Proteomic Analysis Research Articles

Thrombospondin 1 Mediates Autophagy Upon Inhibition of the Rho-Associated Protein Kinase Inhibitor

Age-related macular degeneration (AMD) is a degenerative eye disease leading to central vision loss and is characterized by dysregulated autophagy of the retinal pigment epithelium (RPE) layer. Recent studies have suggested that rho-associated protein kinase (ROCK) inhibitors may enhance autophagy in neurodegenerative diseases and promote the survival of RPE cells. This study investigated the effect of ROCK inhibitors on autophagy gene expression and autophagic vacuole formation in a human RPE (ARPE-19) cell line. The highly selective and potent ROCK inhibitor Y-39983 enhanced the expression of autophagy genes in ARPE-19 cells and increased autophagic vacuole formation. A proteomic analysis using mass spectrometry was performed to further characterize the effects of ROCK inhibition at the protein level. Y-39983 downregulated thrombospondin-1 (THBS1), and suppression of THBS1 in ARPE-19 cells resulted in an increase in autophagic vacuole formation. Our data showed that ROCK inhibitor-induced autophagy was mediated by THBS1 downregulation. We identified ROCK and THBS1 as potential novel therapeutic targets in AMD.

Carotenoid composition and sequestration in cassava (Manihot esculentum Crantz) roots

Cassava (Manihot esculentum Crantz) is a staple food source for many developing countries. Its edible roots are high in starch but lack micronutrients such as β-carotene. In the present study, analysis of pedigree breeding populations has led to the identification of cassava accessions with enhanced β-carotene contents up to 40 μg/g DW. This represents 0.2% of the Recommended Daily Allowance (RDA) for vitamin A. The β-branch of the carotenoid pathway predominates in cassava roots, with dominant levels of β-carotene followed by other minor epoxides of β-ring derived carotenoids. Metabolomic analysis revealed that steady state levels of intermediary metabolism were not altered by the formation of carotenoids, similar to starch and carbohydrate levels. Apocarotenoids appeared to be independent of their carotenoid precursors. Lipidomic analysis provided evidence of a significant positive correlation between carotenoid and lipid content, in particular plastid specific galactolipids. Proteomic analysis of isolated amyloplasts identified the majority of proteins associated with translation and carbohydrate/starch biosynthesis (e.g. glucose-1-phosphate adenylyltransferase). No carotenoid related proteins were detected even in the highest carotenoid containing lines. Carotenoids were associated with fractions typically annotated as plastoglobuli and plastid membranes (particularly the envelope). Proteomic analysis confirmed these structures apart from plastoglobuli, thus potentially amyloplast structures may not contain classical plastoglobuli structures.

Multiomics biomarkers were not superior to clinical variables for pan-cancer screening

Abstract Background Cancer screening tests are considered pivotal for early diagnosis and survival. However, the efficacy of these tests for improving survival has recently been questioned. This study aims to test if cancer screening could be improved by biomarkers in peripheral blood based on multi-omics data. Methods We utilize multi-omics data from 500,000 participants in the UK Biobank. Machine learning is applied to search for proteins, metabolites, genetic variants, or clinical variables to diagnose cancers collectively and individually. Results Here we show that the overall performance of the potential blood biomarkers do not outperform clinical variables for collective diagnosis. However, we observe promising results for individual cancers in close proximity to peripheral blood, with an Area Under the Curve (AUC) greater than 0.8. Conclusions Our findings suggest that the identification of blood biomarkers for cancer might be complicated by variable overlap between molecular changes in tumor tissues and peripheral blood. This explanation is supported by local proteomics analyses of different tumors, which all show high AUCs, greater than 0.9. Thus, multi-omics biomarkers for the diagnosis of individual cancers may potentially be effective, but not for groups of cancers.

Abstract B043: BDNF-TrkB.T1 signaling increases neutrophil recruitment and epithelial-to- mesenchymal transition in gliomas

Abstract Gliomas are the most common type of brain tumor in both children and adults. Communication between glioma cells and the brain tumor microenvironment (TME) is a fundamental aspect of brain cancer pathophysiology. Glioma cells secrete factors that act as chemoattractants, influencing the TME. In addition, neuronal activity within the TME drives the proliferation and growth of gliomas through paracrine signaling, with brain-derived neurotrophic factor (BDNF) being a key mediator. BDNF binds with high affinity to the tropomyosin receptor kinase B (TrkB). Recent work from our group has demonstrated that the TrkB.T1 splice variant is upregulated in human gliomas. Furthermore, TrkB.T1 overexpression enhances tumor aggressiveness in vivo and is associated with the downregulation of genes involved in tumor cell recognition and elimination. However, the role of the TrkB.T1 splice variant in the bidirectional communication between glioma cells and the surrounding microenvironment remains unexplored. Our aim was to investigate whether the increased levels of TrkB.T1 observed in gliomas contribute to modulating immune responses. We investigated the immune cell heterogeneity associated with elevated TrkB.T1 levels in vivo using a glioma mouse model engineered with RCAS/tv-a technology. Tumors overexpressing TrkB.T1 exhibited increased neutrophil recruitment, a phenomenon linked to tumor growth, metastasis and therapeutic resistance. To further dissect the signaling mechanisms underlying the BDNF-TrkB.T1 axis, we conducted in vitro studies using glioma stem cells (GSCs) that express high levels of TrkB.T1. GSCs were treated with BDNF, after which the conditioned media was analyzed using a cytokine multiplex assay, and cell lysates were subjected to proteomic analysis. Our findings revealed that TrkB.T1 significantly upregulates key chemokines previously implicated in neutrophil recruitment. Moreover, BDNF signaling increased the expression of N-cadherin, β- catenin, and Snail, markers indicative of the epithelial-to-mesenchymal transition (EMT) phenotype. To further explore the role of TrkB.T1, we used CRISPR/Cas9-based genome editing to knock down TrkB.T1 in GSCs. In conclusion, our findings suggest that the highly expressed TrkB.T1 receptor in gliomas contributes to creating an immunosuppressive microenvironment by recruiting neutrophils and promoting EMT. These insights highlight TrkB.T1 as a potential therapeutic target for modulating glioma-immune cell interactions, which could be crucial in curbing disease progression. Citation Format: Leyre Merino-Galan, Sergio Ortiz-Espinosa, Hawa L. Jagana, John M. Hemenway, Ashmitha Rajendran, Taylor S. Jackson, Daniel A. Kuppers, Sonali Arora, Patrick J. Paddison, Siobhan S. Pattwell. BDNF-TrkB.T1 signaling increases neutrophil recruitment and epithelial-to- mesenchymal transition in gliomas [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B043.

Elevated Cerebrospinal Fluid Ubiquitin Carboxyl-Terminal Hydrolase Isozyme L1 in Asymptomatic C9orf72 Hexanucleotide Repeat Expansion Carriers.

To identify biochemical changes in individuals at higher risk of developing amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) via C9orf72 hexanucleotide repeat expansion (HRE) heterozygosity. Cross-sectional observational study of 48 asymptomatic C9orf72 HRE carriers, 39 asymptomatic non-carrier controls, 19 people with sporadic ALS, 10 with C9orf72 ALS, 14 with sporadic FTD, and 10 with C9orf72 FTD. Relative abundance of 30 pre-defined cerebrospinal fluid biomarkers of ALS and FTD were compared in asymptomatic C9orf72 HRE carriers and age-matched non-carrier controls. Differential abundance of these proteins was quantified using data independent acquisition mass spectrometry or electro chemiluminescent assay for neurofilament light chain. Unbiased analysis of the entire cerebrospinal fluid proteome was then carried out. Ubiquitin carboxyl-hydrolase isozyme L1 levels were higher in asymptomatic C9orf72 HRE carriers compared with age-matched non-carriers (log2fold change 0.20, FDR-adjusted p-value = 0.034), whereas neurofilament light chain levels did not significantly differ. Ubiquitin carboxyl-hydrolase isozyme L1 levels remained elevated after matching of groups by neurofilament levels (p = 0.011), and after adjusting for age, sex, and neurofilament levels. A significant difference was also observed when restricting analysis to younger participants (<37) matched by neurofilament level (p = 0.007). Elevated cerebrospinal fluid ubiquitin carboxyl-hydrolase isozyme L1 levels in C9orf72 HRE carriers can occur in the absence of increased neurofilament levels, potentially reflecting either compensatory or pathogenic mechanisms preceding rapid neuronal loss. This brings forward the window on changes associated with the C9orf72 HRE carrier state, with potential to inform understanding of penetrance and approaches to prevention. ANN NEUROL 2024.

Integrated proteome and metabolome analysis of the penultimate internodes revealing remobilization efficiency in contrasting barley genotypes under water stress.

The remobilization of stored assimilates from stems to seeds plays a pivotal role in augmenting barley yield, particularly under water stress conditions. This study examines the molecular mechanisms underlying stem reserve utilization by conducting a comparative analysis of the proteome and metabolome across three barley contrasting genotypes: Yousef, Morocco, and PBYT17. Evaluations were performed at 21 and 28 days after anthesis (DAA) under both water stress and control conditions. The results indicate that the Yousef genotype exhibits superior remobilization of stem reserves, thereby demonstrating its potential to thrive even in adverse environmental conditions. Utilizing advanced quantitative proteomics and targeted metabolomics, this investigation identified a significant number of metabolites and proteins exhibiting differential accumulation across the genotypes. Specifically, 17 metabolites and 1580 proteins were catalogued, highlighting the intricate biochemical responses to water stress. Noteworthy enzymes such as sucrose synthase, inositol monophosphatase 3, and galactokinase were found to be closely associated with remobilization efficiency. In the drought-tolerant genotype, these enzymes maintained stable levels, in stark contrast to the decline observed in the susceptible genotype. This stability is crucial for promoting seed development through ascorbic acid synthesis and for mitigating oxidative stress, which is exacerbated by drought conditions. The elevated levels of certain metabolites, including glucose 6-phosphate, and UDP-glucose, in the drought-tolerant genotype suggest a robust mechanism for maintaining signalling molecules for carbon availability, which is then instrumental in regulating plant growth and seed size development. The findings from this study strongly imply that the drought-tolerant genotype, through enhanced antioxidant capacity, can effectively produce energy-rich storage compounds, thereby optimizing carbon allocation under water stress. Such insights are invaluable for future breeding strategies aimed at improving barley resilience in the face of climate variability.

Review and Practical Guide for Getting Started With Single-Cell Proteomics.

Single-cell proteomics (SCP) has advanced significantly in recent years, with new tools specifically designed for the preparation and analysis of single cells now commercially available to researchers. The field is sufficiently mature to be broadly accessible to any lab capable of isolating single cells and performing bulk-scale proteomic analyses. In this review, we highlight recent work in the SCP field that has significantly lowered the barrier to entry, thus providing a practical guide for those who are newly entering the SCP field. We outline the fundamental principles and report multiple paths to accomplish the key steps of a successful SCP experiment including sample preparation, separation, and mass spectrometry data acquisition and analysis. We recommend that researchers start with a label-free SCP workflow, as achieving high-quality and quantitatively accurate results is more straightforward than label-based multiplexed strategies. By leveraging these accessible means, researchers can confidently perform SCP experiments and make meaningful discoveries at the single-cell level.

A novel role of peroxiredoxin 2 in diabetic kidney disease progression by activating the classically activated macrophages.

Diabetic kidney disease (DKD) is the main cause of deaths due to diabetes mellitus (DM). Due to the complexity of its onset, it is difficult to achieve accurate prevention and treatment. The classically activated macrophage (M1) polarization is a crucial proinflammatory mechanism of DKD, while the interaction and cascade effects of oxidative stress and inflammatory response remain to be elucidated. A urine proteomic analysis of patients with DM indicated that peroxiredoxin 2 (PRDX2) had the higher abundance in DKD. We recently found that PRDX of parasitic protozoa Entamoeba histolytica, which was similar to human PRDX2 in amino acid sequence and spatial structure, could activate the inflammatory response of macrophages through toll-like receptor 4 (TLR4). Hence, our study was designed to explore the role of PRDX2 in chronic inflammation during DKD. Combined with in vivo and in vitro experiments, results showed that the PRDX2 was positively correlated with DKD progression and upregulated by high glucose or recombinant tumor necrosis factor-α in renal tubular epithelial cells; Besides, recombinant PRDX2 could promote M1 polarization of macrophages, and enhance the migration as well as phagocytic ability of macrophages through TLR4. In summary, our study has explored the novel role of PRDX2 in DKD to provide a basis for further research on the diagnosis and treatment of DKD.

Double-crosslinked hydrogels and hydrogel beads formed by garlic protein hydrolysates for bioactive encapsulation and gastrointestinal delivery.

Garlic protein is one of the main components of garlic. It has several beneficial characteristics. This study aimed to characterize a double crosslinked hydrogel formed with alginate, calcium ions (Ca2+), and garlic protein hydrolysates (GPH), and to develop hydrogel beads for targeted delivery of bioactive constituents to the gastrointestinal tract. The results indicated that the degree of GPH hydrolysis was approximately 3% following trypsin treatment. The inner structure of the double crosslinked hydrogel showed a honeycomb pattern, with solid-like gel rheology and improved texture properties at a 4% (w/v) GPH concentration. The GPH-based hydrogel beads demonstrated pH sensitivity, swelling in near-neutral and alkaline environments, and the encapsulated paclitaxel (PTX) exhibited an amorphous phase with preferential release in intestinal conditions. The GPH group also achieved greater drug encapsulation efficiency than a soy protein hydrolysate (SPH) group, and proteomic analysis suggested that lower molecular weight and peptide charge favored the formation of peptide-integrated double crosslinking hydrogels. This work indicated that GPH was helpful and could inspire the development of drug delivery systems involving GPH with the required mechanical strength and target-release properties. © 2024 Society of Chemical Industry.

The T4bSS of Legionella features a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors.

To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of all investigated TMD-effectors depended on a C-terminal secretion signal. A deeper analysis of the TMD-effector SidF showed that this signal needed to be presented towards the cytoplasmic side of the inner membrane and that a small periplasmic loop was required for efficient translocation. We propose that strongly hydrophobic TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.

Integrated proteomics and scRNA-seq analyses of ovarian cancer reveal molecular subtype-associated cell landscapes and immunotherapy targets.

Epithelial ovarian cancer (EOC) represents the most lethal gynaecological malignancy, yet understanding the connections between its molecular subtypes and their therapeutic implications remains incomplete. We conducted mass spectrometry-based proteomics analyses of 154 EOC tumour samples and 29 normal fallopian tubes, and single-cell RNA sequencing (scRNA-seq) analyses of an additional eight EOC tumours to classify proteomic subtypes and assess their cellular ecosystems and clinical significance. The efficacy of identified therapeutic targets was evaluated in patient-derived xenograft (PDX) and orthotopic mouse models. We identified four proteomic subtypes with distinct clinical relevance: malignant proliferative (C1), immune infiltrating (C2), Fallopian-like (C3) and differentiated (C4) subtypes. C2 subtype was characterized by lymphocyte infiltration, notably an increased presence of GZMK CD8+ T cells and phagocytosis-like MRC+ macrophages. Additionally, we identified CD40 as a specific prognostic factor for C2 subtype. The interaction between CD40+ phagocytosis-like macrophages and CD40RL+ IL17R CD4+ T cells was correlated with a favourable prognosis. Finally, we established a druggable landscape for non-immune EOC patients and verified a TYMP inhibitor as a promising therapeutic strategy. Our study refines the current immune subtype for EOC, highlighting CD40 agonists as promising therapies for C2 subtype patients and targeting TYMP for non-immune patients.

The use of 4D data-independent acquisition-based proteomic analysis and machine learning to reveal potential biomarkers for stress levels.

Research suggests that individuals who experience prolonged exposure to stress may be at higher risk for developing psychological stress disorders. Currently, psychological stress is primarily evaluated by professional physicians using rating scales, which may be prone to subjective biases and limitations of the scales. Therefore, it is imperative to explore more objective, accurate, and efficient biomarkers for evaluating the level of psychological stress in an individual. In this study, we utilized 4D data-independent acquisition (4D-DIA) proteomics for quantitative protein analysis, and then employed support vector machine (SVM) combined with SHAP interpretation algorithm to identify potential biomarkers for psychological stress levels. Biomarkers validation was subsequently achieved through machine learning classification and a substantial amount of a priori knowledge derived from the knowledge graph. We performed cross-validation of the biomarkers using two batches of data, and the results showed that the combination of Glyceraldehyde-3-phosphate dehydrogenase and Fibronectin yielded an average area under the curve (AUC) of 92%, an average accuracy of 86%, an average F1 score of 79%, and an average sensitivity of 83%. Therefore, this combination may represent a potential approach for detecting stress levels to prevent psychological stress disorders.

Characterization of exosome-mediated propagation of systemic inflammatory responses into the central nervous system.

The mechanisms through which systemic inflammation exerts its effect on the central nervous system (CNS) are still not completely understood. Exosomes are small (30 to 100nm) membrane-bound extracellular vesicles released by most of the mammalian cells. Exosomes play a vital role in cell-to-cell communication. This includes regulation of inflammatory responses by shuttling mRNAs, miRNAs, and cytokines, both locally and systemically to the neighboring as well as distant cells to further modulate the transcriptional and/or translational states and affect the functional phenotype of those cells that have taken up these exosomes. The role of circulating blood exosomes leading to neuroinflammation during systemic inflammatory conditions was hereby characterized. Serum-derived exosomes from LPS-challenged mice (SDEL) were freshly isolated from the sera of the mice that were earlier treated with LPS and used to study the effects on neuroinflammation. Exosomes isolated from the sera of the mice injected with saline were used as a control. In-vitro studies showed that the SDEL upregulate pro-inflammatory cytokine gene expression in the murine cell lines of microglia (BV-2), astrocytes (C8-D1A), and cerebral microvascular endothelial cells (bEnd.3). To further study their effects in-vivo, SDEL were intravenously injected into normal adult mice. Elevated mRNA expression of pro-inflammatory cytokines was observed in the brains of SDEL recipient mice. Proteomic analysis of the SDEL confirmed the increased expression of inflammatory cytokines in them. Together, these results demonstrate and strengthen the novel role of peripheral circulating exosomes in causing neuroinflammation during systemic inflammatory conditions.

A Novel UBM/SIS Composite Biological Scaffold for 2-Year Abdominal Defect Repairing and Strength Recovery in Canine Model.

Biological scaffolds are widely utilized in hernia treatment due to their exceptional pro-regenerative properties, which mitigate scar formation. However, serious complications occurred, caused by inflammatory response, premature degradation, and mechanical failure. Consequently, improvements of the biological scaffold are necessary to mitigate these risks. In this study, a novel biological scaffold integrating basement membrane-containing urinary bladder matrix (UBM) and small intestinal submucosa (SIS) is developed, and its safety and effectiveness are assessed in comparison to a commercial SIS (c-SIS) scaffold. The introduction of UBM as top surface layers significantly promotes cell adhesion, facilitating rapid formation of isolated regeneration zone. Proteomic analysis has demonstrated a more efficientdecellularization of the UBM/SIS scaffold, which subsequently mitigates inflammation in murine models, and promotes the polarization of macrophages toward the pro-healing M2 phenotype in a rat model of abdominal wall muscle defect. Furthermore, a two-year repair trial is conducted on a full-thickness abdominal wall muscle defect in canine model and confirmed that the UBM/SIS scaffold exhibits reduced seroma occurrences and enhanced tissue repair performances. Overall, the efficacyof this novel biological scaffold suggests its potential to minimize hernia recurrence in clinical practice and mitigatepatient suffering from severe inflammatory responses.

Key Proteins in Rat Cerebral Cortex: Application of Cornu aspersum Extract as a Neuroprotective Agent in Alzheimer’s Type Dementia

Alzheimer’s disease (AD) is the most widespread neurodegenerative disorder. Recently, it was found that mucus extract from Cornu aspersum has beneficial effects on memory and cognitive processes in a rat scopolamine model of AD. The present study elucidated the mechanisms of action of standardized mucus snail extract (SE) enriched with a fraction above 20 kDa on Alzheimer-type dementia in rats. Using proteomic analysis on two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) on rat cortex extracts, we compared protein expression in both groups: the first group was treated intraperitoneally with scopolamine (Sco, 2 mg/kg, 11 days) and the second (Sco + SE) group was treated intraperitoneally with Sco (Sco, 2 mg/kg) and protected by SE (0.5 mL/100 g bw) applied daily orally for 11 days. Brain cortex was separated and the expressions of various proteins related to memory and cognitive functions were identified. We found that the expression of Ubiquitin carboxyl-terminal hydrolase isozyme L1, Calbindin, Vacuolar ATP synthase catalytic subunit A, Tropomyosin beta chain, 14-3-3 zeta/delta, Kinesin-1 heavy chain, and Stathmin-4 significantly differs in SE-protected rats as compared to dement animals treated only by Sco, and these brain proteins might be potential therapeutic targets for Alzheimer’s-type dementia treatment.

Abstract B017: Discovery of cancer-specific small RNAs using a new unbiased analysis of cohort sequence data

Abstract It is well-established that small RNAs such as microRNAs can have important roles as oncogenes or tumor suppressors. The majority of studies of small RNAs in cancer have focused on examining the often subtle deregulation of specific, well-characterized molecules, aiming to understand their role in tumorigenesis. However, these efforts have predominantly concentrated on known, well-annotated RNA sequences, potentially overlooking novel, cancer-specific molecules. In this study, we sought to apply a new computational approach to uncover small RNA molecules that are uniquely associated with cancer and are completely absent from all non- cancerous tissues. To achieve this, we developed an unbiased approach that can detect cancer- specific sequences even if they are lowly abundant, do not overlap with existing genome annotations and even if they do not map to the human reference genome. We applied our framework to hundreds of small RNA-seq samples across a diverse range of cancer types from two large-scale public resources: The Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Through this comprehensive analysis, we identified a series of small RNA molecules that are exclusively present in tumor samples and completely absent in adjacent normal tissues across multiple cancer types. Furthermore, these molecules were not detected in a large cohort comprising over 2,500 non-cancerous public samples, underscoring their specificity to cancerous conditions. Some of these molecules are also associated with different patient metadata. For example, one of these candidates exhibited significant correlation with poor patient survival in both Lung Adenocarcinoma and Endometrial Carcinoma within both the TCGA and CPTAC datasets. The discovery of such cancer-specific small RNAs holds immense potential for clinical applications. These molecules could serve as highly specific diagnostic and prognostic biomarkers, aiding in the early detection and stratification of cancers. Moreover, their unique presence in cancer cells, positions them as promising targets for the development of novel therapeutic interventions. In summary, our findings highlight the importance of exploring the full spectrum of small RNAs in cancer research. By moving beyond the constraints of known annotations, we have uncovered previously unknown molecules that could improve cancer diagnostics, prognostics, and treatment. Citation Format: Panagiotis Kalogeropoulos, Maria Alejandra Ulloa, Marc Friedländer. Discovery of cancer-specific small RNAs using a new unbiased analysis of cohort sequence data [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B017.

Proteomic analysis of human Wharton's jelly mesenchymal stem/stromal cells and human amniotic epithelial stem cells: a comparison of therapeutic potential.

Perinatal stem cells have prominent applications in cell therapy and regenerative medicine. Among them, human Wharton's jelly mesenchymal stem/stromal cells (hWJMSCs) and human amniotic epithelial stem cells (hAESCs) have been widely used. However, the distinction in the therapeutic potential of hWJMSCs and hAESCs is poorly understood. In this study, we reported the phenotypic differences between these two distinct cell types and provided the first systematic comparison of their therapeutic potential in terms of immunomodulation, extracellular matrix (ECM) remodelling, angiogenesis and antioxidative stress using proteomics. The results revealed that the two cell types presented different protein expression profiles and were both promising candidates for cell therapy. Both types of cells demonstrated angiogenic and antifibrotic potential, whereas hAESCs presented superior immunological tolerance and antioxidant properties, which were supported by a series of relevant in vitro assays. Our study provides clues for the selection of appropriate cell types for diverse indications in cell therapy, which contributes to the advancement of their clinical translation and application.

Identifying putative allergens from Cenostigma pluviosum pollen using proteomic bioinformatics.

Routinely, signs and symptoms of pollinosis worsen during the flowering period of the Cenostigma pluviosum var. peltophoroides (Sibipiruna) trees. This study aimed to determine whether Sibipiruna pollen contains allergenic proteins. The pollen from mature Sibipiruna anthers was morphologically characterized using optical, scanning electron and emission microscopy. Additionally, biochemical characterization was conducted through Shotgun Label-Free Proteomic Analysis. Three hundred and forty-nine proteins were identified using the UNIPROT database and thirty-six were confirmed as homologous allergenic proteins in the Allergome database. At the first time, Sibipiruna pollen allergenic proteins have been described and this study provides a deeper understanding of the allergenic proteins present in this pollen. These preliminary findings can be useful to assist in the development of targeted extracts for the study of sensitization and potential immunotherapy in the future.

CD40 agonist on patient-derived xenograft mice for the treatment of B-cell acute lymphoblastic leukemia.

Cluster of differentiation 40 (CD40) is expressed on B-cell acute lymphoblastic leukemia (B-ALL) cases. However, the effect of CD40 activation on B-ALL cells has never been tested in vivo. The aim of our preclinical study was to investigate the therapeutic potential of a CD40 agonist in the treatment of B-ALL using patient-derived xenograft (PDX) mouse models. Intravenous administration of the CD40 agonist significantly impeded B-ALL cell proliferation and growth in vivo, accompanied by rapid activation of the extracellular signal-regulated kinase (ERK) pathway, leading to the induction of apoptosis and disruption of cell cycle progression. Co-treatment with a specific inhibitor of ERK further demonstrated that CD40 stimulation induced the pro-apoptosis of B-ALL cells in an ERK-dependent manner. Proteomic analysis revealed alterations in key signaling pathways associated with B-ALL expansion and maintenance. Moreover, the CD40 agonist markedly reduced the frequency of leukemia-initiating cells and leukemia development in PDX mice. Our study showed that the CD40 agonist can be associated with chemotherapeutic agents such as vincristine and dexamethasone, and this combination showed improved effectiveness. Additionally, the CD40 agonist was more effective on pre-B-ALL (EGIL B-III) that expressed CD40, than on common B-ALL (EGIL B-II) that lacked CD40 expression. These findings suggest that CD40 agonists are promising immunotherapeutic candidates for pediatric B-ALL, warranting further clinical investigations to improve patient outcomes in CD40-expressing B-ALL.

Inducible global knockout of surfeit locus protein 4 in adult mice results in hypolipidemia, intestinal lipid accumulation, liver injury, and increased mortality.

Surfeit locus protein 4 (SURF4) acts as a cargo receptor to mediate endoplasmic reticulum export of various cargos. We have shown that SURF4 is essential for secretion of hepatic very low-density lipoprotein and intestinal chylomicron. Knockdown of hepatic Surf4 also significantly reduces the development of atherosclerosis and liver fibrosis without causing overt liver damage. However, constitutive global Surf4 knockout results in embryonic lethality. To further understand the physiological role of Surf4, we generated tamoxifen-inducible global Surf4 knockout mice. We found that conditional knockout of Surf4 in adult mice (Surf4ig-ko) significantly reduced mouse body weight. Male and female Surf4ig-ko mice died approximately 30 and 50 days after tamoxifen administration, respectively. Triglyceride secretion and serum levels of total cholesterol, triglycerides, free fatty acids, apolipoprotein B-100, and apolipoprotein-48 were significantly reduced in Surf4ig-ko mice compared with Surf4flox mice. Proteomics analysis of mouse serum samples revealed 308 proteins with significantly altered expression in Surf4ig-ko mice that have unique functions and are involved in various biological processes. In addition, Surf4ig-ko mice exhibited lipid accumulation in the intestine but not in the liver. However, in Surf4ig-ko mice, liver weight was significantly reduced, and serum transaminase activity was significantly increased, indicating liver damage. Therefore, SURF4 is essential for survival in adult mice, suggesting that the therapeutic use of SURF4 requires precise tissue/cell type-specific targeting.