Shotgun Proteomics Research Articles

Abstract 816: Aurora kinase A inhibition overcomes tolerance to panHER inhibitors in HPV positive HNSCC

Abstract The incidence of HPV-associated (HPV+) head and neck squamous cell carcinoma (HNSCC) has been increasing in recent decades. The EGFR inhibitor cetuximab (CTX) is less active in HPV+ than HPV- HNSCC. HPV oncoproteins E6 and E7 induce HER3 overexpression, and EGFR-HER3 heterodimer-driven survival signaling via PIK3, AKT and ERK1-2 contributes to resistance to cetuximab and the EGFR inhibitor erlotinib. We demonstrate that although panHER inhibitors afatinib and dacomitinib more effectively inhibit growth of HPV+ HNSCC cells than do erlotinib or cetuximab, adaptive signaling through the AURKA/PLK1 axis mediates resistance to panHER inhibitors. We hypothesized that combined panHER and AURKA inhibition would synergize to overcome persistence of HPV+ HNSCC. We find that HPV+ HSNCC cells UM-SCC47 and UPCI:SCC154 express both EGFR and HER3. Indeed, cell viability experiments and clonogenic survival assays demonstrate strong synergy between panHER and AURKA inhibitors, confirmed in xenografted tumor models treated with the second generation AURKA inhibitor VIC-1911 and dacomitinib. We then performed RNAseq and shotgun proteomics to understand the cellular mechanisms behind our combination. Dacomitinib had expected effects downregulating Hallmark G2M and EGFR phosphorylation targets, upregulating EMT markers and cell death markers. In contrast, addition of AURKA inhibition upregulated PLK1 and pPLK1 and cell death markers. These findings indicate that combination therapy with AURKA inhibition will mitigate the adaptability to EGFR and panHER inhibitors previously described for HPV+ HNSCC. Citation Format: Sebastian Cruz-Gomez,Jong Woo Lee,Julian Barrantes,Richard Ivey,Sara Savage,Pratima Chaurasia,Gunner Dickson,Dickson Adah,Bing Zhang,Amanda Paulovich,Barbara Burtness. Aurora kinase A inhibition overcomes tolerance to panHER inhibitors in HPV positive HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 816.

Comprehensive Identification of Lipid-Membrane Protein Interactions via Advanced Proteomics and Extended Lipid-Immobilized Bead Technology.

In biological membranes, lipids interact with membrane proteins (MPs) and play important roles in allosterically regulating their structure and function. Analyzing lipid-MP interactions is necessary for understanding these regulatory mechanisms; however, there have been few comprehensive and systematic studies to date. To address this, we developed a high-sensitivity, high-throughput platform that integrates lipid-immobilized beads with advanced proteomics to analyze lipid-MP interactions in detail. We prepared six types of lipid-immobilized beads, including sphingomyelin (SM), ceramide (Cer), dihydrosphingomyelin (DHSM), dihydroceramide (DHCer), phosphatidylcholine (PC), and cholesterol (Chol). In addition, we introduced a novel type of beads that immobilized SM and Chol (SM/Chol beads) to mimic lipid rafts. We first demonstrated that SM/Chol beads coprecipitated with Nakanori, a protein that specifically recognizes and binds to SM/Chol complexes, whereas beads immobilized with SM or Chol alone did not coprecipitate. This indicates the effectiveness of SM/Chol beads for the identification of raft-associated proteins. Next, the cell lysates were incubated with the seven types of lipid-immobilized beads and the recovered proteins were analyzed using shotgun proteomics. This approach successfully identified over 7000 lipid-binding proteins. Filtering based on fold-change values and subsequent enrichment analysis revealed distinct binding protein profiles for each lipid, highlighting the functional diversity of lipid-MP interactions and their roles in cellular processes. In summary, our methodology enables an unprecedented large-scale exploration of lipid-MP interactions. This platform provides a versatile tool for examining the lipid-mediated regulation of MPs and offers new insights into the physiological significance of the lipidome and its implications for health and disease.

New potential susceptibility factors contributing to tomato bacterial spot disease.

New potential susceptibility factors contributing to tomato bacterial spot disease.

Plasma proteome analysis of rheumatic patients reveals differences in fingerprints based on cardiovascular history: a pilot study

The risk of cardiovascular disease (CVD) in patients with rheumatoid arthritis (RA) is much higher than that in the general population. As its etiology is not fully understood, we performed a pilot study using a shotgun proteomic approach to investigate whether the plasma signature in RA patients with CVD might show an altered profile. Subjects with RA were compared to a group of RA patients with a previous cardiovascular event (CVE). The cohort consisted of an RA control group (n = 10) and a group (n = 10) of RA patients with a history of CVD. Samples were collected at least 6 months before the CVE and 3–6 months after the CVE. All subjects were matched to controls for age, sex, and medication use. Plasma depletion of the 14 most abundant proteins was followed by bottom-up shotgun proteomics analysis (LC‒MS/MS). Relative changes in protein/peptide abundance were investigated using classical statistical analyses with Perseus and XG-Boost machine learning to compare between groups and to determine the relative importance of identified proteins, respectively. Principal component analysis (PCA) revealed no difference in the global protein and peptide signatures between the control and CVE groups. A total of 150, 239 and 74 protein ID’s showed in comparison between Post Event vs. controls, Event vs. no Event and Pre event vs. Post Event respectively a statistically difference in relative abundance (p < 0.05). Remarkedly a total of 236 proteins ID’s showed a statistical significant difference in relative abundance in the PRE-Event group compared to the control group which could also be confirmed by XGboost machine learning. Here, we demonstrated potential differences in the plasma proteome signature of rheumatic patients with cardiovascular events. Interestingly, this signature may be present prior to CVE’s. However the conclusions must be drawn with caution, since this is a pilot study and further investigation with larger cohorts is warranted to identify potential risk markers that may predict the relative risk of CVEs in rheumatic diseases.

Persistent Postirradiation Skeletal Muscle Protein and Insulin Sensitivity Changes in Nonhuman Primates.

Increased incidence of diabetes has been reported after whole-body irradiation in cancer survivors and in the years after exposure in research studies of nonhuman primates. Type 2 diabetes presents in the absence of obesity and suggests that skeletal muscle, the predominant organ responsible for minute-to-minute glucose disposal, is persistently dysfunctional. We evaluated skeletal muscle (SkM) from control (CTL, n = 8) and irradiated (IRRAD, n = 16) male rhesus macaques (Macaca mulatta) that had been exposed to an average whole-body dose of 6.5 Gy after an average of 4 years of follow-up. Irradiated animals had deficient SkM basal and insulin-stimulated receptor activation that was unrelated to histologically assessed fiber size, extracellular matrix and endothelial components. Protein extracted from irradiated muscle showed that Akt2, downstream of insulin receptor activation, was sulfenyl-modified and thus a target for radiation-related glycemic dysregulation. Shotgun proteomics identified upregulation of many mitochondrial and peroxisome-associated proteins, and increases were confirmed by immunoblotting of select protein targets. Proteomic pathway enrichment mapping showed distinct protein clustering between CTL and IRRAD groups. Mitochondrial proteins were surveyed and confirm that mitochondrial turnover may be increased after irradiation with higher fission and fusion markers. The results indicate that irradiated muscle is persistently insulin resistant, with evidence of intracellular protein oxidation and shifts in mitochondrial dynamics and function.

Proteomic profiling of the serological response to a chemically-inactivated nodavirus vaccine in European sea bass Dicentrarchus labrax.

The analysis of animal responses to immunization is fundamental to vaccine development, enabling the evaluation of immune responses and the elucidation of key mechanisms underlying protective immunity. Such insights are essential for advancing vaccines through clinical trial stages and regulatory approval. Furthermore, understanding the molecular signatures of approved vaccines not only deepens our knowledge of their function but also guides the rational design and improvement of future vaccines. This study aims to elucidate alterations in protein abundance patterns in the sera of European sea bass, D. labrax, following immunization with a chemically-inactivated nodavirus vaccine. The shotgun proteome comparison revealed that in vaccinated animals, compared to controls, there is a modulation of the redox balance favouring reactive oxygen species (ROS), an intricate interaction between coagulation and the immune system resulting in the lower abundance of hematopoiesis-related FLT3, and indications of functional adaptive immunity demonstrated by the lower abundance of pentraxin fusion protein-like and the over-representation of myosins. To the best of our knowledge, this study represents the inaugural investigation of the immune response in fish using a proteomics approach, employing D. labrax as the host and nodavirus as the pathogen used for vaccination and challenge.

Fluxomics combined with shotgun proteomics reveals a differential response of bovine kidney cells to extracellular palmitic and α-linolenic acid.

Pyruvate carboxylase (PC) catalyzes the formation of oxaloacetate, a TCA cycle intermediate and gluconeogenic substrate. Altering saturated to unsaturated fatty acid ratio alters PC expression, suggesting a central role in mediating carbon flow through metabolic pathways. Herein, we describe changes in metabolic flux of TCA cycle intermediates and proteome in Madin-Darby bovine kidney (MDBK) cells with PC expression knocked-down (PC-KD), overexpressed (PC-OE), unaltered using a Scramble control, or cells pretreated for 21 h with vehicle control bovine serum albumin (BSA) or different ratios of palmitic acid (P) and α-linolenic acid (L) ranging from 1 mM P:0 mM L (1P:0L) to 0P:1L. All cells were collected for proteome analysis and to measure [U-13C] pyruvate flux or oxidation of [1-14C] palmitic acid and [U-14C] lactate. Compared with Scramble, 13C enrichment of all TCA cycle intermediates was greater in PC-OE, but all were reduced in PC-KD except succinate. Proteins greater in abundance in both cell lines included solute transporters, propionyl CoA carboxylase, and fatty acid binding protein 3. Relative to BSA, 1P:0L increased cell death and increased 13C flux to citrate but decreased enrichment of succinate. Abundance of citrate synthase, aconitase, glutamine aminotransferases, and succinyl CoA synthetases was greater in 1P:0L, but not different in other pretreatments. Results indicate preferential utilization of pyruvate and amino acids by 1P:0L cells whereas 0P:1L treated cells show preference for α-linolenic acid metabolism. PC regulates metabolic flux, C18:3n - 3 cis prevents lipotoxicity, and both alterations in PC and the addition of C18:3n - 3 cis promote oxidation of fatty acids.NEW & NOTEWORTHY PC overexpression increases the capacity for fatty acid oxidation, whereas PC knockdown requires extracellular amino acids to support TCA cycle intermediates. Cells incubated in palmitic acid demonstrated dependency of pyruvate and amino acids as substrates for the TCA cycle. Exposure to α-linolenic acid reduces the dependency of pyruvate as a substrate likely because carbon from α-linolenic acid can be used to supply TCA cycle intermediates whereas palmitic acid carbon is not used.

Identification of Subtle Differences in the Physiological Quality of Commercial Soybean Seed Lots Using Shotgun Proteomics During Germination

Soybean seeds with similar germination rates may exhibit subtle differences in physiological quality, influencing field performance and storage longevity. This study used a shotgun proteomics approach to characterize the proteomic profile of two commercial soybean seed lots (higher- and lower-quality) during germination, aiming to identify biomarkers associated with vigor and deterioration. Proteins were analyzed across three germination phases: imbibition (Phase I, 0.5 h), metabolic activation (Phase II, 20 h), and radicle protrusion (Phase III, 51 h). A total of 777 proteins were identified, and of these differentially abundant proteins (DAPs), the following totals were detected: 12 in Phase I, 17 in Phase II, and 28 in Phase III. In Phase I, ribosomal proteins were more abundant in high-quality seeds, indicating efficient translation and preparation for germination. Conversely, in Phase III, low-quality seeds showed increased levels of storage proteins and stress-response proteins, including alcohol dehydrogenase (ADH), heat shock proteins, and annexins, reflecting delayed germination and more deterioration. These findings highlight the dynamic nature of protein expression during germination and demonstrate the potential of proteomics to detect subtle differences in physiological quality. The identified biomarkers provide insights for seed quality assessment and offer practical applications for improving classification and management of commercial soybean seed lots.

CSF protein biomarkers are associated with response to multi-agent intraventricular chemotherapy in patients with CNS lymphoma

Abstract Background Central nervous system lymphoma (CNSL), is a rare subtype of non-Hodgkin lymphoma, primarily affecting the brain and spinal cord. Most therapeutic systemic agents have limited penetration of the blood-brain and blood-cerebrospinal fluid barrier, with the latter potentially promoting a treatment “sanctuary” for cancer cells. Evaluation of occult disease, particularly in the cerebrospinal fluid (CSF), is challenging. In limited clinical experience the addition of multi-agent intraventricular chemotherapy (MAIVC), delivered through intracranially-implanted CSF reservoirs, to systemic therapy has demonstrated encouraging outcomes, enhancing both progression-free survival and overall survival. However, given the potential morbidity associated with MAIVC, identification of minimally-invasive biomarkers for guiding patient selection and management are necessary. Leveraging the longitudinal, large volume of CSF, the objective of this study was to identify CSF-based proteomic biomarkers that can serve as reliable indicators of CSF clearance in response to MAIVC and CNSL treatment outcome. Methods 115 CSF samples from 59 CNSL patients receiving MAIVC were profiled using a high-throughput protocol coupled with mass-spectrometry that only requires 30 μL of CSF. Results More than 2000 unique proteins were detected using shotgun proteomics. CSF proteomics revealed key proteins (SGCE, LCP1, AGRN, OLFML3, HRSP12) distinguishing early from never responders to MAIVC, with AUROC 0.86 (95% CI: 0.696-1). By integrating tumor volume from brain MRI scans with proteomic data, we identified potential intraventricular tumor burden markers for CNSL management, in particular LCP1. Conclusions The study identified CSF-based proteomic biomarkers, particularly LCP1, that can classify MAIVC response and indicate tumor burden in CNSL patients.

Kinetic Redox Shotgun Proteomics Reveals Specific Lipopolysaccharide Effects on Intestinal Epithelial Cells, Mitigated by a Mn Superoxide Dismutase Mimic.

Overproduction of reactive oxygen species and antioxidant superoxide dismutases (SOD1, SOD2) dysregulation contribute to chronic inflammation such as generated in inflammatory bowel diseases (IBD). A kinetic redox shotgun proteomic strategy (OcSILAC for Oxidized cysteine Stable Isotope Labelling by Amino acids in Cell culture) was used to explore the lipopolysaccharide (LPS) effects including LPS-induced oxidation and inflammation cascades on a dedicated intestinal epithelial cell line (HT29-MD2) together with the potential mitigating role of a Mn-based SOD-mimic Mn1. While LPS induced transient oxidative damages at early times (15 min.), cells incubated with Mn1 showed, in this time frame, a significantly reduced cysteine oxidation, highlighting Mn1 antioxidant properties. Over time, cysteine oxidation of LPS treated cells was counteracted by an overexpression of antioxidant proteins (SOD1, NQO1) and a late (6h) preponderant increased in SOD2 level. Mn1, when co-incubated with LPS, attenuated the level of most LPS-modified proteins, i.e. proteins involved in the inflammatory response. Our results highlight Mn1 as a potentially effective antioxidant and anti-inflammatory agent to consider in the treatment of IBD, as well as a useful tool for exploring the interconnection between oxidative stress and inflammation.

Discovery of Cysteine Carboxyalkylations by Real-Time Isotopic Signature Targeted Profiling.

Data-dependent acquisition (DDA) is widely applied in shotgun proteomics. However, restricted by the scanning speed of mass spectrometry (MS) instruments, it remains challenging for DDA to directly detect peptides with low abundance. Herein, we developed a real-time targeted MS data acquisition method, "isoSTAR", which identifies target peptides by their unique isotopic signatures during the stage of full-MS scanning and subjects them to targeted MS/MS scans immediately. The method showed dramatic improvement in sensitivity in identifying target peptides with low abundance compared to traditional MS acquisition methods. Using this method, we discovered a series of carboxyalkylations on cysteines during fatty acid metabolism and verified their modification structures using synthetic peptide standards. We envision that isoSTAR will become a powerful and versatile tool to enhance shotgun proteomics applications in profiling protein-centric modifications.

Characterization of Flexusin A, a Novel Circular Bacteriocin Produced by Marine Bacterium Bacillus flexus R29-2.

Circular bacteriocins are potent antimicrobials against pathogenic Gram-positives. In searching for marine bacteriocins, an antibacterial peptide (flexusin A) was purified from the fermentation broth of marine bacterium Bacillus flexus R29-2. Genome sequencing and gene annotation revealed the chromosome contained an unknown circular bacteriocin gene cluster. Approaches including shot-gun proteomics analysis, AntiSMASH and BAGEL4 predication as well as the comprehensive sequence alignment, were then conducted, respectively, to verify the correlation of flexusin A with the gene-encoded precursor peptide. The results confirmed that flexusin A was the mature circular bacteriocin of the predicated precursor peptide with six amino acids as leader peptide. Flexusin A was 6098.4 Da in size, with a net charge of +3 and PI of 9.60. It shared the typical saposin-like fold spatial conformation features as commonly found in other circular bacteriocins. Flexusin A was pH, thermal, and protease tolerant. It exhibited a narrow antimicrobial spectrum against Gram-positives, and it can strongly inhibit Staphylococcus aureus by causing cell destruction via membrane destabilization. Taken together, a novel circular bacteriocin flexusin A was identified in this work. The characterization of flexusin A has extended circular bacteriocins family to 26 members. This is also the first report on bacteriocin production by B. flexus.

Exoproteomic Evidence for BcTSPO-Mediated Regulation of Virulence Factors in Bacillus cereus.

Bacillus cereus translocator protein (BcTSPO) is a transmembrane protein that plays a regulatory role in various Bacillus cereus phenotypes, potentially including its virulence. Given that the exoproteome is a major determinant of B. cereus virulence, particularly during the late stages of active growth, its analysis is crucial for understanding the regulatory functions of BcTSPO. In this study, we conducted a comparative analysis of the exoproteomes and cellular proteomes of a Δtspo mutant and the wild-type ATCC 14579 strain during the late exponential and early stationary growth phases. Shotgun proteomics revealed that the absence of BcTSPO significantly depleted the exoproteome of secreted virulence factors, including the HBL, NHE, and CytK enterotoxins. Interestingly, the cholesterol-dependent cereolysin O (CLO) was more abundant in the Δtspo exoproteome, indicating a distinct regulatory mechanism. The cellular proteome analysis further confirmed that BcTSPO positively regulates, likely indirectly, the synthesis of enterotoxins and negatively regulates the production of CLO. Additionally, the Δtspo exoproteome was enriched in moonlighting proteins, reflecting the impact of BcTSPO absence on cellular metabolism and stress responses. Overall, these findings highlight the critical role of BcTSPO in shaping the composition of both the exoproteome and cellular proteome, with significant implications for the virulence of B. cereus.

Endothelial-mesenchymal crosstalk drives osteogenic differentiation of human osteoblasts through Notch signaling

BackgroundAngiogenesis and osteogenesis are closely interrelated. The interaction between endothelial and bone-forming cells, such as osteoblasts, is crucial for normal bone development and repair. Juxtacrine and paracrine mechanisms play key roles in cell differentiation towards the osteogenic direction, assuming the direct effect of endothelium on osteogenic differentiation. However, the mechanisms of this interplay have yet to be thoroughly studied.MethodsIsolated endothelial cells (EC) from human umbilical vein and human osteoblasts (OB) from the epiphysis of the femur or tibia were cultured in direct and indirect (separated by membrane) contact in vitro under the osteogenic differentiation conditions. Osteogenic differentiation was verified by RT-PCR, and alizarin red staining. Shotgun proteomics and RNA-sequencing were used to compare both EC and OB under different co-culture conditions to assess the mechanisms of EC-OB interplay. To verify the role of Notch signaling, experiments with Notch modulation in EC were performed by EC lentiviral transduction with further co-cultivation with OB. Additionally, the effect of Notch modulation in EC was assessed by RNA-sequencing.ResultsEC have opposite effects on osteogenic differentiation depending on the co-culture conditions with OB. In direct contact, EC enhance osteogenic differentiation, but in indirect cultures, EC suppress it. Our proteotranscriptomic analysis revealed that the osteosuppressive effect is related to the action of paracrine factors secreted by EC, while the osteoinductive properties of EC are mediated by the Notch signaling pathway, which can be activated only upon a physical contact of EC with OB. Indeed, in the direct co-culture, the knockdown of Notch1 and Notch3 receptors in EC has an inhibitory effect on the OB osteogenic differentiation, whereas activation of Notch by intracellular domain of either Notch1 or Notch3 in EC has an inductive effect on the OB osteogenic differentiation.ConclusionThe data indicate the dual role of the endothelium in regulating osteogenic differentiation and highlight the unique role of the Notch signaling pathway in inducing osteogenic differentiation during cell-to-cell interactions. The findings of the study emphasize the importance of intercellular communication in the regulation of osteoblast differentiation during bone development and maintenance.Graphical

Changes in Protein Expression in Warmed Human Lens Epithelium Cells Using Shotgun Proteomics.

Background and Objectives: In previous studies, we reported that the assessment of the cumulative thermal dose in the crystalline lens, conducted through computational modeling utilizing a supercomputer and the biothermal transport equation, exhibited a significant association with the incidence of nuclear cataracts. In this study, we have investigated the types of proteins that expressed underlying 35.0 °C (normal-temp) and 37.5 °C (warming-temp) by using the shotgun liquid chromatography (LC) with tandem mass spectrometry (MS/MS)-based global proteomic approach. Materials and Methods: We have discussed the changes in protein expression in warmed iHLEC-NY2 cells using Gene Ontology analysis and a label-free semiquantitative method based on spectral counting. Results: In iHLEC-NY2, 615 proteins were detected, including 307 (49.9%) present in both lenses cultured at normal-temp and warming-temp, 130 (21.1%) unique to the lens cultured at normal-temp, and 178 (29.0%) unique to the lens cultured at warming-temp. Furthermore, LC-MS/MS analysis showed that warming decreased the expression of actin, alpha cardiac muscle 1, actin-related protein 2, putative tubulin-like protein alpha-4B, ubiquitin carboxyl-terminal hydrolase 17-like protein 1, ubiquitin-ribosomal protein eL40 fusion protein, ribosome biogenesis protein BMS1 homolog, histone H2B type 1-M, and histone H2A.J. in iHLEC-NY2. Conclusions: The decreases in the specific protein levels of actin, tubulin, ubiquitin, ribosomes, and histones may be related to cataract development under warming conditions. This investigation could provide a critical framework for understanding the correlation between temperature dynamics and the development of nuclear cataracts.

Query Mix-Max Method for FDR Estimation Supported by Entrapment Queries.

Estimating the false discovery rate (FDR) is one of the key steps in ensuring appropriate error control in the analysis of shotgun proteomics data. Traditional estimation methods typically rely on decoy sequence databases or spectral libraries, which may not always provide satisfactory results due to limitations of decoy construction methods. This study introduces the query mix-max (QMM) method, a decoy-free alternative for FDR estimation in proteomics. The QMM framework builds upon the existing mix-max procedure but replaces decoy matches with entrapment queries to estimate the number of false positive discoveries. Through simulations and real data set analyses, the QMM method was demonstrated to provide reasonably accurate FDR estimation across various scenarios, particularly when smaller sample-to-entrapment spectra ratios were achieved. The QMM method tends to be conservatively biased, particularly at higher FDR values, which can ensure stringent FDR control. While flexible, the protocol's effectiveness may vary depending on the evolutionary distance between the sample and entrapment organisms. It also requires a sufficient number of entrapment queries to provide stable FDR estimates, especially for low FDR values. Despite these limitations, the QMM method is a promising alternative as one of the first query-based FDR estimation approaches in shotgun proteomics.

PyViscount: Validating False Discovery Rate Estimation Methods via Random Search Space Partition.

Validating false discovery rate (FDR) estimation is an essential but surprisingly understudied aspect of method development in shotgun proteomics. Currently available validation protocols mostly rely on ground truth data sets, which typically involve manipulating the properties of the search space or query spectra used. As a result, comparing estimated FDR and ground truth-based false discovery proportion values may not be representative of the scenarios involving natural data sets encountered in practice. In this study, we introduce PyViscount─a Python tool implementing a novel validation protocol based on random search space partition, which enables generating a quasi ground-truth using unaltered search spaces of unique candidate peptides and generic data sets of experimental query spectra. Furthermore, validation of existing FDR estimation methods by PyViscount is consistent with alternative validation protocols. The presented novel approach to validation free from the need for synthetic data sets or dubious manipulation of the data may be an attractive alternative for proteomics practitioners, allowing them to obtain deeper insights into the performance of existing and new FDR estimation methods.

Exploring the exoproteome of the parasitic nematode Anisakis simplex (s. s.) and its impact on the human host - an in vitro cross-talk proteomic approach.

Anisakis simplex sensu stricto (s. s.) is one of the most widespread parasitic nematodes of marine organisms, with humans as accidental hosts. While many studies have explored nematode biology and host interactions, the role of extracellular vesicles (EVs) as signaling molecules in parasitic nematodes is less understood. Therefore, the proteins present in the EVs of A. simplex (s. s.) (Anis-EVs) were identified. In addition, a cross-talk proteomic approach was used to identify differentially regulated proteins (DRPs) in the proteome of the human intestinal epithelial cell line (Caco-2) co-cultured with L3 larvae of A. simplex (s. s.) or directly exposed to two concentrations (low or high) of Anis-EVs. In addition, DRPs were identified in the proteome of A. simplex (s. s.) larvae affected by co-culture with Caco-2. To achieve this goal, the shotgun proteomics method based on isobaric mass labeling (via tandem mass tags; TMT) was used with a combination of nano high-performance liquid chromatography (nLC) coupled with an LTQ-Orbitrap Elite mass spectrometer. In addition, ELISA assays were used to demonstrate if Caco-2 respond to A. simplex (s. s.) larvae and Anis-EVs with significant changes in selected cytokines secretion. The results of this study indicate the anti-inflammatory character of Anis-EVs in relation to Caco-2. At the same time, direct treatment with Anis-EVs resulted in more significant changes in the Caco-2 proteome than co-culture with L3 larvae. The results obtained should lead to a better understanding of the molecular mechanisms underlying the development of A. simplex (s. s.) infection in humans and will complement the existing knowledge on the role of EVs in host-parasite communication.

Proteomic and lipidomic analyses reveal novel molecular insights into oat (Avena sativa L.) lipid regulation and crosstalk with starch synthesis during grain development.

Proteomic and lipidomic analyses reveal novel molecular insights into oat (Avena sativa L.) lipid regulation and crosstalk with starch synthesis during grain development.

Characterization and Proteomic Profiling of Hepatocyte-like Cells Derived from Human Wharton's Jelly Mesenchymal Stromal Cells: De Novo Expression of Liver-Specific Enzymes.

End-stage liver disease (ESLD), affecting millions worldwide, represents a challenging issue for clinical research and global public health. Liver transplantation is the gold standard therapeutic approach but shows some drawbacks. Hepatocyte transplantation could be a reliable alternative for patient treatment. Mesenchymal stromal cells derived from Wharton's jelly of the umbilical cord (WJ-MSCs) can differentiate into hepatocyte-like cells (HLCs) and show immunomodulatory functions. Due to the increasing demand for fully characterized cell therapy vehicles warranting both the safety and efficacy of treatments, in this work, we extensively characterized WJ-MSCs before and after the application of a hepatocyte-directed differentiation protocol. HLCs exhibited a morphology resembling that of hepatocytes, expressed early and late hepatic markers (α-fetoprotein, albumin, CK18, HNF4-α), and acquired hepatic functions (glycogen synthesis, xenobiotics detoxification), as also revealed by the shotgun proteomics approach. HLCs maintained the same pattern of immunomodulatory molecule expression and mesenchymal markers, other than displaying specific enzymes, suggesting these cells as promising candidates for cellular therapy of ESLD. Our work shed new light on the basic biology of HLCs, suggesting new therapeutic approaches to treat ESLD.