Proteomic Analysis Research Articles

Characterizing Excretory-Secretory Products Proteome Across Larval Development Stages in Ascaris suum.

Ascaris lumbricoides and Ascaris suum are parasitic nematodes that primarily infest the small intestines of humans and pigs, respectively. Ascariasis poses a significant threat to human health and swine health. Understanding Ascaris larval development is crucial for developing novel therapeutic interventions that will prevent ascariasis in both humans and pigs. This study aimed to characterize the excretory-secretory (ES) proteome of different Ascaris suum larval stages (L3-egg, L3-lung, L3-trachea) to identify potential targets for intervention to prevent Ascaris -induced global morbidity. Stage-specific larvae were isolated, cultured in vitro and ES-product was collected. Third-stage Ascaris larvae (L3) were isolated from embryonated eggs (L3-egg), isolated from the lungs of Balb/c mice infected with Ascaris suum eggs at day 8 post infection (L3-lungs) and isolated from the trachea of Balb/c mice infected with Ascaris suum eggs at day 12 post infection (L3-trachea). ES products were obtained by culturing larvae. Proteomic analysis was conducted using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatic tools including MaxQuant, Perseus, and Andromeda, following a detailed protocol available on GitHub. The analysis encompassed peptide identification, scoring, and quantification against an organism-specific database, with subsequent quality control, correlation assessment, and differential abundance determination using the Amica algorithm. A total of 58 unique proteins were identified in the ES products. Fourteen proteins were common across all stages, while others were stage-specific. Principal component analysis revealed distinct protein profiles for each stage, suggesting qualitatively different proteomes. Gene ontology analysis indicated stage-specific GO enrichment of specific protein classes, such as nuclear proteins in L3-egg ES products and metabolic enzymes in L3-lung and L3-trachea ES products. This study revealed stage-specific differences in the composition of Ascaris ES products. Further investigation into the functional roles of these proteins and their interactions with host cells is crucial for developing novel therapeutic and diagnostic strategies against ascariasis.

Integrative proteome and metabolome unveil the central role of IAA alteration in axillary bud development following topping in tobacco

Axillary bud is an important aspect of plant morphology, contributing to the final tobacco yield. However, the mechanisms of axillary bud development in tobacco remain largely unknown. To investigate this aspect of tobacco biology, the metabolome and proteome of the axillary buds before and after topping were compared. A total of 569 metabolites were differentially abundant before and 1, 3, and 5 days after topping. KEGG analyses further revealed that the axillary bud was characterized by a striking enrichment of metabolites involved in flavonoid metabolism, suggesting a strong flavonoid biosynthesis activity in the tobacco axillary bud after topping. Additionally, 9035 differentially expressed proteins (DEPs) were identified before and 1, 3, and 5 days after topping. Subsequent GO and KEGG analyses revealed that the DEPs in the axillary bud were enriched in oxidative stress, hormone signal transduction, MAPK signaling pathway, and starch and sucrose metabolism. The integrated proteome and metabolome analysis revealed that the indole-3-acetic acid (IAA) alteration in buds control dormancy release and sustained growth of axillary bud by regulating proteins involved in carbohydrate metabolism, amino acid metabolism, and lipid metabolism. Notably, the proteins related to reactive oxygen species (ROS) scavenging and flavonoid biosynthesis were strongly negatively correlated with IAA content. These findings shed light on a critical role of IAA alteration in regulating axillary bud outgrowth, and implied a potential crosstalk among IAA alteration, ROS homeostasis, and flavonoid biosynthesis in tobacco axillary bud under topping stress, which could improve our understanding of the IAA alteration in axillary bud as an important regulator of axillary bud development.

O-264 Profiling Tumor-Initiating Cells in Uterine Leiomyoma through Single-Cell Multi-omics

Abstract Study question What are the molecular and cellular features that contribute to the transformation of myometrial stem cells into tumor-initiating cells within uterine leiomyoma? Summary answer Transformative changes in differentiation and genetic processes, in a distinct subset of M/LM cells drives their progression into a tumorigenic state. What is known already Uterine leiomyomas (LM), which are benign smooth muscle-derived tumours of myometrium, impact approximately 75% of women, causing significant physical and psychological challenges and imposing substantial healthcare costs. The widespread consensus postulates a monoclonal origin for LM, implying their derivation from a dysregulated single multipotent stem cell that could give rise to tumor-initiating cells (TICs). While previous studies have shown cellular heterogeneity in both myometrium (M) and LM, the precise identity of the originating cells is unknown. Study design, size, duration Prospective, observational, and biomedical study conducted at Hospital La Fe (Valencia, Spain) for one year. Eight sample pairs of LM and M underwent single-nuclei RNAseq (snRNA-seq; n = 16) and single-cell proteomic (scP; n = 16) analyses, to generate a detailed transcriptomic and proteomic map decoupled from cell type, state, and spatial location. We further employed single-cell RNA velocity inference for differentiation trajectory [BR1] analysis of mesenchymal cells, followed by the identification of potential driver genes in LM tumorigenesis. Participants/materials, setting, methods Upon obtaining informed consent, we procured LM and M samples from eight patients aged 35-50 undergoing hysterectomies. Part of these samples were preserved in paraffin for spatial transcriptomics utilizing VISIUM (10x Genomics). The remaining tissues were dissociated into single-cell suspensions and underwent scRNA-seq and scP using Chromium Controller and Orbitrap Eclipse Tribid mass spectrometry, respectively. The analysis of the collected data was carried out using publicly accessible R/Python tools. Main results and the role of chance Analysis of snRNAseq (∼52,000 cells) revealed similar cellular compositions in M and LM, including endothelial, perivascular, smooth muscle cells (SMC), fibroblasts, and immune cells. Further trajectory analysis unveiled a subset of myometrial cells with potential as TICs, exhibiting reduced expression of hormone receptors like PGR. Analysis of these cells [BR1] suggested differentiation into two distinct cell types -fibroblasts and SMCs- with elevated hormone expression over pseudotime. Differential analysis between M and LM also showed a dysregulated transcriptomic profile in the TICs within the tumor, as evidenced by upregulation of RAD51B and HMGA2 and downregulation PGR among other genes. These genes are indicative markers, underscoring their significant roles in the tumorigenesis of LM by influencing hormone response and disrupting DNA repair mechanisms. Spatial transcriptomic analysis confirmed these findings and suggested that the distribution of TICs could be ubiquitous, since the expression of potential myometrial-stem markers, was markedly increased only on certain spots within the tissue across both LM and M. Lastly, scProt profiling (∼5,000 cells) also allowed the identification of putative TICs, which showed upregulation in LM of specific proteins involved in replication, transcription, and translation. Limitations, reasons for caution Our study sets the basis for TICs isolation in LM, yet functional validation studies need to be performed to address the clinical potential of our findings. Further studies including more patients, and addressing racial disparities will help to generalize these findings to a broader population. Wider implications of the findings Our findings indicate the presence of a particular subset of M/LM cells whose changes in differentiation path and genetic processes seem to drive the transformation of these cells into a tumorigenic state to develop LM. Targeting these markers might show promise for effectively treating these tumors. Trial registration number NCT04214457

P-309 Injectable “homing-like” bioactive short-fibers for endometrial repair and efficient live births

Abstract Study question To develop effective therapeutic interventions to promote endometrial growth and facilitate the restoration of the damaged endometrium of rats with intrauterine adhesion (IUA). Summary answer We have developed injectable “homing-like” bioactive decellularized extracellular matrix short-fibers which can achieve efficient endometrial repair and live births of rats with IUA. What is known already The prevalence of infertility caused by endometrial defects has been steadily increasing, posing a significant challenge to women’s reproductive health. Various factors, such as infection and intrauterine surgery, can lead to damage to the basal layer of the endometrium, which hinders functional endometrial repair and results in the pathological development of IUA. However, there is currently no effective clinical treatment strategy for IUA. Therefore, it is imperative to develop effective therapeutic interventions to promote endometrial growth and receptivity, ameliorate endometrial fibrosis, and facilitate the restoration of the damaged endometrium that ultimately reverses the poor pregnancy outcomes of patients with IUA. Study design, size, duration We have developed an innovative method for creating injectable acellular extracellular matrix short fibers (DEFs) from porcine skin. We investigated the effects of DEFs by in vitro and in vivo experiments over a period of two years. Participants/materials, setting, methods In vitro, we investigated the pro-proliferative, anti-fibrosis and angiogenesis effects of DEFs in human primary endometrial stromal cells (HESCs) and human umbilical vein endothelial cells (HUVECs). In vivo, we constructed a rat IUA model to investigate the promotion of endometrial repair and fertility remodeling by DFEs. Main results and the role of chance The DEFs can effectively attract endometrial cells, enabling cell adhesion, spreading, and proliferation on their surface. DEFs are rich in bioactive growth factors, which creates a nourishing microenvironment for endometrial recovery while attracting and recruiting endogenous endometrial cells for in situ regeneration. Initially, DEFs formed stable bonds with the surface of endometrial cells via electrostatic interactions after injection into the uterine cavity, which acted as a physical barrier to prevent intrauterine adhesions. Subsequently, DEFs effectively facilitated the proliferation and angiogenesis of HESCs and HUVECs, and inhibited fibrosis of pretreated HESCs. Next, DEFs could decrease endometrial collagen deposition and promote endometrial repair and endometrial angiogenesis by releasing growth factors. Therefore, bioactive DEFs could efficiently improve endometrial receptivity, promote endometrial repair, and achieve efficient live births in endometrium-injured rats. Limitations, reasons for caution The composition of DEFs is complex. The mechanism by which DEFs promotes endometrial repair remains unclear. In our future endeavors, our primary aim is to conduct a proteomic analysis of the DEFs to pinpoint the vital proteins responsible for their ability to induce tissue growth. Wider implications of the findings The DEFs can achieve efficient live births and the preparation method is suitable for mass production. In addition, the injectability of DEFs can achieve non-invasive intrauterine injection, which can reduce the risk of endometrial re-injury. Therefore, the DEFs system provides a new promising clinical strategy for endometrial factor infertility. Trial registration number enter ‘not applicable

P-235 Lipid profile changes during neurodevelopment (ND) of mouse embryos cultured in vitro

Abstract Study question Does in vitro culture (IVC) induce changes in lipid profile of the embryo? Summary answer IVC induces changes in embryonic lipid homeostasis that subsequently affect placenta-brain axis (PBA) function and are then maintained in the brain of adult mice. What is known already During ART, embryos are exposed to oxidative stress (OS)-prone environment. Cellular lipids are particularly sensitive to OS. Lipids are a crucial component for proper brain development and any aberrancies in lipid profile of the embryo can affect neurodevelopment (ND) through the PBA, leading to functional and behavioural deficits during adulthood. In particular, deficiencies of ether-linked glicerophospolipids (e.g. plasmalogens), which are highly abundant in the brain, and polyunsaturated fatty acids (PUFAs), have been associated with psychiatric and ND disorders. Study design, size, duration C57BL6 mouse embryos were cultured in vitro for 72hs, and analysed or transferred to 25 recipient females for further development. From 6 pregnant mice, conceptuses (placentae and brain) were collected at near-term pregnancy (19dpc, n > 8/group). Remaining 8 pregnant mice were allowed to deliver and raise litters. Subsequently, brain was collected from male offspring at 4 month of age for lipid homeostasis evaluation. Control group consisted of embryos and offspring obtained from naturally mated C57BL6 mice. Participants/materials, setting, methods C57BL6 blastocysts (n > 15/group) were fixed and subjected to lipid analysis by Fourier transform infra-red (FTIR) and Coherent antistokes Raman spectroscopies (CARS). To reveal lipid changes indicative for ND disorders, selected PUFAs were analyzed by liquid chromatography-mass spectrometry (LC-MS) (n > 8/group) in placenta and brains from prenatal and adult mice. Additionally, placental expression of genes regulating biosynthesis of plasmalogens and proteome of adult males brain, was performed. Values were considered different when p < 0.05, Mean±SEM; Mann-Whitney test. Main results and the role of chance Our results show that IVC induced lipid composition changes in blastocyst, characterised by increased proportion of phospholipids, fatty amides and cholesterol (59±8.2 vs.44.7±8.8; 149±4.5vs. 104±12; 44±2.9vs. 27±4.6 sum of absorbance, p < 0.05); and lipid droplets (LDs) distribution changes, such as, increase size (2.3±0.11 vs 1.8±0.07 µm2, p = 0.0021), and number of large LDs (5.5±0.7 vs. 3.3±0.2, p = 0.006). LC-MS revealed increased levels of linolenic (LA), arachidonic (AA) and docosahexaenoic (DHA) acids in placenta (LA:15.49±1.7 vs. 6.92±2; AA:263.6±29.1 vs. 62.7±14.7 DHA:0±2.5 vs. 2.7±0.8 pmol/mg; p < 0.03) and foetal brain (LA:13.6±5.5 vs. 1.5±0.9; AA:240.5±100 vs. 39.3±9.6; 13.9±6.9 vs. 2.11±0.6 pmol/mg; p < 0.03) of IVC conceptuses, which may condition brain development and function. Conversely, levels of LA and AA were low in adult brain from IVC offspring (LA:4.4±1.5 vs 14.5±3.8; AA: 581±95 vs 1842±454 pmol/mg, p < 0.04). Additionally, the expression of genes regulating biosynthesis of plasmalogens was reduced in IVC placentae (Gnpat:0.2±0.04 vs 1.2±03; Tmem189:0.3±0.1 vs. 1.3±0.3 fold change; p ≤ 0.01). Proteome analysis demonstrated that pathways regulating lipid homeostasis, such as fatty acid oxidation and phospholipid binding and those involved in IL-17a signaling and neurogenesis were upregulated in brain of adult IVC offspring. Limitations, reasons for caution Our results may not entirely reflect what occurs in human embryo, as mouse oocytes have a higher lipid content than human oocyte. Differences in lipid content may cause an increased or decreased penetrance and exhibition of developmental changes of the embryos. Wider implications of the findings Being of non-genetic background, lipid changes induced by IVC may similarly threaten embryos regardless of species causing ND disorders. Our study sheds new light on non-genetic origins of ND disorders and will likely ignite further studies on embryonic lipid changes and their consequences, relevant for human pregnancy and neuropsychiatry. Trial registration number not applicable

P-802 Exploring the effects of phthalate mixtures on human endometrial organoids: Insights into transcriptomic and proteomic profiles throughout the menstrual cycle

Abstract Study question Does the exposure of human endometrial organoids (hEOs) to phthalates influence their transcriptomic and proteomic profiles, particularly during the window of implantation? Summary answer The mixture of phthalates altered the expression of several genes which are essential during the window of implantation. What is known already hEOs display a robust responsiveness to steroid hormones and accurately reproduce essential morphological features observed in the receptive endometrium in vivo. The conventional approach to evaluating chemical health risks has predominantly been centered on the assessment of individual substances, overlooking the reality that individuals are regularly exposed to diverse mixtures of phthalates, a class of Endocrine Disrupting Chemicals (EDCs). There is a limited number of studies indicating that phthalates and their combinations can have a negative impact on the endometrium. To this purpose, we explore the effect of this mixture on endometrial receptivity during the Window Of Implantation (WOI). Study design, size, duration Endometrial tissues (n = 6) used for this study were obtained, with written informed consent from all participants. The endometrial organoids were generated from the biopsies and treated with hormones to mimic the WOI. Samples were then exposed with a mixture of phthalates in order to study the transcriptomic and proteomic changes on these cells throughout the menstrual cycle, especially the WOI. Participants/materials, setting, methods hEOs were generated from endometrial biopsies of healthy volunteers (n = 6). Organoids were cultured for 4 days and then divided into: untreated without any hormonal treatment; stimulated with 10 nM estrogen (E2); treated with 10 nM estrogen, 1μM progesterone (P4) and 1μM cyclic adenosine monophosphate (cAMP). EOs were further on exposed to the phthalate mixture. Proteomic profiling was performed using 2-D electrophoresis and Mass spectrometry. Samples were also subject to transcriptomic analysis, by RNA sequencing. Main results and the role of chance hEOs present a suitable in vitro model for the screening of the impact of phthalate mixtures on the endometrium throughout the menstrual cycle. Cytotoxicity test confirmed that the chosen concentration (100x of the value referring to the geometrical means in urine) of the mixture is not toxic for the cells. The transcriptomic and proteomic analyses revealed several up-regulated or down-regulated genes which are involved into pivotal pathways implicated to the WOI. Limitations, reasons for caution This study represents a preliminary experiment. All results need to be validated on an enlarged number of samples to provide more statistical strength. Wider implications of the findings hEOs offer a robust in vitro model to simulate and gain a more profound comprehension of the consequences arising from the combinatory effects of EDCs, on the endometrium. This model proves valuable in examining the impact of various EDCs across different phases of the menstrual cycle. Trial registration number not applicable

P-658 Identification of novel biomarkers for the detection of polycystic ovary syndrome (PCOS) in adolescents and adult women

Abstract Study question Can novel biomarkers be identified to distinguish between PCOS-positive cases and healthy PCOS-negative controls in women as early as adolescence? Summary answer Meteorin-like protein (METRNL), fibroblast growth factor binding protein-1 (FGFBP1) and leukotriene A4 hydrolase (LTA4H) identified PCOS as early as adolescence, independent of PCOS phenotype. What is known already International evidence-based guidelines recommend use of the Rotterdam criteria for PCOS diagnosis. Despite this guidance, diagnosing PCOS can be extremely challenging due to the heterogeneity of clinical presentation, and further complicated in adolescence and young adulthood due to overlap between signs of physiologic changes during puberty and common symptoms of PCOS (e.g., menstrual irregularity, excess male hormones/androgens, multiple follicles in ovaries). Timely diagnosis of PCOS allowing for appropriate monitoring, follow up and early intervention may reduce the risk of associated comorbidities. An unmet need exists for biomarkers/alternative tools to support earlier diagnosis of PCOS in adolescents/adult women of reproductive age. Study design, size, duration This was a retrospective study (October 2020–June 2023) using banked samples from women with PCOS (confirmed per Rotterdam criteria) and healthy controls who had participated in previous studies sponsored by/in collaboration with Roche Diagnostics. Candidate biomarkers identified by exploratory proteomics analysis (Olink Proximity Extension Assay) were verified by ELISA and receiver operating characteristic-area under the curve (ROC-AUC) analysis for their ability to discriminate PCOS cases and controls in women aged 15-45 years. Participants/materials, setting, methods Top-performing biomarkers from Olink discovery (n = 51 PCOS phenotype A; n = 37 controls) were selected for further validation based on AUC >0.970, biological relevance for PCOS and commercial ELISA availability. Serum biomarker concentrations/biomarker ratios were determined and ROC-AUC analysis conducted in two validation cohorts (Cohort 1: n = 90 cases [phenotype A, n = 30; B, n = 20; C, n = 20; D, n = 20]; n = 47 controls; Cohort 2: n = 240 cases [15-19 years, n = 70; 20-24 years, n = 99; 25-40 years, n = 71]; n = 48 controls, median 26.0 years). Main results and the role of chance Serum METRNL concentrations were numerically lower, and FGFBP1 and LTA4H concentrations numerically higher, in PCOS cases compared with controls. All three biomarkers distinguished PCOS cases and controls, regardless of PCOS phenotype A-D (Cohort 1: AUCs 0.91-0.99 [METRNL], 0.84-0.88 [FGFBP1] and 0.76-0.90 [LTA4H]), and across different age groups in women of reproductive age (Cohort 2: 15-19, 20-24 and 25-40 years; AUCs 0.88, 0.91 and 0.96 [METRNL], respectively; 0.90, 0.88 and 0.91 [FGFBP1], respectively; 0.74, 0.70 and 0.78 [LTA4H], respectively). Compared with results for the individual biomarkers, combining FGFBP1 with METRNL, and LTA4H with METRNL, as biomarker ratios further improved the ability to distinguish PCOS cases and controls, independent of PCOS phenotype A-D (Cohort 1: AUCs 0.95-1.00 [FGFBP1:METRNL] and 0.97-0.99 [LTA4H:METRNL]) and across age groups (Cohort 2: 15-19, 20-24 and 25-40 years; AUCs 0.91, 0.96 and 0.98 [FGFBP1:METRNL], respectively, and 0.91, 0.91 and 0.97 [LTA4H:METRNL], respectively). Findings were not impacted by adjustment for baseline body mass index. METRNL, FGFBP1 and LTA4H are linked to biological mechanisms associated with clinical manifestations and underlying characteristics of PCOS, i.e., metabolic pathways, inflammation and ovarian function. While METRNL has been described previously in adult PCOS, FGFBP1 and LTA4H findings are novel in this context. Limitations, reasons for caution These findings in retrospective samples warrant validation in additional independent prospective cohorts, particularly in adolescents and young adult women. Wider implications of the findings Reliable diagnosis of PCOS in adolescents and young adult women represents an unmet medical need. METRNL, FGFBP1 and LTA4H have potential use as biomarkers to support the detection of PCOS in adolescents and adult women, potentially shortening time to diagnosis and allowing for earlier and targeted intervention. Trial registration number not applicable

Effects of Copper on Legionella pneumophila Revealed via Viability Assays and Proteomics.

Cu is an antimicrobial that is commonly applied to premise (i.e., building) plumbing systems for Legionella control, but the precise mechanisms of inactivation are not well defined. Here, we applied a suite of viability assays and mass spectrometry-based proteomics to assess the mechanistic effects of Cu on L. pneumophila. Although a five- to six-log reduction in culturability was observed with 5 mg/L Cu2+ exposure, cell membrane integrity only indicated a <50% reduction. Whole-cell proteomic analysis revealed that AhpD, a protein related to oxidative stress, was elevated in Cu-exposed Legionella relative to culturable cells. Other proteins related to cell membrane synthesis and motility were also higher for the Cu-exposed cells relative to controls without Cu. While the proteins related to primary metabolism decreased for the Cu-exposed cells, no significant differences in the abundance of proteins related to virulence or infectivity were found, which was consistent with the ability of VBNC cells to cause infections. Whereas the cell-membrane integrity assay provided an upper-bound measurement of viability, an amoebae co-culture assay provided a lower-bound limit. The findings have important implications for assessing Legionella risk following its exposure to copper in engineered water systems.

O-267 Extracellular vesicles as biomarkers of endometriosis

Abstract Study question Can extracellular vesicles serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound? Summary answer Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes. What is known already Endometriosis is characterised by intraperitoneal lesions of endometrial tissue. Although ultrasound detection of severe endometriosis is possible, the diagnosis of peritoneal lesions currently relies on invasive laparoscopy, which leads to a delay of 8-10 years. We have previously shown that peritoneal fluid (PF) of women with endometriosis contains endometriosis specific EVs. Traced in peripheral blood (PB), EVs could serve as biomarkers of endometriosis, particularly for stages I and II, which are difficult to detect by ultrasound. Study design, size, duration Women 18-45 years of age undergoing laparoscopic surgery for endometriosis or unrelated conditions were invited to participate in our study (HREC 20-159A/HREC 20-882A). Following informed consent, we collected PF and PB samples from n = 101 participants (n = 47 controls, n = 54 cases). Exclusion criteria were pregnancy, malignancy, and menopause. Participants/materials, setting, methods We processed PF and PB samples by differential ultracentrifugation and validated the presence of EVs by nanoparticle tracking analysis (NTA), Western blotting and transmission electron microscopy (TEM). EVs were analysed by untargeted, label-based quantitative proteomics using Tandem Mass Tags (TMT). Data analysis was performed using Proteome Discoverer v2.4 (Thermo Fisher) followed by statistical analysis using R. Candidate biomarker proteins were tested by traditional and automated Western blotting (WES, ProteinSimple). Main results and the role of chance We ascertained the identity of EVs by TEM and NTA (mode size 121.8 ± 18.0 nm (blood, n = 5) and 155.9 ± 37.2 (PF, n = 6)), and signals for syntenin and ALIX in immunoblots as well as expression of CD9, CD63 and CD81 in capture bead-flow cytometry. Proteomic analysis identified 9145 protein groups across all sample groups, with 602 significantly regulated between comparisons (adjusted P-value &amp;lt;0.05). In PF, 533 proteins changed significantly in abundance (245 up/288 down), while in blood, only four proteins changed in abundance. We tested the presence/absence of two of these as candidate markers on PB EV protein preparations from n = 32 samples to date; our best marker showed a sensitivity of 67% and a specificity of 90%; upon exclusion of severe endometriosis cases, sensitivity improved to 80%. Limitations, reasons for caution Because of the changes in vesicle composition and amounts with the menstrual cycle, we have to group the samples by menstrual cycle phase and disease severity. This leaves relatively small numbers per group per condition. Another limitation concerns the relatively small amount of sample liquid and thus, EVs available for analysis. Wider implications of the findings Our findings suggest the feasibility of defining a protein signature of endometriosis based on EV cargoes. A non-invasive clinical test would allow for earlier diagnosis and thus treatment of endometriosis. It would also allow patients to initiate fertility treatment early. Trial registration number not applicable

Identification of purinergic system components in the venom of Bothrops mattogrossensis and the inhibitory effect of specioside extracted from Tabebuia aurea.

Snake bites are a severe problem in the countryside of Brazil and are usually attributed to snakes of the genera Bothrops, Crotalus, and Lachesis. Snake venom can release ectoenzymes and nucleotidases that modulate the purinergic system. In addition to serum therapy against snake poisoning, medicinal plants with anti-inflammatory activities, such as Tabebuia aurea, is empirically applied in accidents that occur in difficult-to-access areas. This study aimedwas to verify the presence and activity of nucleotidases in the crude venom of Bothrops mattogrossensis (BmtV) in vitro and characterize the modulation of purinergic components, myeloid differentiation, and inflammatory/oxidative stress markers by BmtV in vivo and in vitro. Moreover, our study assessed the inhibitory activities of specioside, an iridoid isolated from Tabebuia aurea, against the effects of BmtV. Proteomic analysis of venom content and nucleotidase activity confirm the presence of ectonucleotidase-like enzymes in BmtV. In in vivo experiments, BmtV altered purinergic component expression (P2X7 receptor, CD39 and CD73), increased neutrophil numbers in peripheral blood, and elevated oxidative stress/inflammatory parameters such as lipid peroxidation and myeloperoxidase activity. BmtV also decreased viability and increased spreading index and phagocytic activity on macrophages. Specioside inhibited nucleotidase activity, restored neutrophil numbers, and mediate the oxidative/inflammatory effects produced by BmtV. We highlight the effects produced by BmtV in purinergic system components, myeloid differentiation, and inflammatory/oxidative stress parameters, while specioside reduced the main BmtV-dependent effects.

A hot and cold tumor‑related prognostic signature for stage II colorectal cancer.

Globally, colorectal cancer (CRC) is one of the most lethal and prevalent malignancies. Based on the presence of immune cell infiltration in the tumor microenvironment, CRC can be divided into immunologically 'hot' or 'cold' tumors, which in turn leads to the differential efficacy of immunotherapy. However, the immune characteristics of hot and cold CRC tumors remain largely elusive, prompting further investigation of their properties regarding the tumor microenvironment. In the present study, a predictive model was developed based on the differential expression of proteins between cold and hot CRC tumors. First, the differentially expressed proteins (DEPs) were identified using digital spatial profiling and mass spectrometry-based proteomics analysis, and the pathway features of the DEPs were analyzed using functional enrichment analysis. A novel eight-gene signature prognostic risk model was developed (IDO1, MAT1A, NPEPL1, NT5C, PTGR2, RPL29, TMEM126A and TUBB4B), which was validated using data obtained from The Cancer Genome Atlas. The results revealed that the risk score of the eight-gene signature acted as an independent prognostic indicator in patients with stage II CRC (T3-4N0M0). It was also found that a high-risk score in the eight-gene signature was associated with high immune cell infiltration in patients with CRC. Taken together, these findings revealed some of the differential immune characteristics of hot and cold CRC tumors, and an eight-gene signature prognostic risk model was developed, which may serve as an independent prognostic indicator for patients with stage II CRC (T3-4N0M0).

Microglia Morphological Response to Mesenchymal Stromal Cell Extracellular Vesicles Demonstrates EV Therapeutic Potential for Modulating Neuroinflammation.

Mesenchymal stromal cell derived extracellular vesicles (MSC-EVs) are a promising therapeutic for neuroinflammation. MSC-EVs can interact with microglia, the resident immune cells of the brain, to exert their immunomodulatory effects. In response to inflammatory cues, such as cytokines, microglia undergo phenotypic changes indicative of their function e.g. morphology and secretion. However, these changes in response to MSC-EVs are not well understood. Additionally, no disease-relevant screening tools to assess MSC-EV bioactivity exist, which has further impeded clinical translation. Here, we developed a quantitative, high throughput morphological profiling approach to assess the response of microglia to neuroinflammation-relevant signals and whether this morphological response can be used to indicate the bioactivity of MSC-EVs. Using an immortalized human microglia cell-line, we observed increased size (perimeter, major axis length) and complexity (form factor) upon stimulation with interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Upon treatment with MSC-EVs, the overall morphological score (determined using principal component analysis) shifted towards the unstimulated morphology, indicating that MSC-EVs are bioactive and modulate microglia. The morphological effects of MSC-EVs in TNF-γ/IFN-α stimulated cells were concomitant with reduced secretion of 14 chemokines/cytokines (e.g. CXCL6, CXCL9) and increased secretion of 12 chemokines/cytokines (e.g. CXCL8, CXCL10). Proteomic analysis of cell lysates revealed significant increases in 192 proteins (e.g. HIBADH, MEAK7, LAMC1) and decreases in 257 proteins (e.g. PTEN, TOM1, MFF) with MSC-EV treatment. Of note, many of these proteins are involved in regulation of cell morphology and migration. Gene Set Variation Analysis revealed upregulation of pathways associated with immune response, such as regulation of cytokine production, immune cell infiltration (e.g. T cells, NK cells) and morphological changes (e.g. Semaphorin, RHO/Rac signaling). Additionally, changes in microglia mitochondrial morphology were measured suggesting that MSC-EV modulate mitochondrial metabolism. This study comprehensively demonstrates the effects of MSC-EVs on human microglial morphology, cytokine secretion, cellular proteome, and mitochondrial content. Our high-throughput, rapid, low-cost morphological approach enables screening of MSC-EV batches and manufacturing conditions to enhance EV function and mitigate EV functional heterogeneity in a disease relevant manner. This approach is highly generalizable and can be further adapted and refined based on selection of the disease-relevant signal, target cell, and therapeutic product.

O-266 Multi-omics landscape of uterine fluid-derived extracellular vesicles across the menstrual cycle

Abstract Study question Do extracellular vesicles (EVs) mirror the cyclic physiological changes in endometrial tissue, reflecting the endometrial maturation along the entire menstrual cycle? Summary answer Multi-omics data highlights the importance of EVs during the mid-secretory phase and EVs cargo reflects the state of the tissue throughout the menstrual cycle. What is known already Extracellular vesicles isolated from uterine fluid (UF) during the mid-secretory phase have been characterized for their pivotal role in embryo-maternal communication, particularly in the context of microRNAs (miRNAs) and mRNA. EVs have been posited as potential biomarkers in a minimally invasive liquid biopsy approach for assessing endometrial receptivity, hopefully replacing the endometrial tissue biopsy-based testing. However, the dynamics of EVs and their cargo throughout the distinct phases of the menstrual cycle remain unknown. This is the first study to elucidate UF-EV content throughout the maturation of the endometrium. Study design, size, duration Whole-exome RNA and small-RNA sequencing was performed on 15 paired samples, comprising UF and endometrial biopsy (EB), obtained from healthy female subjects across the menstrual cycle. This initial dataset was expanded with 35 additional UF samples. Cumulatively, the analysis encompassed samples (EB and UF) across the menstrual cycle’s four distinct phases: proliferative (n = 10), early-secretory (ES) (n = 17), mid-secretory (MS) (n = 20), and late-secretory (LS) (n = 18). Additionally, 20 UF-EV samples were used for flow cytometry surface proteome analysis. Participants/materials, setting, methods Participants were excluded when infertility-associated conditions were found and included when they had at least one child born, with no hormonal medications used for three months before the sampling. EVs were extracted from UF with size-exclusion chromatography (SEC) and subsequently with glycosaminoglycan-based precipitation. The EVs were characterized with nanoparticle tracking analysis, western blot, transmission electron microscopy and flow-cytometry surface proteome analysis (MACSPlex) for 37 surface markers. Single-end RNA and small-RNA sequencing were performed on NextSeq1000. Main results and the role of chance The transcriptome, miRNome, and protein surface markers of UF-EVs were characterized throughout the menstrual cycle. Transcriptome profiling was performed in comparison to the preceding phase. During the ES phase, 5,507 differentially expressed genes (DEGs) were observed, most of them being downregulated and associated with G-coupled-protein signaling, while in the MS phase, 867 DEGs were identified associated with nucleosome assembly and cell differentiation. Interestingly in the LS phase, the histone genes associated with nucleosome assembly were upregulated compared to MS, highlighting that histone dynamics change corresponding to the menstrual cycle. The exploration of gene ontology revealed consistent enrichment of genes related to extracellular vesicles in UF-EV and EBs across the menstrual cycle. The miRNA analysis of UF-EVs and EB showed significant overlap in miRNA expression level especially in the MS phase. Furthermore, we found 16 unique miRNAs in the mid-secretory phase whose targets were associated with embryo morphogenesis and cell adhesion. MACSPlex analysis revealed that CD9 and CD63, canonical EV markers, were significantly abundant during the MS phase, indicative of an enriched secretion of EVs during the preparation of the endometrium for embryo implantation. These insights underscore that the cargo of UF-EVs reflects the dynamic changes in the endometrial tissue. Limitations, reasons for caution The research on UF-EVs faced notable challenges due to the irregular and non-standardized collection methods of UF in gynecological practice. This variability in UF collection introduces potential confounding factors. The study also operated with a limited sample size, underscoring the need for further validation. Wider implications of the findings Our study indicates that the UF-EVs reflect the menstrual cycle-related molecular changes and UF-EV analysis could be potentially used to follow the menstrual cycle-related molecular processes in the uterine environment. Trial registration number not applicable

Designing an mRNA Vaccine against P. jirovecii Involved in Fatal Pneumonia Infections via Comparative Proteomics and Reverse Vaccinology Approaches.

Pneumocystis jirovecii is the most emerging life-threating health problem that causes acute and fatal pneumonia infection. It is rare and more contagious for patients with leukemia and immune-deficiency disorders. Until now there is no treatment available for this infection therefore, it is needed to develop any treatment against this pathogen. In this work, we used comparative proteomics, robust immune-informatics, and reverse vaccinology to create an mRNA vaccine against Pneumocystis jirovecii by targeting outer and transmembrane proteins. Using a comparative subtractive proteomic analysis of two Pneumocystis jirovecii proteomes, a distinct non-redundant Pneumocystis jirovecii (strain SE8) proteome was chosen. Seven Pneumocystis jirovecii transmembrane proteins were chosen from this proteome based on hydrophilicity, essentiality, virulence, antigenicity, pathway interaction, protein-protein network analysis, and allergenicity. The reverse vaccinology approach was used to predict the immunogenic and antigenic epitopes of major histocompatibility complex (MHC) I, II and B-cells from the selected proteins on the basis of their antigenicity, toxicity and allergenicity. These immunogenic epitopes were linked together to construct the mRNA-based vaccine. To enhance the immunogenicity, suitable adjuvant, linkers (GPGPG, KK, and CYY), and PRDRE sequences were used. Through predictive modeling and confirmation via the Ramachandran plot, we assessed secondary and 3D structures. The adjuvant RpfE was incorporated to enhance the vaccine construct's immunogenicity (GRAVY index: -0.271, instability index: 39.53, antigenicity: 1.0428). The physiochemical profiling of vaccine construct was predicted it an antigenic, efficient, and potential vaccine. Notably, strong interactions were observed between the vaccine construct and TLR-3/TLR-4 (-1301.7 kcal/mol-1 and -1374.7 kcal/mol-1). The results predicted that mRNA-based vaccines trigger a cellular and humoral immune response, making the vaccine potential candidate against Pneumocystis jirovecii and it is more suitable for in-vitro analysis and validation to prove its effectiveness.

Glutenin from the Ancient Wheat Progenitor Is Intrinsically Allergenic as It Can Clinically Sensitize Mice for Systemic Anaphylaxis by Activating Th2 Immune Pathway.

Wheat allergy is a major type of food allergy with the potential for life-threatening anaphylactic reactions. Common wheat, Triticum aestivum (hexaploid, AABBDD genome), was developed using tetraploid wheat (AABB genome) and the ancient diploid wheat progenitor (DD genome)-Aegilops tauschii. The potential allergenicity of gluten from ancient diploid wheat is unknown. In this study, using a novel adjuvant-free gluten allergy mouse model, we tested the hypothesis that the glutenin extract from this ancient wheat progenitor will be intrinsically allergenic in this model. The ancient wheat was grown, and wheat berries were used to extract the glutenin for testing. A plant protein-free colony of Balb/c mice was established and used in this study. The intrinsic allergic sensitization potential of the glutenin was determined by measuring IgE response upon transdermal exposure without the use of an adjuvant. Clinical sensitization for eliciting systemic anaphylaxis (SA) was determined by quantifying the hypothermic shock response (HSR) and the mucosal mast cell response (MMCR) upon intraperitoneal injection. Glutenin extract elicited a robust and specific IgE response. Life-threatening SA associated and a significant MMCR were induced by the glutenin challenge. Furthermore, proteomic analysis of the spleen tissue revealed evidence of in vivo Th2 pathway activation. In addition, using a recently published fold-change analysis method, several immune markers positively and negatively associated with SA were identified. These results demonstrate for the first time that the glutenin from the ancient wheat progenitor is intrinsically allergenic, as it has the capacity to elicit clinical sensitization for anaphylaxis via activation of the Th2 pathway in vivo in mice.

Proteomic analysis of granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex by MALDI imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

P-620 Oocyte competence in Vitro Fertilization: relation between serum anti-Müllerian hormone (AMH) concentrations and complement C4 protein expression in follicular fluid

Abstract Study question To investigate if serum AMH concentration variations are correlated with protein expression in follicular microenvironment affecting IVF outcomes. Summary answer Serum AMH concentrations were associated with altered levels of intrafollicular protein involvement of the complement pathway in immunity response in follicular fluid (FF). What is known already Anti-Müllerian hormone (AMH) plays a crucial role in regulating growth and development of ovarian follicles. The follicular fluid is produced during folliculogenesis and contains a variety of proteins that play important roles in follicle development and oocyte maturation. AMH concentration in follicular fluid can be a marker of ovarian reserve status and oocyte competence. AMH elevated levels may indicate a higher ovarian reserve and are sometimes associated with a greater ovarian response to ovarian stimulation used in IVF. However, the exact relationship between AMH levels in follicular fluid and IVF outcomes may vary among women and depend on various factors. Study design, size, duration The study included FF collected from 75 patients underwent IVF from December 2018 to June 2019.All patients enrolled underwent GnRH antagonist stimulation protocol with r-FSH according to clinical/ovarian features.Patients were sorted into three groups according to serum AMH concentrations: AMH≤1.0 ng/ml, n = 25 (A group); AMH=1.1-2.0 ng/ml, n = 29 (B Group); AMH&amp;gt;2.0 ng/ml, n = 21 (C group).Cutoffs for defining low(A), average(B), and high(C) AMH serum concentrations corresponded to round values of 30th and 70th centiles of each measurement. Participants/materials, setting, methods Exclusion criteria: endometriosis, metabolic/endocrinology diseases and oligoasthenoteratozoospermia. FF aspirates from individual follicles (diameter ≥18 mm) were collected on the day of oocyte retrieval. The FF was analyzed by two-dimensional gel electrophoresis (2-DE). Oocyte number and quality, fertilization rate are IVF outcome measures. Data were analyzed using the unpaired Student’s t-test by IBM SPSS-STATISTICS versions 20.0 and considered significant if p-value ≤ 0.05. Main results and the role of chance 180 protein spots were highlighted in three groups: 80% is represented in all the samples analyzed, whereas 19 protein spots out of 180 are group-specific. Five unique upregulated proteins were identified in the A group and five in C group respect to control group (B). These were identified IG, CO4, ALBU, TRFE, IGLC in A group, while A1AT, ANT3 A2HS, HPT and CO4 in C group. A and C group showed worse oocyte quality associated with a lower fertilization rate than B group. On the basis of the proteome analysis, the component of the complement cascade C4 was found more plentiful in patient with low and high AMH serum concentration compared to average AMH serum concentration ones. The presence of high levels of the unprocessed precursor C4 correlated to serum AMH fluctuations is an indication of non-activation of the complement pathways fundamental for follicle maturation and ovulation compromising IVF outcome. Limitations, reasons for caution Limited number of patients. Age and lifestyle factors (i.e. smoking, diet) are not considered. Wider implications of the findings Results showed an increase of CO4 in FF of patients with low and high AMH serum concentrations. CO4 FF high levels could be indicative of defective complement activation during ovulation with consequent impaired processes and lower standard oocyte competence. Thus, CO4 could be identify as biomarker to predict IVF success. Trial registration number Not Applicable

O-265 Mapping decidualization resistance at multi-omic level

Abstract Study question What are the cellular and molecular features of decidualization resistance (DR) at single-cell resolution? Summary answer There is an absence of the stromal-epithelial transition with aberrant crosstalk among cell types in essential pathways (e.g., endoglin, WNT) confirmed at the protein level. What is known already DR refers to the inability of the endometrium to undergo decidualization changes preparing the endometrium for a conceptus, noted in disorders like endometriosis, recurrent pregnancy loss, miscarriage and preeclampsia. The molecular mechanisms causing this disorder remains unknown. It has been demonstrated that there is a transcriptomic signature of DR in the endometrium of former preeclampsia patients, during and years after suffering from the disease. Our objective is to dilucidated what is DR at single-cell resolution. Study design, size, duration This cross-sectional study compares late secretory phase endometrial biopsies from patients that previously have suffered severe Preeclampsia (sPE). The sample size consisted of 11 sPE patients and 12 control participants for single-cell RNA sequencing (scRNA-seq), 8 sPE patients and 8 controls for spatial transcriptomic mapping covering 95 tissue regions, and 10 sPE patients and 10 controls for proteomic analysis. The study spanned 2 years, from sample collection to omics analysis. Participants/materials, setting, methods Late secretory phase endometrial biopsies from sPE patients and controls underwent molecular analyses. Methods included 10x Genomics protocol for single-cell RNA sequencing, MiloR to calculate cell abundance, CellChat to infer cell-to-cell communication and ClusterProfiler for gene ontology. We solved spatial mapping at transcriptomic level using Nanostring. Proteomics data was collected by laser-capture microdissection coupled mass spectrometry. Protein-protein networks, involving PR and ESR1 receptors as master regulators of decidualization, were built using String. Main results and the role of chance Initial morphological analyses of endometrial biopsies collected during the late secretory phase revealed gross anatomical defects in glandular morphology that were histologically confirmed. scRNA-seq revealed sPE-associated alterations in cell composition involving stromal and epithelial subtypes, denoting a higher proportion of cells expressing proliferative markers in the stroma (MMP11, SFRP1) and epithelium (IHH, EMID), and a near absent epithelial-stromal transition that is normally linked to decidualization. Further, abnormal abundance and gene dysregulation were detected in the three decidualized stages identified. Mapping cell-cell communication networks confirmed aberrant crosstalk among cell types involved in essential pathways (e.g., endoglin, WNT, SPP1). Spatial transcriptomic in a replication cohort of samples revealed a landscape of dysregulated genes in stromal, luminal and glandular enriched regions, corroborating sPE-associated DR. Laser capture microdissection/mass spectrometry of samples revealed differential protein expression at a global level and confirmed many of the scRNA-seq results, such as sPE-exclusive proteins related to proliferation and the absence of a stromal to epithelial transition at a pathway level. These data lend insights into potential molecular mechanisms underlying the pathogenesis of DR. Limitations, reasons for caution DR analysed in this work is associated with sPE but we do not know if this decidualization defect would be the same in other DR associated conditions such as endometriosis, recurrent pregnancy loss, or miscarriage. Wider implications of the findings Here we present the most comprehensive snapshot of DR to date, providing a full range of markers potentially responsible for this decidualization disorder linked to sPE. We concluded that stromal and epithelial cell populations are altered revealing a near absence of the stromal-epithelial transition, cellular crosstalk and glandular constitution. Trial registration number not aplicable

Wheat TaNACα18 functions as a positive regulator of high-temperature adaptive responses and improves cell defense machinery.

Global wheat production amounted to >780 MMT during 2022-2023 whose market size are valued at >$128 billion. Wheat is highly susceptible to high-temperature stress (HTS) throughout the life cycle and its yield declines 5-7% with the rise in each degree of temperature. Previously, we reported an array of HTS-response markers from a resilient wheat cv. Unnat Halna and described their putative role in heat acclimation. To complement our previous results and identify the key determinants of thermotolerance, here we examined the cytoplasmic proteome of a sensitive cv. PBW343. The HTS-triggered metabolite reprograming highlighted how proteostasis defects influence the formation of an integrated stress-adaptive response. The proteomic analysis identified several promising HTS-responsive proteins, including a NACα18 protein, designated TaNACα18, whose role in thermotolerance remains unknown. Dual localization of TaNACα18 suggests its crucial functions in the cytoplasm and nucleus. The homodimerization of TaNACα18 anticipated its function as a transcriptional coactivator. The complementation of TaNACα18 in yeast and overexpression in wheat demonstrated its role in thermotolerance across the kingdom. Altogether, our results suggest that TaNACα18 imparts tolerance through tight regulation of gene expression, cell wall remodeling and activation of cell defense responses.

Design, synthesis, antimicrobial activity, and mechanism of novel 3-(2,4-dichlorophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole derivatives.

Plant pathogens cause substantial crop losses annually, posing a grave threat to global food security. Fungicides have usually been used for their control, but the rapid development of pesticide resistance renders many ineffective, therefore the search for novel and efficient green pesticides to prevent and control plant diseases has become the top priority in crop planting. The results of bioassay studies indicated that most of the target compounds showed certain antimicrobial activity in vitro. In particular, compound X7 showed high inhibitory activity against Xanthomonas oryzae pv. oryzae (Xoo), with an EC50 value of 27.47 μg mL-1, surpassing conventional control agents such as thiazole zinc (41.55 μg mL-1) and thiodiazole copper (53.39 μg mL-1). Further studies on molecular docking showed that X7 had a strong binding affinity with 2FBW. The morphological change observed by scanning electron microscopy indicated that the surface of Xoo appears wrinkled and cracked under X7 treatment and a total of 2662 proteins were identified by label-free proteomic analysis. Three experiments have elucidated the mechanism whereby X7 induced considerable changes in the physiological and biochemical properties of Xoo, which in turn affected the reproduction and growth of bacteria. This work represents a pivotal advancement, offering important reference for the research and development therapeutics in combating plant pathogens. © 2024 Society of Chemical Industry.