset of proteins that differentiate between spatial regions of infection over two time points. These proteins appear involved in tricarboxylic acid metabolomic pathways, calcium-dependent processes, and cytoskeletal organization. Gene ontology analysis further uncovered relationships to tissue damage/repair and calcium-related defense mechanisms. Applying our analysis in infectious disease highlighted differential proteomic changes across abscess regions over time, reflecting the dynamic nature of host-pathogen interactions.

Serum Proteomic Profile of Asthmatic Patients after Six Months of Benralizumab and Mepolizumab Treatment.

Severe eosinophilic asthma is characterized by chronic airway inflammation, oxidative stress, and elevated proinflammatory cytokines, especially IL-5. Mepolizumab and benralizumab are both humanized IgG antibodies directed against IL-5 signaling, directly acting on eosinophils count. Together with the complexity of severe asthma classification and patient selection for the targeted treatment, there is also the urgency to clarify the follow-up of therapy to identify biomarkers, in addition to eosinophils, for the optimal duration of treatment, persistence of effectiveness, and safety. To this purpose, here we performed a follow-up study using differential proteomic analysis on serum samples after 1 and 6 months of both therapies and sera from healthy patients. Statistical analysis by PCA and heatmap analyses were performed, and identified proteins were used for enrichment analysis by MetaCore software. The analysis highlighted 82 differences among all considered conditions. In particular, 30 referred to benralizumab time point (T0, T1B, T6B) and 24 to mepolizumab time point (T0, T1M, T6M) analyses. t-SNE and heatmap analyses evidence that the differential serum protein profile at 6 months of both treatments is more similar to that of the healthy subjects. Among the identified proteins, APOAI, APOC-II, and APOC-III are upregulated principally after 6 months of benralizumab treatment, plasminogen is upregulated after 6 months of both treatments and ceruloplasmin, upregulated already after 1 month of benralizumab, becoming higher after 6 months of mepolizumab. Using enrichment analysis, identified proteins were related to lipid metabolism and transport, blood coagulation, and ECM remodeling.

Exosomal CD44 Transmits Lymph Node Metastatic Capacity Between Gastric Cancer Cells via YAP-CPT1A-Mediated FAO Reprogramming.

BackgroundLymph node metastasis (LNM) commonly occurs in gastric cancer (GC) and is tightly associated with poor prognosis. Exosome-mediated lymphangiogenesis has been considered an important driver of LNM. Whether exosomes directly transmit the LNM phenotype between GC cells and its mechanisms remain elusive.MethodsA highly lymphatic metastatic GC cell line (HGC-27-L) was established by serial passage of parental HGC-27 cells in BALB/c nude mice. The capacities of migration, invasion and LNM; fatty acid oxidation (FAO) levels; and the role of exosome-transferred LNM phenotype were compared among HGC-27-L, HGC-27 and primary GC cell line AGS. Exosomes derived from GC cells and sera were separately isolated using ultracentrifugation and ExoQuick exosome precipitation solution, and were characterized by transmission electron microscopy, Nanosight and western blotting. Transwell assay and LNM models were conducted to evaluate the capacities of migration, invasion and LNM of GC cells in vitro and in vivo. β-oxidation rate and CPT1 activity were measured to assess FAO. CPT1A inhibitor etomoxir was used to determine the role of FAO. Label-free LC-MS/MS proteome analysis screened the differential protein profiling between HGC-27-exosomes and AGS-exosomes. Small interference RNAs and YAP inhibitor verteporfin were used to elucidate the role and mechanism of exosomal CD44. TCGA data analysis, immunochemistry staining and ELISA were performed to analyze the expression correlation and clinical significance of CD44/YAP/CPT1A.ResultsFAO was increased in lymphatic metastatic GC cells and indispensable for sustaining LNM capacity. Lymphatic metastatic GC cell-exosomes conferred LNM capacity on primary GC cells in an FAO-dependent way. Mechanistically, CD44 was identified to be enriched in HGC-27-exosomes and was a critical cargo protein regulating exosome-mediated transmission, possibly by modulating the RhoA/YAP/Prox1/CPT1A signaling axis. Abnormal expression of CD44/YAP/CPT1A in GC tissues was correlated with each other and associated with LNM status, stages, invasion and poor survival. Serum exosomal CD44 concentration was positively correlated with tumor burden in lymph nodes.ConclusionsWe uncovered a novel mechanism: exosomal CD44 transmits LNM capacity between GC cells via YAP-CPT1A-mediated FAO reprogramming from the perspective of exosomes-transferred LNM phenotype. This provides potential therapeutic targets and a non-invasive biomarker for GC patients with LNM.

Study of Dimorphism Transition Mechanism of Tremella fuciformis Based on Comparative Proteomics.

Tremella fuciformis is a dimorphic fungus that can undertake a reversible transition between yeast-like conidia and hyphal forms. The transformation mechanism and proteomic differences between these two forms have not been reported. Therefore, in this study, we attempted to explore the differential protein profiles of dikaryotic yeast-like conidia from fruiting bodies and mycelia (FBMds) and dikaryotic mycelia (DM) by synthetically applying high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) full proteomics and parallel reaction monitoring (PRM) targeted proteomics. The results showed that a total of 5687 proteins were quantified, and 2220 of them (39.01%) showed more than a two-fold change in expression. The functional analysis of the differentially expressed proteins (DEPs) confirmed that the DEPs were mainly located in the membrane and nucleus. The FBMds tended to express proteins involved in biosynthesis, metabolism, DNA replication and transcription, and DNA damage repair. At the same time, DM exhibited an increased expression of proteins involved in signal transduction mechanisms such as the mitogen-activated protein kinase (MAPK) signaling pathway and the Ras signaling pathway. Further, phosphorylation analysis confirmed the importance of the MAPK signaling pathway in T. fuciformis dimorphism, and comparative metabolism analysis demonstrated the metabolic difference between FBMds and DM. The information obtained in the present study will provide new insights into the difference between FBMds and DM and lay a foundation for further research on the dimorphism formation mechanism of T. fuciformis.

Abstract P2-12-24: Exosomal metabolic signatures are associated with differential response to neoadjuvant chemotherapy in patients with breast cancer

Abstract Background: Neoadjuvant chemotherapy (NAC) is becoming the standard of care for aggressive breast cancer (BC). In patients with BC, NAC can increase eligibility for breast-conserving surgery, lower the excision volume, and assist in evaluating the disease course. NAC may shrink (residual disease, RD) or even eliminate (pathologic complete response, pCR) the tumor. Patients with RD often have a poor long-term prognosis. In contrast, pCR is associated with better long-term survival outcomes. Approximately 30% of patients with BC show pCR after NAC, while the rest exhibit RD. A critical barrier to improving NAC outcomes in patients with BC is the lack of precise molecular targets and limited understanding of the molecular mechanisms underlying differential treatment outcomes in patients with BC receiving NAC. Thus, there is an urgent clinical need to elucidate the underlying molecular regulation associated with NAC resistance. Studies have shown that cellular stress induces the release of small extracellular vesicles known as exosomes, contributing to drug resistance. In this study, we evaluated the ability of exosomal metabolic profiles to predict NAC response in BC patients. Methods: We collected blood samples of 16 patients with BC who received NAC at Grady Memorial Hospitals, USA. Further, total exosomes were isolated from plasma by an ultracentrifugation method and characterized for their concentration (number/ml), size distribution, and exosomal content. Exosome sizes were validated using nanoparticle tracking analysis. RNA, proteins, and metabolites were isolated from exosomes to perform deep multiplatform analyses (viz. miRNA seq, proteomics, and metabolomics) of exosomal cargo to identify various potential biological determinants of NAC response and evaluate their contributions to the risk of RD in patients with BC after NAC. KEGG and HMDB IDs were used for metabolomics data analyses. Various analyses viz. enriched metabolomic pathway analysis, MS peak to pathway analysis (using GSEA and Mummichog), and network explorer analysis were performed using Metaboanalyst online portal to reveal the metabolic signatures in exosomes of BC patients who exhibited pCR and RD after NAC treatment. Results: Among the 16 patients with BC who received NAC, eight patients attained pCR, and eight had RD. Interestingly, we found that patients with pCR secreted 61% fewer exosomes but had 24% larger-sized exosomes than patients with RD following NAC. Furthermore, significant differences in the loading of exosomal cargo were observed between the two groups. Based on these results, we proposed that the differential RNA, protein, and metabolite profile of exosomes might contribute to the higher drug sensitivity in patients with pCR. Our proteomics and miRNA-seq analyses of exosomes from patients with pCR and RD after NAC did not show any significant differences between the groups. However, metabolite set enrichment analysis using MetaboAnalyst revealed that compared to exosomes from patients with pCR, patients with RD were significantly enriched in various metabolic pathways. These pathways included valine, leucine, and isoleucine biosynthesis; phenylalanine metabolism; one-carbon pool folate; and inositol phosphate metabolism. Additionally, MS peak to pathway analysis revealed differential enrichment of various other metabolic pathways, particularly in patients with RD.Conclusions: These data strongly suggest that exosomal metabolic signatures are associated with differential NAC outcomes in patients with BC. We believe that in-depth studies of these metabolite signatures will give essential clues about differential NAC response in patients with BC. Citation Format: Shriya Joshi, Chakravarthy Garalapati, Shristi Bhattarai, Darshan Shimoga Chandrashekar, Sooryanarayana Varambally, Gagan Deep, Ritu Aneja. Exosomal metabolic signatures are associated with differential response to neoadjuvant chemotherapy in patients with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-12-24.

Revealing the differential protein profiles behind the nitrogen use efficiency in popcorn (Zea mays var. everta)

We investigated the proteomic profiles of two popcorn inbred lines, P2 (N-efficient and N-responsive) and L80 (N-inefficient and nonresponsive to N), under low (10% of N supply) and high (100% of N supply) nitrogen environments, associated with agronomic- and physiological-related traits to NUE. The comparative proteomic analysis allowed the identification of 79 differentially accumulated proteins (DAPs) in the comparison of high/low N for P2 and 96 DAPs in the comparison of high/low N for L80. The NUE and N uptake efficiency (NUpE) presented high means in P2 in comparison to L80 at both N levels, but the NUE, NUpE, and N utilization efficiency (NUtE) rates decreased in P2 under a high N supply. DAPs involved in energy and carbohydrate metabolism suggested that N regulates enzymes of alternative pathways to adapt to energy shortages and that fructose-bisphosphate aldolase may act as one of the key primary nitrate responsive proteins in P2. Proteins related to ascorbate biosynthesis and nitrogen metabolism increased their regulation in P2, and the interaction of l-ascorbate peroxidase and Fd-NiR may play an important role in the NUE trait. Taken together, our results provide new insights into the proteomic changes taking place in contrasting inbred lines, providing useful information on the genetic improvement of NUE in popcorn.