Protein Metabolism Research Articles

[Effect of Polyethylene Microplastics on the Microbial Community of Saline Soils].

Mulching to conserve moisture has become an important agronomic practice in saline soil cultivation, and the effects of the dual stress of salinity and microplastics on soil microbes are receiving increasing attention. In order to investigate the effect of polyethylene microplastics on the microbial community of salinized soils, this study investigated the effects of different types (chloride and sulphate) and concentrations (weak, medium, and strong) of polyethylene (PE) microplastics (1% and 4% of the dry weight mass of the soil sample) on the soil microbial community by simulating microplastic contamination in salinized soil environments indoors. The results showed that:PE microplastics reduced the diversity and abundance of microbial communities in salinized soils and were more strongly affected by sulphate saline soil treatments. The relative abundance of each group of bacteria was more strongly changed in the sulphate saline soil treatment than in the chloride saline soil treatment. At the phylum level, the relative abundance of Proteobacteria was positively correlated with the abundance of fugitive PE microplastics, whereas the relative abundances of Bacteroidota, Actinobacteriota, and Acidobacteria were negatively correlated with the abundance of fugitive PE microplastics. At the family level, the relative abundances of Flavobacteriaceae, Alcanivoracaceae, Halomonadaceae, and Sphingomonasceae increased with increasing abundance of PE microplastics. The KEGG metabolic pathway prediction showed that the relative abundance of microbial metabolism and genetic information functions were reduced by the presence of PE microplastics, and the inhibition of metabolic functions was stronger in sulphate saline soils than in chloride saline soils, whereas the inhibition of genetic information functions was weaker than that in chloride saline soils. The secondary metabolic pathways of amino acid metabolism, carbohydrate metabolism, and energy metabolism were inhibited. It was hypothesized that the reduction in metabolic functions may have been caused by the reduced relative abundance of the above-mentioned secondary metabolic pathways. This study may provide a theoretical basis for the study of the effects of microplastics and salinization on the soil environment under the dual pollution conditions.

Investigation of the Alternations in the Muscle Quality of Swimming Crab (Ovalipes punctatus) during Cold-Chain Transportation Using Physicochemical and TMT-Based Quantitative Proteomic Analysis.

Physicochemical properties and protein alterations in Ovalipes punctatus during cold-chain transportation were examined via sensory scores, water-holding capacity (WHC), glucose (GLU) content, catalase (CAT) activity, urea nitrogen (UN) content, and tandem mass tag (TMT)-based proteomic analysis. The results revealed that sensory characteristics and texture of crab muscle deteriorated during transportation. Proteomic analysis revealed 442 and 470 different expressed proteins (DEPs) in crabs after 18 h (FC) and 36 h (DC) of transportation compared with live crabs (LC). Proteins related to muscle structure and amino acid metabolism significantly changed, as evidenced by the decreased WHC and sensory scores of crab muscle. Glycolysis, calcium signaling, and peroxisome pathways were upregulated in the FC/LC comparison, aligning with the changes in GLU content and CAT activity, revealing the stress response of energy metabolism and immune response in crabs during 0-18 h of transportation. The downregulated tricarboxylic acid (TCA) cycle and carcinogenesis-reactive oxygen species pathways were correlated with the decreasing trend in CAT activity, suggesting a gradual retardation in both energy and antioxidant metabolism in crabs during 18-36 h of transportation. Furthermore, the regulated purine nucleoside metabolic and nucleoside diphosphate-related processes, with the increasing changes in UN content, revealed the accumulation of metabolites in crabs.

PSI-17 Effect of timing of high dietary zinc supplementation on the microbiome of gestating sows and their piglets

Abstract The objective of this study was to assess if high dietary zinc (Zn) fed to gestating sows modulates the microbiome of the sow and that of their offspring. Sows were fed 1 of 3 dietary treatments: 1) Control: a corn-soybean meal-based diet containing 125 ppm total supplemental zinc, 2) Breed-to-Farrow: as Control + 141 ppm supplemental Zn as ZnSO4 fed from 5 d post-breeding to farrowing; and 3) Day 110-to-Farrow: as Control + 2,715 ppm supplemental Zn as ZnSO4 starting on d 110 of gestation until farrowing. A subset of third parity sows (n = 30; 10 per treatment) were selected to assess the microbiome of colostrum, milk, and rectal and vaginal surfaces of sows. At farrowing, 4 pigs per litter (n = 120) were selected based on birthweight (BiW; 2 average BiW pigs and 2 pigs with BiW below the litter average) were selected to assess the piglet gut microbiome on the day of birth (d 0) and d5 of age. 16S rRNA sequencing was implemented for colostrum and milk samples while all other sample types were sequenced using shotgun metagenomics to determine taxonomic and functional profiles. Measures of alpha and beta diversity were conducted along with determination of differentially abundant taxa and pathways in tandem with generalized linear mixed models. Greater dietary concentrations of Zn fed to gestating sows minimally affected their colostrum, milk, and vaginal microbiome. However, fecal samples of Breed-to-Farrow sows showed decreased abundance of potentially pathogenic bacteria such as Salmonella enterica and Shigella flexneri and greater abundance of bacteria typically involved in fiber fermentation compared with Control sows (Figure 1a). Piglets born to Day 110-to-Farrow sows also showed greater abundance of some taxa typically involved in fiber breakdown and short chain fatty acid (SCFA) generation on d 5 compared with Control pigs (Figure 1b). At d 0, Breed-to-Farrow piglets showed less abundance of methanogenic archaea and differential abundance of taxa with potentially pathogenic roles, compared with Control pigs (Figure 1c). Gene families and pathways playing roles in central metabolic functions (starch, pyruvate, sucrose, amino acid metabolism) were more abundant in Breed-to-Farrow piglets compared with pigs born to Control sows on d 0 and 5 of age. In conclusion, greater Zn fed to gestating sows impacted the abundance of gut taxa with potential roles in SCFA-production and pathogenicity in both sow and piglet, despite overall minor effects. However, the physiological significance of these findings for the sow and piglet remains unclear.

1 The role of gut microbiota on regulation of growth and metabolism by branched-chain amino acids

Abstract Branched-chain amino acids (BCAA) including leucine, isoleucine and valine are essential amino acids, which are involved in energy homeostasis and regulation of many physiological paradigms including growth. The BCAA serve not only as an essential substrate in the synthesis of body proteins, but also have an important role in regulation of protein turnover. The effects of BCAA on growth and metabolism are likely mediated through different pathways including shifts in metabolic profile in peripheral organs, oxidative capacity of muscle, gut development, and protein metabolism in muscle. Although it has not been appropriately studied or fully dissected, the impact of BCAA on growth and metabolism is thought to lie in the alteration of gut microbiota by BCAA. It has long been believed that gut microbiota, more specifically, the activity of the microbiome and its metabolic products, directly influence a variety of aspects in metazoan physiology. For example, deregulation of bacterial community structure has been linked with host longevity and stress resistance, the pathogenesis of several disorders, regulation of appetite. Multiple studies have demonstrated that modulation of dietary BCAA levels could elicit clear shifts in gut microbial community structure. As part of “Physiology Symposium: The Microbiome and the Host: Functional Role in Metabolism, Immunity, and Gut-Brain Axis”, the possible involvement of intestinal microbiota on regulatory roles of BCAA on growth and metabolism will be discussed.

Plasma proteomic biomarkers and the association between poor cardiovascular health and incident dementia: The UK Biobank study.

The study examined how plasma proteome indicators may explain the link between poor cardiovascular health (CVH) and dementia risk. The present study involved 28,974 UK Biobank participants aged 50-74y (2006-2010) at baseline (2006-2010) who were followed-up for ≤ 15 y for incidence of dementia. CVH was calculated using Life's Essential 8 (LE8) total scores. The scores were standardized and reverse coded to reflect poor CVH (LE8z_rev). OLINK proteomics was available on this sample (k = 1,463 plasma proteins). The study primarily tested the mediating effects of the plasma proteome in LE8z_rev-dementia effect. The total effect was decomposed into "mediation only" or pure indirect effect (PIE), "interaction only" or interaction referent (INTREF), "neither mediation nor interaction" or controlled direct effect (CDE), and "both mediation and interaction" or mediated interaction (INTMED). The study found poorer CVH assessed by LE8z_rev increased the risk of all-cause dementia by 11 % [per 1 SD, hazard ratio, (HR) = 1.11, 95 % CI: 1.03-1.20, p = 0.005). The study identified 11 plasma proteins with strong mediating effects, with GDF15 having the strongest association with dementia risk (per 1 SD, HR = 1.24, 95 % CI: 1.16, 1.33, P < 0.001 when LE8z_rev is set at its mean value) and the largest proportion mediated combining PIE and INTMED (62.6 %; 48 % of TE is PIE), followed by adrenomedullin or ADM. A first principal component with 10 top mediators (TNFRSF1A, GDF15, FSTL3, COL6A3, PLAUR, ADM, GFRAL, ACVRL1, TNFRSF6B, TGFA) mediated 53.6 % of the LE8z_rev-dementia effect. Using all the significant PIE (k = 526) proteins, we used OLINK Insight pathway analysis to identify key pathways, which revealed the involvement of the immune system, signal transduction, metabolism, disease, protein metabolism, hemostasis, membrane trafficking, extracellular matrix organization, developmental biology, and gene expression among others. STRING analysis revealed that five top consistent proteomic mediators were represented in two larger clusters reflecting numerous interconnected biological gene ontology pathways, most notably cytokine-mediated signaling pathway for GDF15 cluster (GO:0019221) and regulation of peptidyl-tyrosine phosphorylation for the ADM cluster (GO:0050730). Dementia is linked to poor CVH mediated by GDF15 and ADM among several key proteomic markers which collectively explained ∼ 54 % of the total effect.

126 A nutritional solution based on calcidiol (25-OH-D3, HyD) and triterpenoid affects muscle protein synthesis pathways in grower-finisher pigs

Abstract To boost profitability in pig production, it is important to achieve increased lean growth rates and maximize efficiency, especially in grower-finisher pigs where feed intakes are high. Molecular research, especially the study of gene expression, is a proven tool for understanding the precise mechanisms behind muscle tissue growth, including a recent focus on pathways such as the mammalian target of rapamycin (mTOR). In the current work, gene expression analyses were used to study the mode of action of the combination of calcidiol (25-OH-D3, HyD) and triterpenoid, which has shown significant growth and efficiency improvements in grower-finisher pigs (McCormack et al., 2024). Three experimental diets were evaluated for 7 wk; Positive control: 0.7% total Ca, 0.36% total P, vit D3 at 1500IU/kg feed (PC), negative control: PC diet + 30% reduced Ca and 10% reduced P (NC), and NC diet with 1,500IU D3 in calcidiol form (25-OH-D3, HyD) and triterpenoid (NC+). At 19 wk of age, 16 animals per treatment were sacrificed and tissue samples were taken from the longissimus muscle and preserved in RNA later solution. The RNA was extracted, quality control checked, and mRNA sequenced using the Illumina platform with 150 base pair paired-end sequencing and 30M read depth. The sequenced data were aligned to the Sus scrofa reference genome (Sscrofa11.1) using Kallisto. Data were normalized and analyzed for differentially expressed genes (DEGs) between treatments using DeSeq2. Genes were assigned to biological pathways using Biofractal’s customisation of the Reactome pathway database. For statistical analysis, RNA integrity number and treatment were included in the model. In the PC compared with the NC, there were 71 DEGs using a false discovery rate (FDR) of &amp;lt; 0.10. Altered pathways included the inhibition of cellular stress pathways and increased cell turnover in the muscle of PC compared with NC pigs. In the NC+ compared with the NC, there were 155 DEGs (FDR &amp;lt; 0.10). The most significantly upregulated gene in the NC+ treatment was adenylate cyclase (FDR &amp;lt; 0.001), an upstream regulator of several pathways related to energy metabolism and muscle protein synthesis. This mechanism was confirmed in the pathway analysis, which found that the NC+ treatment activated PKA-mediated phosphorylation of CREB, a pathway that inhibits muscle atrophy. Furthermore, the NC+ treatment activated several pathways related to muscle growth, including the mammalian target of rapamycin complex 1 (mTORC1). In this study the addition of calcidiol (25-OH-D3, HyD) and triterpenoid to the diet was found to specifically affect molecular pathways in the muscle of grower-finisher pigs involved in both the inhibition of muscle breakdown as well as activating muscle growth.

PSV-16 Effect of precision feeding and functional amino acids on immune response and protein metabolism of growing pigs raised in poor sanitary conditions

Abstract The activation of the immune system reduces the nutrient availability for growth and increases the variability among pigs. Functional amino acids (AA) supplementation can reduce the negative effects of immune activation. Additionally, providing daily tailored diets using individual precision feeding (IPF) was shown to account for individual variability. Therefore, we hypothesized that the association of IPF and functional AA supplementation could reduce the negative effect of immune-challenged pigs fed with daily tailored diets. The aim of this study was to assess performance and blood parameters variation of growing pigs under poor sanitary conditions while being fed functional AA 120% above the estimated INRA AA ratios within IPF, compared with a conventional group-phase (GP) feeding system. The sanitary condition was characterized by oral inoculation of 2 × 109 cfu of Salmonella typhimurium and manure spreading on the pen floor on d 0. Sixty gilts (23 ± 2.8 kg body weight; BW) were distributed in a complete randomized block design in a 2 x 2 factorial arrangement composed of 2 diets: CN with 100% INRA AA ratio or AA+ with 120% of the ratio for Met, Thr and Trp; and 2 feeding systems: GP or IPF. GP pigs received a single diet matching the SID Lys requirement of the 80th-percentile pig at the beginning of the trial whereas IPF pigs received a diet tailored daily to each pig requirement. All pigs were group-housed and fed ad libitum for 28 d by automatic feeders. Body weight was measured at d 0 and 28 and feed intake was recorded daily. Blood samples were collected at d 0, 7 and 28. Data were analyzed with diet and feeding systems and their interactions as fixed effects and BW blocks as random effect. Blood parameters were analyzed as repeated measurements over time. The pig was considered the experimental unit. No diet effect or interactions were observed for any variable (P &amp;gt; 0.05), suggesting that only supplementing Met, Trp, and Thr is not enough to support growth during a sanitary challenge (Table 1). Haptoglobin and IgG increased linearly whereas IgA, urea, and creatinine had a quadratic increase over time (P &amp;lt; 0.05; Table 2). The IPF decreased growth performance and increased serum IgA and creatinine (P &amp;lt; 0.05), mostly due to the fact that IPF reduces AA provision compared with GP system. The IPF model, as most mathematical models, does not account for the AA and energy costs related to the immune challenge. Therefore, IPF might have underestimated the requirements of the pigs, and limited the amount of nutrients provided. In conclusion, the combined effect of IPF and functional AA supplementation was not able to reduce the negative effect of the sanitary challenge.

Proteomic characterization of the different stages of seed germination in Cupressus gigantea

Abstract Seed germination is a pivotal period of plant growth and development. This process can be divided into four major stages, swelling absorption, seed coat dehiscence, radicle emergence and radicle elongation. Cupressus gigantea, a tree native to Tibet, China, is characterized by its resistance to stresses such as cold, and drought, and has a high economic and ecological value. Nevertheless, given its unique geographic location, its seeds are difficult to germinate. Therefore, it is crucial to explore the mechanisms involved in seed germination in this species to improve the germination efficiency of its seeds, thereby protecting this high-quality resource. Here, our findings indicate that seed germination was enhanced when exposed to a 6-h/8-h light/dark photoperiod, coupled with a temperature of 20°C. Furthermore, the application of exogenous GA3 (1 mg/ml, about 2.9 mM) stimulated the germination of C. gigantea seeds. Subsequently, proteomics was used to detect changes in protein expression during the four stages of seed germination. We identified 34 differentially expressed proteins (DEPs), including 13 at the radicle pre-emergence stage, and 17 at the radicle elongation stage. These DEPs were classified into eight functional groups, cytoskeletal proteins, energy metabolism, membrane transport, stress response, molecular chaperones, amino acid metabolism, antioxidant system and ABA signalling pathway. Most of them were found to be closely associated with amino acid metabolism. Combined, these findings indicate that, along with temperature and light, exogenous GA3 can increase the germination efficiency of C. gigantea seeds. Our study also offers insights into the changes in protein expression patterns in C. gigantea seeds during germination.

MtPIN4 plays critical roles in amino acid biosynthesis and metabolism of seed in Medicago truncatula.

The regulation of seed development is critical for determining crop yield. Auxins are vital phytohormones that play roles in various aspects of plant growth and development. However, its role in amino acid biosynthesis and metabolism in seeds is not fully understood. In this study, we identified a mutant with small seeds through forward genetic screening in Medicago truncatula. The mutated gene encodes MtPIN4, an ortholog of PIN1. Using molecular approaches and integrative omics analyses, we discovered that auxin and amino acid content significantly decreased in mtpin4 seeds, highlighting the role of MtPIN4-mediated auxin distribution in amino acid biosynthesis and metabolism. Furthermore, genetic analysis revealed that the three orthologs of PIN1 have specific and overlapping functions in various developmental processes in M. truncatula. Our findings emphasize the significance of MtPIN4 in seed development and offer insights into the molecular mechanisms governing the regulation of seed size in crops. This knowledge could be applied to enhance crop quality by targeted manipulation of seed protein regulatory pathways.

Combining Transcriptome- and Metabolome-Analyzed Differentially Expressed Genes and Differential Metabolites in Development Period of Caoyuanheimo-1 (Agaricus sp.) from Inner Mongolia, China

Caoyuanheimo-1 (Agaricus sp.) is a delectable mushroom native to Inner Mongolia, China, belonging to the Agaricus genus and valued for both its edible and medicinal properties. Although it has been cultivated to a certain extent, the molecular mechanisms regulating its development remain poorly understood. Building on our understanding of its growth and development conditions at various stages, we conducted transcriptomic and metabolomic studies to identify the differentially expressed genes (DEGs) and metabolites throughout its growth cycle. Simultaneously, we analyzed the synthesis pathways and identified several key genes involved in the production of terpenoids, which are secondary metabolites with medicinal value widely found in mushrooms. A total of 6843 unigenes were annotated, and 449 metabolites were detected in our study. Many of these metabolites and differentially expressed genes (DEGs) are involved in the synthesis and metabolism of amino acids, such as arginine, cysteine, methionine, and other amino acids, which indicates that the genes related to amino acid metabolism may play an important role in the fruiting body development of Caoyuanheimo-1. Succinic acid also showed a significant positive correlation with the transcriptional level changes of nine genes, including laccase-1 (TRINITY_DN5510_c0_g1), fruiting body protein SC3 (TRINITY_DN3577_c0_g1), and zinc-binding dihydrogenase (TRINITY_DN2099_c0_g1), etc. Additionally, seventeen terpenoids and terpenoid-related substances were identified, comprising five terpenoid glycosides, three monoterpenoids, two diterpenoids, one sesquiterpenoid, one sesterterpenoid, two terpenoid lactones, and three triterpenoids. The expression levels of the genes related to terpenoid synthesis varied across the three developmental stages.

Acetylation proteomics and metabolomics analyses reveal the involvement of starch synthase undergoing acetylation modification during UV-B stress resistance in Rhododendron Chrysanthum Pall

BackgroundRhododendron chrysanthum Pall. (R. chrysanthum) is a plant that lives in high mountain with strong UV-B radiation, so R. chrysanthum possess resistance to UV-B radiation. The process of stress resistance in plants is closely related to metabolism. Lysine acetylation is an important post-translational modification, and this modification process is involved in a variety of biological processes, and affected the expression of enzymes in metabolic processes. However, little is known about acetylation proteomics during UV-B stress resistance in R. chrysanthum.ResultsIn this study, R. chrysanthum OJIP curves indicated that UV-B stress damaged the receptor side of the PSII reaction center, with a decrease in photosynthesis, a decrease in sucrose content and an increase in starch content. A total of 807 differentially expressed proteins, 685 differentially acetylated proteins and 945 acetylation sites were identified by quantitative proteomic and acetylation modification histological analysis. According to COG and subcellular location analyses, DEPs with post-translational modification of proteins and carbohydrate metabolism had important roles in resistance to UV-B stress and DEPs were concentrated in chloroplasts. KEGG analyses showed that DEPs were enriched in starch and sucrose metabolic pathways. Analysis of acetylation modification histology showed that the enzymes in the starch and sucrose metabolic pathways underwent acetylation modification and the modification levels were up-regulated. Further analysis showed that only GBSS and SSGBSS changed to DEPs after undergoing acetylation modification. Metabolomics analyses showed that the metabolite content of starch and sucrose metabolism in R. chrysanthum under UV-B stress.ConclusionsDecreased photosynthesis in R. chrysanthum under UV-B stress, which in turn affects starch and sucrose metabolism. In starch synthesis, GBSS undergoes acetylation modification and the level is upregulated, promotes starch synthesis, making R. chrysanthum resistant to UV-B stress.

Utilizing transcriptomics and proteomics to unravel key genes and proteins of Oryza sativa seedlings mediated by selenium in response to cadmium stress

BackgroundCadmium (Cd) pollution has declined crop yields and quality. Selenium (Se) is a beneficial mineral element that protects plants from oxidative damage, thereby improving crop tolerance to heavy metals. The molecular mechanism of Se-induced Cd tolerance in rice (Oryza sativa) is not yet understood. This study aimed to elucidate the beneficial mechanism of Se (1 mg/kg) in alleviating Cd toxicity in rice seedlings.ResultsExogenous selenium addition significantly improved the toxic effect of cadmium stress on rice seedlings, increasing plant height and fresh weight by 20.53% and 34.48%, respectively, and increasing chlorophyll and carotenoid content by 16.68% and 15.26%, respectively. Moreover, the MDA, ·OH, and protein carbonyl levels induced by cadmium stress were reduced by 47.65%, 67.57%, and 56.43%, respectively. Cell wall metabolism, energy cycling, and enzymatic and non-enzymatic antioxidant systems in rice seedlings were significantly enhanced. Transcriptome analysis showed that the expressions of key functional genes psbQ, psbO, psaG, psaD, atpG, and PetH were significantly up-regulated under low-concentration Se treatment, which enhanced the energy metabolism process of photosystem I and photosystem II in rice seedlings. At the same time, the up-regulation of LHCA, LHCB family, and C4H1, PRX, and atp6 functional genes improved the ability of photon capture and heavy metal ion binding in plants. Combined with proteome analysis, the expression of functional proteins OsGSTF1, OsGSTU11, OsG6PDH4, OsDHAB1, CP29, and CabE was significantly up-regulated under Se, which enhanced photosynthesis and anti-oxidative stress mechanism in rice seedlings. At the same time, it regulates the plant hormone signal transduction pathway. It up-regulates the expression response process of IAA, ABA, and JAZ to activate the synergistic effect between each cell rapidly and jointly maintain the homeostasis balance.ConclusionOur results revealed the regulation process of Se-mediated critical metabolic pathways, functional genes, and proteins in rice under cadmium stress. They provided insights into the expression rules and dynamic response process of the Se-mediated plant resistance mechanism. This study provided the theoretical basis and technical support for crop safety in cropland ecosystems and cadmium-contaminated areas.

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Abstract Treatment of triple-negative breast cancer (TNBC) remains challenging. Cancer stem cells (CSCs) are the most intractable subpopulation of TNBC cells, which has associated with a high risk of relapse and poor prognosis. However, eradication of CSCs continues to be difficult. Here, we integrated the multiomics data of our large TNBC cohort (n=360) to identify vital markers of CSCs. We discovered that EMSY, inducing a BRCAness phenotype, was preferentially expressed in breast CSCs, promoted the enrichment of ALDH+ cells and was positively correlated with poor relapse-free survival. Mechanistically, EMSY competitively bound to the Jmjc domain, which was critical for the enzyme activity of the KDM5B-specific H3K4 demethylase to reshape methionine metabolism and promote CSC self-renewal and tumorigenesis in an H3K4 methylation-dependent manner. Moreover, EMSY accumulation in TNBC cells sensitized them to PARP inhibitors against bulk cells and methionine deprivation against CSCs. These findings indicate that clinically relevant eradication of CSCs could be achieved with a strategy that targets CSC-specific vulnerabilities in amino acid metabolism. EMSY was highly expressed in BCSCs and positively correlated with TNBC malignancy a. Heatmap with mRNA expression characteristics of non-CSC and CSC subtypes. The top 2,000 most differentially expressed genes used for clustering are plotted. Samples are also annotated on top by the FUSCC subtypes5. b. Volcano plot showing genes targeted by a siRNA inducing a significant change (Fold change (FC) represented the proportion of ALDH+ cells compared to the control)34. c. Overlap analysis of the upregulated genes in CSC subtype (Gene cluster in A) and the genes via siRNA (Gene cluster in B) inducing a significant reduction of the ALDH+ cell ratio. d. The mRNA expression of EMSY in non-CSC and CSC TNBC transcriptomic subtypes (n = 360, Mann-Whitney’s test). Each boxplot showed the median and 95% confidence intervals (95% CI). e. The CNV frequency of EMSY in non-CSC and CSC subtypes. f, g. EMSY mRNA expression of samples with different EMSY copy number in FUSCC (n=302) and TCGA (n=71) TNBC cohorts. h, i. Immunohistochemistry analysis of EMSY expression in specimens of patients between non-CSC (n=45) and CSC (n=20) subtypes. Representative images (h) and the graph of H Scores (i, mean ± SEM, Mann-Whitney’s test) were shown. Scale bar, 4μm (left), 20μm (right). j, k. Kaplan–Meier RFS plots of EMSY in FUSCC (n=360) and GSE234669 (n=442) TNBC (https://kmplot.com/analysis/). Accumulation of EMSY enhanced CSC self-renewal and tumorigenesis a. The mRNA (left) and protein (right) expression of EMSY in FACS sorted ALDH+ and ALDH− cells. b-d. ALDH activity detected by ALDEFLUOR assay and shown as mean ± SEM (n=3/group, one-way ANOVA’s test) in EMSY-knockdown cells (b, c) and EMSY-overexpressing cells (d). e, f. Self-renewal ability determined by primary mammosphere formation (e, n=6 biological independent experiments, one-way ANOVA’s test) and secondary mammosphere formation (f, n=6 biological independent experiments, one-way ANOVA’s test) in EMSY-knockdown SUM149 and MDA-MB-453 cells. Scale bar, 200 μm. g. Self-renewal ability determined by primary mammosphere formation and secondary mammosphere formation in EMSY-overexpressing SUM159 (n=6 biological independent experiments, one-way ANOVA’s test). Scale bar, 200 μm. h-l. EMSY-knockdown SUM149 were injected into mammary fat pads with extreme limiting dilutions (5 x 104, 5 x 103 cells/fat pad). Tumor growth curves (n=5/group, two-way ANOVA’s test) (h, i) were shown. Tumor cell ALDH activity was determined by ALDEFLUOR assay and the bar plot was shown as mean ± SEM (n=5/group, one-way ANOVA test) (j). Frequency of breast CSC was calculated based on the positive tumor sites per group by ELDA (ELDA: Limiting Dilution Analysis for stem cell research (wehi.edu.au)) (k, l). Dairy methionine deprivation, alone or in combination with PARPi, as a therapeutic strategy for EMSY-amplification tumors. a. Immunohistochemistry analyses of EMSY expression in specimens of patients and representative images were shown. b. Patient-derived organoid (PDO) models were performed with fresh isolated primary tissues derived from four independent TNBC patients according to the mRNA level of EMSY. PDO was cultured with fresh medium with (Complete) or without methionine (methionine deprivation, MD) and tumor sphere numbers were determined (n=6/group, two-way ANOVA test). c-e. Effect of MD and olaparib on CSCs and tumor progression. Mice bearing patient-derived xenograft model (PDX, FD-009) were treated with PARPi (50mg/kg, i.p., once every day), dairy MD, and their combination. Tumor volume (c), tumor cell ALDH activity (d) and absolute ALDH+ cancer cells in tumor tissue (e) were assessed (n=5/group, one/two-way ANOVA test). f. Single breast cancer cells from the primary tumors were implanted for a second tumor formation in a limited dilution assay (1000 and 10,000 cells/fat pad); frequency of breast CSC was calculated based on the positive tumor sites per group by ELDA (ELDA: Limiting Dilution Analysis for stem cell research (wehi.edu.au)). Citation Format: Cui-Cui Liu, Ke-da Yu. EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-06-07.

Abstract PO5-13-03: Early Detection of Breast Cancer using Targeted Plasma Metabolomic Profiling

Abstract Background: Breast cancer (BC) is the second leading cause of cancer death among women. Accordingly, early diagnosis is key to the successful treatment, management, and care of BC. Recent studies confirmed that plasma metabolites could be reliable cancer biomarkers, allowing for the development of a minimally invasive routine blood test that can be used for screening, as well as for monitoring disease evolution in patients. The exact metabolic pathways involved in early BC development remain unclear. Metabolomic profiling of women with BC may help to identify new biomarkers to predict breast cancer long before symptoms appear. The purpose of this study was to validate a plasma metabolomic biomarker panel for an improved risk assessment for early detection BC in 241 patients, and to understand the potential role and the relationship between BC subtypes and hormone receptor status. Methods: Our study included a total of 185 plasma samples from women with biopsy-confirmed BC and 56 plasma samples from healthy controls. A targeted, quantitative mass spectrometry (MS)-based metabolomics approach was used to analyze 138 metabolites in plasma samples using a combination of direct injection (DI) MS and reverse-phase high performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). The sample set was split into a discovery set and validation set. Metabolite concentration data, clinical data, and hormones receptor status were used to determine optimal biomarker sets. The same biomarkers and regression models were used and assessed on the validation models. The area under the receiver operator characteristic curves (AUROC), sensitivities and specificities at selected cut off points were calculated for each subgroup. Results: A large proportion of BC patients were at an early stage, with 98 at stage I (53.0 %), 70 at stage II (37.8%), and 17 at stage III (9.2%). The BC patients were of all subtypes: 138 luminal A (76,2%), 23 luminal B (12.7%), 5 hormone receptor-negative/HER2-positive (2.8%), and 15 triples negative (8.3%). A feature selection was performed on the training set using Partial Least-Squares Discriminant Analysis (PLS-DA), and the top performing metabolites were identified as the most important in discriminating BC from healthy subjects. The impact of age was also investigated. Features with &amp;gt;80% missing values were removed. To predict BC, the best signature comprised 9 variables implicated in fatty acid metabolism, amino acid metabolism, polyamine biosynthesis and related signaling pathways We further developed and validated a logistic regression with AUROC &amp;gt; 0.9 using these metabolites and other clinical data for detecting different stages and subtypes of BC. Conclusion: This study identified and validated a simple, high-performing, metabolite-based test for the early detection of BC. After confirmation in other independent cohort studies, our findings could provide the foundations for the development of a blood-based routine test for women at the highest risk for BC that is cost-effective, accurate and reliable. In addition, this approach could be used to complement other modalities especially for BC patients with dense breasts. Citation Format: Jean-Francois Haince, Lun Zhang, Rashid Ahmed Bux, Paramjit S. Tappia, Bram Ramjiawan, David Wishart, Andrew Maksymiuk. Early Detection of Breast Cancer using Targeted Plasma Metabolomic Profiling [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-13-03.

Impact of the Oral Administration of Polystyrene Microplastics on Hepatic Lipid, Glucose, and Amino Acid Metabolism in C57BL/6Korl and C57BL/6-Lepem1hwl/Korl Mice

The impact of microplastics (MPs) on the metabolic functions of the liver is currently unclear and not completely understood. To investigate the effects of the administration of MPs on the hepatic metabolism of normal and obese mice, alterations in the lipid, glucose (Glu), and amino acid regulation pathways were analyzed in the liver and adipose tissues of C57BL/6Korl (wild type, WT) or C57BL/6-Lepem1hwl/Korl mice (leptin knockout, Lep KO) orally administered polystyrene (PS) MPs for 9 weeks. Significant alterations in the lipid accumulation, adipogenesis, lipogenesis, and lipolysis pathways were detected in the liver tissue of MP-treated WT and Lep KO mice compared to the vehicle-treated group. These alterations in their liver tissues were accompanied by an upregulation of the serum lipid profile, as well as alterations in the adipogenesis, lipogenesis, and lipolysis pathways in the adipose tissues of MP-treated WT and Lep KO mice. Specifically, the level of leptin was increased in the adipose tissues of MP-treated WT mice without any change in their food intake. Also, MP-induced disruptions in the glycogenolysis, Glu transporter type 4 (GLUT4)-5′ AMP-activated protein kinase (AMPK) signaling pathway, levels of lipid intermediates, and the insulin resistance of the liver tissues of WT and Lep KO mice were observed. Furthermore, the levels of seven endogenous metabolites were remarkably changed in the serum of WT and Lep KO mice after MP administrations. Finally, the impact of the MP administration observed in both types of mice was further verified in differentiated 3T3-L1 adipocytes and HepG2 cells. Thus, these results suggest that the oral administration of MPs for 9 weeks may be associated with the disruption of lipid, Glu, and amino acid metabolism in the liver tissue of obese WT and Lep KO mice.

Abstract PO5-27-09: Pharmacological eIF4E inhibition suppresses anti-tumor activity in ER+ breast cancer

Abstract Elevated levels of eukaryotic initiation factor 4E (eIF4E) are found in a broad range of cancers, including breast, and have been associated with aggressive, drug resistant tumors. Furthermore, elevated eIF4E activity is sufficient to cause transformation in various cancers, while inhibition suppresses tumor growth. eIF4E, the main regulator and rate limiting factor of protein synthesis, is the downstream integrator of several important oncogenic signaling pathways (PI3K/AKT/mTOR, Ras/Raf/MEK, and Myc). Activation of eIF4E results in increased protein levels of key proliferation and metabolism proteins such as Cyclin D1 (CCND1) and Ornithine Decarboxylase 1 (ODC1), as well as an overall increase in cellular synthesis machinery, causing cellular growth and proliferation. Here, we describe the development and characterization of novel, potent, and selective eIF4E inhibitors. Compounds from this chemical series have nanomolar activity in multiple biochemical and biophysical m7G cap-competition assays as well as potent inhibition of translation in cellular and biochemical assays. In cells, these compounds rapidly and reversibly decrease protein levels of several oncogenes, including CCND1, leading to a G1 cell cycle arrest. We demonstrate that eIF4E inhibitors cause growth inhibition in a variety of breast cancer cell lines, including ER+ breast cancer. These inhibitors also inhibit growth of breast cancer lines with acquired resistance to Palbociclib with the same potency as the parental cell lines. Additionally, eIF4E inhibition shows an increased sensitivity in resistant cell lines when combined with standard of care (SOC), including Palbociclib. Select analogs from the series demonstrate favorable ADMET/PK properties with good oral bioavailability and low safety risk. Lastly, these compounds have demonstrated near complete tumor growth inhibition in non-breast cancer in vivo models. Ongoing experiments will address in vivo efficacy in ER+ breast cancer in both monotherapy and in combination with SOC. Citation Format: Matthew Friedersdorf, Devon Blake, Sarah Thompson, Krista Marran, Jessica Sorrentino. Pharmacological eIF4E inhibition suppresses anti-tumor activity in ER+ breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-09.

Desorption Separation Ionization Mass Spectrometry (DSI-MS) for Rapid Analysis of COVID-19.

During the coronavirus disease 2019 (COVID-19) pandemic, which has witnessed over 772 million confirmed cases and over 6 million deaths globally, the outbreak of COVID-19 has emerged as a significant medical challenge affecting both affluent and impoverished nations. Therefore, there is an urgent need to explore the disease mechanism and to implement rapid detection methods. To address this, we employed the desorption separation ionization (DSI) device in conjunction with a mass spectrometer for the efficient detection and screening of COVID-19 urine samples. The study encompassed patients with COVID-19, healthy controls (HC), and patients with other types of pneumonia (OP) to evaluate their urine metabolomic profiles. Subsequently, we identified the differentially expressed metabolites in the COVID-19 patients and recognized amino acid metabolism as the predominant metabolic pathway involved. Furthermore, multiple established machine learning algorithms validated the exceptional performance of the metabolites in discriminating the COVID-19 group from healthy subjects, with an area under the curve of 0.932 in the blind test set. This study collectively suggests that the small-molecule metabolites detected from urine using the DSI device allow for rapid screening of COVID-19, taking just three minutes per sample. This approach has the potential to expand our understanding of the pathophysiological mechanisms of COVID-19 and offers a way to rapidly screen patients with COVID-19 through the utilization of machine learning algorithms.

Antifungal mechanisms of binary combinations of volatile organic compounds produced by lactic acid bacteria strains against Aspergillus flavus.

Aspergillus flavus（A. flavus）, a common humic fungus known for its ability to infect agricultural products, served as the subject of investigation in this study. The primary objective was to assess the antifungal efficacy and underlying mechanisms of binary combinations of five volatile organic compounds (VOCs) produced by lactic acid bacteria, specifically in their inhibition of A. flavus. This assessment was conducted through a comprehensive analysis, involving biochemical characterization and transcriptomic scrutiny. The results showed that VOCs induce notable morphological abnormalities in A. flavus conidia and hyphae. Furthermore, they disrupt the integrity of the fungal cell membrane and cell wall, resulting in the leakage of intracellular contents and an increase in extracellular electrical conductivity. In terms of cellular components, VOC exposure led to an elevation in malondialdehyde content while concurrently inhibiting the levels of total lipids, ergosterol, soluble proteins, and reducing sugars. Additionally, the impact of VOCs on A. flavus energy metabolism was evident, with significant inhibition observed in the activities of key enzymes, such as Na+/K+-ATPase, malate dehydrogenase, succinate dehydrogenase, and chitinase. And they were able to inhibit aflatoxin B1 synthesis. The transcriptomic analysis offered further insights, highlighting that differentially expressed genes (DEGs) were predominantly associated with membrane functionality and enriched in pathways about carbohydrate and amino acid metabolism. Notably, DEGs linked to cellular components and energy-related mechanisms exhibited down-regulation, thereby corroborating the findings from the biochemical analyses. In summary, these results elucidate the principal antifungal mechanisms of VOCs, which encompass the disruption of cell membrane integrity and interference with carbohydrate and amino acid metabolism in A. flavus.

Ferroptosis and its role in gastric and colorectal cancers.

Ferroptosis is a novel mechanism of programmed cell death, characterized by intracellular iron overload, intensified lipid peroxidation, and abnormal accumulation of reactive oxygen species, which ultimately resulting in cell membrane impairment and demise. Research has revealed that cancer cells exhibit a greater demand for iron compared to normal cells, indicating a potential susceptibility of cancer cells to ferroptosis. Stomach and colorectal cancers are common gastrointestinal malignancies, and their elevated occurrence and mortality rates render them a global health concern. Despite significant advancements in medical treatments, certain unfavorable consequences and drug resistance persist. Consequently, directing attention towards the phenomenon of ferroptosis in gastric and colorectal cancers holds promise for enhancing therapeutic efficacy. This review aims to elucidate the intricate cellular metabolism associated with ferroptosis, encompassing lipid and amino acid metabolism, as well as iron metabolic processes. Furthermore, the significance of ferroptosis in the context of gastric and colorectal cancer is thoroughly examined and discussed.

Structural insights on anti-biofilm mechanism of heated slightly acidic electrolyzed water technology against multi-resistant Staphylococcus aureus biofilm on food contact surface

Slightly acidic electrolyzed water (SAEW) has proven to be an efficient and novel sanitizer in food and agriculture field. This study assessed the efficacy of SAEW (30 mg/L) at 40 °C on the inactivation of foodborne pathogens and detachment of multi-resistant Staphylococcus aureus (MRSA) biofilm. Furthermore, the underlying mechanism of MRSA biofilm under heated SAEW at 40 °C treatment on metabolic profiles was investigated. The results showed that the heated SAEW at 40 °C significantly effectively against foodborne pathogens of 1.96–7.56 (lg (CFU/g)) reduction in pork, chicken, spinach, and lettuce. The heated SAEW at 40 °C treatment significantly reduced MRSA biofilm cells by 2.41 (lg (CFU/cm2)). The synergistic effect of SAEW treatment showed intense anti-biofilm activity in decreasing cell density and impairing biofilm cell membranes. Global metabolic response of MRSA biofilms, treated by SAEW at 40 °C, revealed the alterations of intracellular metabolites, including amino acids, organic acid, fatty acid, and lipid. Moreover, signaling pathways involved in amino acid metabolism, energy metabolism, nucleotide synthesis, carbohydrate metabolites, and lipid biosynthesis were functionally disrupted by the SAEW at 40 °C treatment. As per our knowledge, this is the first research to uncover the potential mechanism of heated SAEW treatment against MRSA biofilm on food contact surface.