Metabolic Pathways Research Articles

Sex-specific molecular signature of mouse podocytes in homeostasis and in response to pharmacological challenge with rapamycin

BackgroundSex differences exist in the prevalence and progression of major glomerular diseases. Podocytes are the essential cell-type in the kidney which maintain the physiological blood-urine barrier, and pathological changes in podocyte homeostasis are critical accelerators of impairment of kidney function. However, sex-specific molecular signatures of podocytes under physiological and stress conditions remain unknown. This work aimed at identifying sexual dimorphic molecular signatures of podocytes under physiological condition and pharmacologically challenged homeostasis with mechanistic target of rapamycin (mTOR) inhibition. mTOR is a crucial regulator involved in a variety of physiological and pathological stress responses in the kidney and inhibition of this pathway may therefore serve as a general stress challenger to get fundamental insights into sex differences in podocytes.MethodsThe genomic ROSAmT/mG-NPHS2 Cre mouse model was used which allows obtaining highly pure podocyte fractions for cell-specific molecular analyses, and vehicle or pharmacologic treatment with the mTOR inhibitor rapamycin was performed for 3 weeks. Subsequently, deep RNA sequencing and proteomics were performed of the isolated podocytes to identify intrinsic sex differences. Studies were supplemented with metabolomics from kidney cortex tissues.ResultsAlthough kidney function and morphology remained normal in all experimental groups, RNA sequencing, proteomics and metabolomics revealed strong intrinsic sex differences in the expression levels of mitochondrial, translation and structural transcripts, protein abundances and regulation of metabolic pathways. Interestingly, rapamycin abolished prominent sex-specific clustering of podocyte gene expression and induced major changes only in male transcriptome. Several sex-biased transcription factors could be identified as possible upstream regulators of these sexually dimorphic responses. Concordant to transcriptomics, metabolomic changes were more prominent in males. Remarkably, high number of previously reported kidney disease genes showed intrinsic sexual dimorphism and/or different response patterns towards mTOR inhibition.ConclusionsOur results highlight remarkable intrinsic sex-differences and sex-specific response patterns towards pharmacological challenged podocyte homeostasis which might fundamentally contribute to sex differences in kidney disease susceptibilities and progression. This work provides rationale and an in-depth database for novel targets to be tested in specific kidney disease models to advance with sex-specific treatment strategies.

Abstract C043: Hyperinsulinemia promotes pancreatic cancer progression by altering tumor metabolism

Abstract Metabolic diseases, such as type 2 diabetes (T2D), insulin resistance, and obesity, often accompany pancreatic ductal adenocarcinoma (PDAC), and they are associated with reduced survival. Hyperinsulinemia is a common hallmark symptom shared by those disorders and is independently associated with reduced survival of PDAC patients. While it has been established that endogenous hyperinsulinemia accelerates PDAC initiation by promoting the formation of KRAS-driven pre-cancerous lesions, its role in the progression of established tumors remains poorly understood. This represents a major knowledge gap in our understanding of whether insulin needs to be monitored and controlled during PDAC treatment, as it is not typically considered in current treatment paradigms. We hypothesized that hyperinsulinemia promotes the progression of PDAC tumors. Using patient-derived PDAC organoids (PDOs) and a mouse model of metabolic disorders, we found that insulin may promote PDOs’ growth by reprogramming tumor metabolism. A High-fat diet (HFD) treatment that induces hyperinsulinemia, but not hyperglycemia in mice accelerated the growth of PDO-derived orthotopic xenografts. Importantly, the fasting insulin level showed a significant positive correlation with the endpoint tumor volume, suggesting that endogenous insulin may positively contribute to PDOs’ growth in vivo. To assess the direct effect of insulin on the growth and molecular profile of PDOs, we treated an early passage PDO line with different concentrations of insulin and glucose at physiologically relevant levels. We observed a ∼1.2-fold increase in cell number after 7 days of culture in high insulin (10 nM) conditions with high (15 mM) or physiological (6 mM) levels of glucose, relative to the low insulin control. Phosphoproteomic analysis demonstrated significantly greater PI3K/AKT/mTOR, but not RAF/MEK/ERK, activity under high insulin with high or physiological levels of glucose compared to the control, directing the investigation toward the metabolic effects of insulin. Consistently, unbiased proteomics profiling revealed that metabolic proteins were significantly enriched in both high insulin conditions, and interestingly insulin enriched different sets of proteins in various metabolic pathways depending on glucose levels. Together, our preliminary results suggest that hyperinsulinemia may enhance the growth and progression of PDAC by altering tumor metabolism to support cell growth. Further experiments are needed to functionally link the insulin-mediated metabolic changes with increased PDO growth, as well as testing the direct effect of endogenous insulin on PDAC cells in vivo. Citation Format: Jeffrey Lin, James D Johnson, David Schaeffer, Vincent Richard, Christoph Borchers, Janel L Kopp. Hyperinsulinemia promotes pancreatic cancer progression by altering tumor metabolism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C043.

Abstract C017: Targeting KRAS selectively induce metabolic reprogramming in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease largely due to the lack of effective treatments that overcome the tumor drug resistance. Oncogenic KRAS mutation contribute to most PDAC and drive multiple metabolic pathways to promote tumor growth. Despite this overarching role of KRAS, inhibition of KRAS/MAPK pathway often lead to resistance by yet unclear mechanism. Here we find that the pharmacological inhibition of KRAS/MAPK pathway induces distinct responses in various pancreatic cancer cells. Specifically, while KRAS/MAPK inhibition suppressed growth in some cells, the same treatment induced or did not change the growth of other cell lines and also selectively impacted cell migration. In addition, metabolic activities were suppressed in cells where growth was suppressed or not changed but was accelerated where KRAS/MAPK inhibition induced growth. Analysis of gene expression profile of human PDAC and KRAS-driven mouse tumors identified nutrient transporters that are upregulated KRAS induction but also upregulated upon KRAS inhibition. Our study reveals context-dependent response to KRAS inhibition in pancreatic cancer and suggest targeting nutrient transporters to overcome therapeutic resistance. Citation Format: Laiba Shiekh, Matthew Cheung, Linyuhui Zheng, Erica Marlette, Chiamaka J. Ezeh, Don-Gerard Conde, Zeribe C. Nwosu. Targeting KRAS selectively induce metabolic reprogramming in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C017.

Reactive Oxygen Species Mediate the Bactericidal Activity of Chlorine Against Salmonella.

Chlorine and its derivatives have been used as an antibacterial agent to reduce Salmonella contamination in poultry meat during processing. We evaluated the survival of 4 different Salmonella serotypes (Typhimurium, Enteritidis, Heidelberg, and Gaminara) in the presence of 50ppm sodium hypochlorite (NaOCl) alone or with the addition of thiourea (radical scavenger) or Dip (iron chelator) to determine the contribution of reactive oxygen species (ROS) in the bactericidal activity of NaOCl. The result showed that for all four serotypes the addition of thiourea or Dip significantly increased the % survival as compared to the respective NaOCl treatment groups, while it was significantly higher with thiourea as compared to Dip (P < 0.05). We also evaluated the survival of 11 deletion mutants of S. Typhimurium, which were demonstrated to increase (∆atpC, ∆cyoA, ∆gnd, ∆nuoG, ∆pta, ∆sdhC, and ∆zwf) or decrease the production of ROS (∆edd, ∆fumB, ∆pykA, and ∆tktB) in Escherichia coli (E. coli), in the presence of 50ppm. The results showed that only two (∆sdhC and ∆zwf) out of 7 ROS-increasing mutants showed reduced % survival as compared to the wild-type (P < 0.05), while all four deletion ROS-decreasing mutants showed significantly higher % survival as compared to the wild-type (P < 0.05). This work suggests that the production of ROS is a major component of the bactericidal activity of NaOCl against Salmonella serotypes and there might be a significant difference in the metabolic pathways involved in ROS production between Salmonella and E. coli.

Abstract B082: Oncogenic KRAS-mediated epigenetic reprogramming is altered by loss of Activating Transcription Factor 3

Abstract Introduction: Over 90% of PDAC patients harbor an activating KRAS mutation, the most common form being KRASG12D. However, additional genetic mutations and environmental events, including chronic or hereditary pancreatitis, are needed for KRAS mutations to lead to PDAC. Our laboratory showed Activating Transcription Factor 3 (ATF3) was required for repressing genes that stabilize the mature acinar cell phenotype and for KRASG12D-driven progression to advanced PanIN lesions. However, ATF3 contributes to gene activation and repression and has been linked to numerous transcription factors and epigenetic mediators, and how ATF3 affects PDAC progression remains unknown. We hypothesize that KRASG12D promotes ATF3-driven histone acetylation in precursor PanINs lesions. Methods: C57Bl/6l mice allowing for acinar-inducible KRASG12D combined with (Ptf1acreERTKRASG12D) or without (Ptf1acreERTKRASG12DAtf3-/-; APK) Atf3 deletion were treated with tamoxifen to induce KRASG12D. After 10 days, mice were treated with cerulein to induce injury and PanIN progression. Mice were sacrificed after two weeks and 3D organoid lines developed. RNA-seq and chromatin immunoprecipitation for H3K27 acetylation (H3K27ac) followed by sequencing (ChIP-seq) was performed. Bioinformatic analysis was used to identify differentially enriched pathways. Levels of acetyl-CoA, the substrate for acetylation, was compared between lines along with levels of H3K27ac, H3K4me1, H3K4me3, and H3K9me3 through western blots. Results and Discussion: KRASG12D expression promoted epigenetic reprogramming in PanINs, including dysregulation of H3K27ac enrichment. ChIP-seq results showed the absence of ATF3 reduces H3K27 acetylation preferentially at gene promoters and pathway analysis showed differential enrichment and activation of KRAS signaling. Mechanistically, ATF3-deleted mice have lower acetyl-CoA levels, linking ATF3 to metabolic pathways as a possible mechanism for gene regulation. Comparing genes with an ATF3-dependent acetylation pattern to a curated list of KRAS-targeted genes identified SMARCC2 (BAF170), a SWI/SNF complex subunit, and KDM1B, a histone demethylase affecting H3K4, suggesting a potential role for ATF3 in widespread epigenetic regulation. Public sequencing data from PDAC patients, supports these findings as ATF3 expression is positively correlated with expression of several SWI/SNF complex subunits and demethylases. Conclusion: This study highlights a role for ATF3- mediated epigenetic dysregulatio. In oncogenic KRAS-dependent PDAC. Targeting this ATF3- mediated epigenetic landscape could be a promising new therapeutic avenue. Citation Format: Fatemeh Mousavi, Christopher L Pin, Parisa Shooshtari. Oncogenic KRAS-mediated epigenetic reprogramming is altered by loss of Activating Transcription Factor 3 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B082.

Abstract C022: Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma

Abstract Alteration of essential metabolic pathways is a major mechanism by which oncogenic KRAS promotes tumor development and growth in pancreatic ductal adenocarcinoma (PDAC). KRAS- driven PDAC is dependent on nutrient scavenging pathways, including macropinocytosis and autophagy to fuel the metabolic demand of rapid proliferation. Thus, these metabolic processes are attractive targets for the development of treatments for PDAC. Acute KRAS loss results in downregulation of macropinocytosis in PDAC. Additionally, our lab demonstrated that KRAS loss or inhibition of ERK MAPK signaling decreased glucose consumption and glycolysis but increased autophagy, thereby enhancing dependency on autophagy for survival and growth. Accordingly, dual ERK MAPK and autophagy inhibition (via chloroquine) synergistically enhanced anti-tumor efficacy in PDAC. However, early clinical data has demonstrated that resistance to this treatment arises over time through unknown mechanisms. We observed that both autophagy induction and macropinocytosis downregulation are transient following RAS, MEK, or ERK inhibition—with autophagic and macropinocytic activity returning to or surpassing basal levels after within two weeks of treatment. Furthermore, we found that prolonged pharmacological inhibition of RAS, MEK, or ERK pathway consistently resulted in significant upregulation of macropinocytosis. We propose that prolonged MAPK inhibition upregulates macropinocytosis over time, consequently abrogating dependency on autophagy and therefore reducing sensitivity to autophagy inhibition. Furthermore, we found that chloroquine, which is commonly thought of as a lysosomotropic drug, does not prevent degradation of proteins engulfed via macropinocytosis. Together our data suggests that upregulation of macropinocytosis may mediate resistance to the combination of ERK MAPK inhibition and chloroquine. With the recent advance in development RAS inhibitors, the resistance mechanisms by which PDAC can overcome RAS inhibitor treatment is a major focus of the field. Given the dependence of PDAC on macropinocytic uptake for tumorigenic growth, it is imperative to understand how long-term treatment with RAS inhibitors regulate macropinocytosis. We demonstrated that PDAC cells with acquired resistance to RAS inhibition exhibit a 2- to 10-fold increase in macropinocytic uptake. We found that upregulated macropinocytosis following long-term RAS or MAPK inhibition is accompanied by increased albumin uptake and sensitivity to albumin-bound paclitaxel (nab-paclitaxel), a therapeutic that is included in the current standard of care for PDAC patients. These results suggest that RAS inhibitor pretreatment or concurrent treatment with nab-paclitaxel may represent a strategy to improve this current PDAC treatment. We conclude that a more complete understanding of the regulation of essential metabolic pathways following both acute and sustained RAS inhibition will better inform future RAS inhibitor combination therapies. Citation Format: Ryan Robb, Sarah Ackermann, Khalilah Taylor, Runying Yang. Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C022.

PSVII-13 Influence of early maternal gestational body weight gain on liver gene expression across generations in offspring

Abstract Maternal nutrition in the periconceptional period influences gene regulation in the fetal liver across generations, affecting long-term metabolism. This study aimed to examine the effects of the rate of body weight (BW) gain of the dam (F0) during early gestation on the differential gene expression of the liver of first (F1) and second-generation (F2) offspring. Crossbred Angus heifers (F0) were assigned into two groups based on their targeted rate of BW gain during the first 84 d of gestation: low gain (LG; 0.28 kg/d; n = 8) or moderate gain (MG; 0.79 kg/d; n = 8). Following this period, all heifers (F0) were kept on a forage-based diet until the F1 offspring were weaned at 8 mo. The F1 heifers underwent estrus synchronization and were artificially inseminated at 15 mo of age. Liver samples were obtained from the F1 heifers at birth and later when harvested at 84 d of gestation, along with liver samples from their F2 fetuses, for a comprehensive multigenerational RNA-Seq analysis. Differentially expressed genes (DEGs) were identified using the DESeq2 R-package, focusing on significant genes determined by a P-value ≤ 0.05 and a log2 fold change |0.5|. Metabolic pathways and biological processes were analyzed using the WebGestalt tool to understand the implications of the observed gene expression changes. At birth, F1 heifers from MG dams exhibited 281 DEGs, comprising 152 downregulated and 129 upregulated genes compared with the offspring of LG dams. These genes were over-represented in lipid metabolism, cellular homeostasis, and signaling pathways, such as MAPK and chemokine signaling. At harvest, 159 DEGs were identified in F1 heifers, with 67 genes downregulated and 92 genes upregulated in the MG group. These genes were related to cellular morphogenesis and biogenesis processes, with a notable downregulation of metabolic pathways. For the F2 fetuses, 192 DEGs were identified, where 71 genes were downregulated, and 121 genes were upregulated. These genes were involved in biological functions such as peptide secretion to cell proliferation and signaling pathways, including PI3K-Akt and Hippo signaling. The analysis of DEGs shared between F1 heifers and F2 fetuses highlighted the influence of the moderate BW weight gain of the F0 dams. At birth and harvest, F1 heifers shared three genes (NAV2, FAM131C, SH3D21). F1 heifers at birth and F2 fetuses shared five genes (CD3E, H1-12, SLC7A11, PIP4P2, ENSBTAG00000051730). From the harvest at d 84 in F1 heifers and F2 fetuses, five genes were shared (DCDC2, RIN1, ENSBTAG00000008911, ENSBTAG00000040518, ENSBTAG00000048049). No DEGs were shared across all comparisons. These findings emphasize the multigenerational impact of early gestational BW gain of the dam on liver gene expression, influencing metabolic pathways and signaling across generations.

Characterization of natural compounds derived from diatom C. gracilis as potential therapeutic agents: An in-silico networking and docking study

Marine diatom Chaetoceros gracilis have been known as the key player regulating the nutritional content of aquaculture species. Being able to synthesize an array of high value bioactive compounds like amino acids, lipids, terpenoids and polysaccharides, it also serves as potential therapeutic and nutraceutical agent. This in silico-based study elucidates the interactive association of different compounds, proteins and pathways of the diatom C. gracilis through an integrated network pharmacology and molecular docking approach. According to the Network analysis of the 41 compounds detected, saturated hydrocarbons, diterpenoids and phenolic compounds scored the highest degree (score > 140). These compounds were further coded for approximately 349 protein targets and almost 490 different pathways. HSP90AA1, STAT3, HIF1A, MTOR, ESR1, PIK3CA, MAPK1 and PTGS2 secured highest degree of protein-protein interaction according to STRING database. The gene enrichment analysis further revealed that these proteins were closely associated with metabolic pathways like Pathways in cancer, neuroactive ligand-receptor interaction, calcium and cAMP signaling pathway, PI3K-Akt signaling pathway, Alzheimer's disease and pathways of neurodegeneration which played an instrumental role in the metabolism of diseases and disorders like cancers of breast, prostrate, and liver, schizophrenia and other mental and hypertensive disorders. Furthermore, the molecular docking and toxicity assessment of a few novel compounds was done with mTOR and HSP90AA1 which revealed promising and stable interactions. Thus, this study provides the first in silico insight outlining the anti-cancerous and neuroprotective potential of novel bioactive compounds derived from marine diatom C.gracilis.

PSIV-14 Short-term heat stress affects the plasma metabolome of Florida Cracker sheep

Abstract Florida Cracker sheep is a heritage sheep breed and an endangered species that was developed under natural selection in Florida, US. The objective of this study was to evaluate the effect of short-term heat stress on the plasma metabolome of Florida Cracker ewes (2 yr). A total of fourteen ewes were allocated to heat stress (HT, n = 7) or cooling (CTL, n = 7) conditions for 6 wk during the summer season. Treatment groups were grouped in covered concrete floor pens with similar dimensions and characteristics and had access to shelter, feed, and freshwater ad libitum. The HT pen was exposed to regular ambient temperatures, and for the cooling pen, an evaporative cooling fan was used. Data for ambient temperature and relative humidity of HT and CTL pens were recorded every week to estimate the temperature humidity index (THI). Blood samples were collected from each experimental ewe 1 wk before the start of the experiment (baseline) and at wk 1, 2, 3, 4, 5, and 6 post-treatment. Plasma was extracted from the blood and processed for targeted metabolomics. Targeted metabolomics was performed using Nuclear Magnetic Resonance spectroscopy to quantify a total of 50 metabolites, including organic acids, amino acids, hexoses, lipids, and carnitines. Statistical analysis for biomarker and pathway enrichment was performed using Metaboanalyst 5.0. The THI values for the HT and THI groups ranged from 81.3 to 89.5 and from 73.5 to 75.6, respectively. A total of 19 differentially abundant metabolites were identified between HT and CTL. The pathway enrichment analysis showed upregulation of fatty acid and primary bile biosynthesis, and glutathione, porphyrin, glycine, serine, and threonine metabolism (P ≤ 0.05) in HT compared with CTL sheep. These metabolic pathways could have a role in the response of the ewes to short-term HT. Our findings highlight that short-term HT alters the plasma metabolome of Florida Cracker ewes. Understanding these changes can contribute to the development of effective management strategies to mitigate the detrimental impact of HT on sheep operations in the Southern US.

PSV-2 Rumen fluid metabolomics of dairy cows fed combinations of essential oil blend, onion peel extract and prebiotics using the RUSITEC system

Abstract Dairy products have a crucial role in the diets of over 6 billion people worldwide, providing essential nutrients such as energy, high-quality protein, and micronutrients. With dairy cows accounting for nearly 81% of global milk production, strategies to increase milk production sustainably are vital, especially with the U.S. being the second-largest producer. Essential oil blends (EOB), onion peel extract (OPE), and prebiotics such as mannan oligosaccharides (MOS) and galacto-oligosaccharides (GOS) offer promising avenues to enhance bovine milk production. These additives have the potential to modify rumen ecology and fermentation activity, while also mitigating greenhouse gas emissions in dairy cows. Therefore, this study investigated the effects of combining EOB, OPE, GOS, and/or MOS on specific rumen metabolites and metabolic pathways using the rumen simulation technique (RUSITEC) system. Rumen-cannulated non-lactating Holstein Friesian cows (n = 3) were used as rumen fluid donors. Total mixed ration (TMR) consisted of corn silage (60%), alfalfa hay (20%), and concentrate (20%). Using a completely randomized design, 16 fermentation vessels were divided into 4 groups with 4 replicates each over a 9-d period. The treatments included a control group (CON; TMR only), GEO (TMR+GOS+EOB+OPE), MEO (TMR+MOS+EOB+OPE), and OLEO (TMR+OLG+EOB+OPE). The EOB comprised of garlic, lemongrass, cumin, lavender, and nutmeg (4:2:2:1:1), while OLG contained GOS and MOS in equal proportions. EOB was added at 10 µL/g, and OPE, GOS, MOS, and OLG were included at 30 mg/g TMR. Rumen effluents were collected over 5 d for quantitative untargeted metabolome analysis. Analysis of rumen effluents using liquid chromatography-mass spectrometry and MetaboAnalyst 5.0 revealed 495 detected and identified metabolites. Volcano plot analysis showed no significant differences (P ≥ 0.05) in metabolite abundance between the CON and MEO, GEO, or OLEO treatments. Partial least squares-discriminant analysis (PLS-DA) also indicated no clear separation between the CON and treatment groups, suggesting no significant alteration in the metabolome. Compared with CON, the relative concentrations of dopamine 4-O-glucuronide, proline, and histamine were less while pyrocatechol was greater in MEO group. Isomer 1 of 2-hydroxyhepta-2,4-dienedioic acid, histamine, and dopamine 4-O-glucuronide were less while 2-aminomuconic acid and isomer 1 of hulupinic acid were greater in the GEO group. Histamine was less and hydroxypyridine was greater in the OLEO group. Phosphonate and phosphinate metabolism tended to be upregulated in OLEO. Meanwhile, metabolic pathways related to amino acids, vitamins, nitrogen metabolism, and primary bile acid biosynthesis remained unaltered by MEO, GEO and OLEO treatments. In summary, the study demonstrated that the inclusion of MEO, GEO, and OLEO had no adverse effects on rumen metabolites. Future research should focus on combinations of nutraceuticals that could positively impact nutrient metabolism and production performance of dairy cows.

PSIII-7 Effects of Saccharomyces cerevisiae fermentation product on total tract digestibility, ruminal fermentation, and plasma metabolome of beef steers fed a finishing diet

Abstract Saccharomyces cerevisiae fermentation product (SCFP) has been widely studied; however, its mode of action on high-concentrate diets has not been fully elucidated. This study evaluated the impact of SCFP on total tract digestibility, fecal excretions of N, P, Zn, and Cu, rumen fermentation, and plasma metabolome of finishing beef steers. Ruminally cannulated Holstein steers [n = 8; body weight (BW) = 580 ± 29.2 kg] were enrolled in a crossover design study with 25-d treatment periods and a 24-d washout period. Steers were fed ad libitum a finishing diet consisting of 20% corn silage and 80% concentrate (on DM basis). Steers were blocked based on initial BW and randomly assigned to two treatments in period 1 and switched to the other treatment in period 2. The treatments were CON (Control, basal diet only) and SCFP (basal diet top-dressed with 12 gּ steer-1ּ ּ d-1 SCFP, NaturSafe, Diamond V, Cedar Rapids, IA). Feed and fecal samples were collected during d 22 to 24 for determination of digestibility. Blood was collected before morning feeding on d 25. Plasma samples were analyzed for metabolome with liquid chromatography-mass spectrometry (LC-MS). Rumen fluid was collected via the rumen cannula at 0, 4, 8, and 12 h post-morning feeding on d 25 and analyzed for pH, volatile fatty acids (VFA), and NH3-N. Performance and ruminal fermentation characteristics data were analyzed using the GLIMMIX procedure of SAS 9.4. Metabolomic data analysis was conducted with Metaboanalyst 6.0. Supplementing steers with SCFP reduced DMI (10.6 vs. 11.4 kg/d, P = 0.05) without affecting (P &amp;gt; 0.05) average daily gain, feed efficiency, and digestibility of organic matter, crude protein, and neutral detergent fiber. Treatment did not affect (P &amp;gt; 0.10) fecal excretions of N, P, Cu, and Zn. Feeding SCFP increased ruminal pH (6.29 vs. 6.01, P = 0.01), numerically reduced concentrations of NH3-N (5.01 vs 6.25 mM, P = 0.16), and total VFA (96.8 vs. 102.5 mM, P = 0.15). SCFP also reduced the ruminal molar proportion of butyrate (11.2 vs. 12.8 molar %, P = 0.01) and tended to increase those of isobutyrate (1.03 vs. 0.90, molar %, P = 0.06) and isovalerate (2.62 vs. 2.29, molar %, P = 0.10). Supplementation of SCFP enriched (P ≤ 0.05) purine metabolism, amino sugar and nucleotide sugar metabolism, and lysine degradation pathways in the plasma. Additionally, tryptophan metabolism and pyrimidine metabolism pathways tended to be enriched (0.05 &amp;lt; P ≤ 0.10) by SCFP. Our findings indicate that feeding SCFP improved the ruminal pH of finishing cattle, which may be beneficial for preventing subacute ruminal acidosis in feedlot cattle. Additionally, SCFP enriched several important plasma nitrogen metabolism pathways, potentially associated with microbial nitrogen metabolism in the rumen, warranting further research.

Comparative Transcriptome Analysis of Mature Leaves of Dimocarpus longan cv. ‘Sijimi’ Provides Insight into Its Continuous-Flowering Trait

Longan (Dimocarpus longan Lour.) is an important tropical and subtropical fruit, and most of its cultivars bloom once a year (once-flowering, OF). Dimocarpus longan cv. ‘Sijimi’ (SJ) is a tropical ecotype variety that blooms several times a year (continuous-flowering, CF) without the need for low-temperature induction. Several studies have focused on the mechanism of continuous flowering in SJ longan; however, none used leaves as research material. As leaves are a key organ in sensing floral-induction signals, we compared gene-expression differences between mature leaves of CF (SJ) and OF (D. longan cv. ‘Shixia’ (SX) and D. longan cv. ‘Chuliang’ (CL)) longan by transcriptome sequencing. An average of 47,982,637, 43,833,340 and 54,441,291 clean reads were obtained for SJ, SX and CL respectively, and a total of 6745 differentially expressed genes (DEGs) were detected. Following Metabolic pathways, Plant-pathogen interaction and Biosynthesis of secondary metabolites, most of the other genes were assigned to the KEGG classifications of MAPK signaling pathway- plant, Plant hormone signal transduction, Amino sugar and nucleotide sugar metabolism and Starch and sucrose metabolism. WGCNA analysis clustered genes into 27 modules, among which bisque4 and darkorange2 module genes specifically were expressed at low and high levels in SJ, respectively. Different gene-expression patterns were detected between CF and OF longan in bisque4 and darkorange2 modules, especially the high levels of transcription factor (TF) expression and the large number of gibberellic acid (GA)-signaling-pathway-specific genes expressed at high levels in CF longan (SJ). Floral-induction-gene expression levels in CF longan, such as levels of GA-signaling-related and FT genes, were always high. In CF longan, after vegetative-growth accumulation, flowers could be directly induced, thereby eliminating the need for low-temperature induction.

PSIV-24 Evaluation of different colostrum feeding volumes on serum immunoglobulin G concentration and metabolic profiles of calves in high altitude area

Abstract Qinghai-Tibet Plateau is the highest plateau in the world, reputed as “the roof of the world” and “the world’s third pole”. Its ecological environment is characterized by low atmospheric oxygen pressure, cold, and limited feed supplies. This study aimed to investigate the serum immunoglobulin G (IgG) concentration and metabolic profiles of calves in high altitude area (3,650 m) fed different volumes of colostrum [8, 12, or 15% of birth body weight (BW)] using untargeted metabolomics strategies. Neonatal Holstein hybrid heifer calves (n = 44) were randomly divided into 1 of the 3 colostrum feeding amounts: 8% of birth BW (Low volume, L), 12% of birth BW (Middle volume, M), and 15% of birth BW (High volume, H) in colostrum. To safeguard the efficiency of colostrum intake, the colostrum was fed to calves within 0.5 h and 6 h after birth. The serum samples at 24 h after birth were collected for IgG concentration determination by indirect enzyme-linked immunosorbent assay (ELISA) and for LC-MS/MS metabonomic analysis. As the volumes of colostrum increased, there was a linearly increased in the serum IgG concentration (P &amp;lt; 0.01). Differential metabolites were selected according to the following 3 conditions: minimum fold change (FC) &amp;gt; 2, P-value &amp;lt; 0.01 in single-dimensional statistical analysis, and variable importance in projection (VIP) &amp;gt; 1 in the OPLS-DA model. Multivariate statistical analyses were performed to visualize differences among groups. Based on the screening criteria, a total of 3 differential metabolites (22 alpha-Hydroxy-campesterol, N-Myristoyl Histidine, and Calcitetrol) were detected between L and M, of which all differential metabolites were down-regulated. Six differential metabolites were detected between M and H, of which PC[P-18:1(11Z)/18:1(12Z)-2OH(9,10)] was up-regulated and 5 differential metabolites including 1-arachidonoyl-2-hydroxy-sn-glycero-3-phosphate, propanoyl phosphate, ascorbic acid 6-palmitate, 20-hydroxyecdysone, and 6’’’-deamino-6’’’-hydroxyparomomycin II were down-regulated. There were 28 differed metabolites (including leucyl-tryptophan, calcitetrol, 3’’-Oxoribostamycin) between L and H, of which 9 differential metabolites were up-regulated and 19 differential metabolites were down-regulated (Figure 1). Based on KEGG metabolic enrichment pathway analysis and pathway topology analysis, porphyrin metabolism was identified as the most impacted pathway between L and H (Figure 2). In conclusion, increasing colostrum volumes significantly increased serum IgG concentrations at 24 h after birth, favoring the success of the transfer of passive immunity. Alterations in serum metabolome of calves in the Qinghai-Tibet Plateau fed different volumes of colostrum were successfully revealed by untargeted metabolomics. The role of the identified differential metabolites and enriched pathway in the transfer of passive immunity in calves at high altitude deserves further investigation. Keywords: calf, colostrum volumes, high altitude area

Dysregulation of sphingolipid metabolism in liver fibrosis

The dysregulation of sphingolipid metabolism emerges as a pivotal factor in the development and progression of liver fibrosis, a condition marked by the overproduction and buildup of extracellular matrix proteins that can lead to liver cirrhosis and failure. Sphingolipids, a diverse class of lipids essential for cellular structure and signaling, are integral to numerous biological functions such as cellular proliferation, morphological differentiation, and programmed cell death. In the context of liver fibrosis, changes in sphingolipid metabolism have been associated with the activation of hepatic stellate cells, the primary cells responsible for fibrogenesis in the liver. These metabolic disruptions lead to an imbalance between profibrotic and antifibrotic sphingolipids, notably sphingosine-1-phosphate and ceramide, contributing to the pathophysiological mechanisms that drive fibrosis. The intricate relationship between sphingolipid metabolism and fibrotic pathways underscores the potential of targeting sphingolipid metabolic enzymes and receptors as therapeutic strategies to mitigate liver fibrosis. The core of this review delves into how disruptions in sphingolipid metabolism contribute to liver fibrosis, exploring biomarkers and potential therapeutic targets. Challenges in research and future directions for comprehensively understanding sphingolipid roles in liver fibrosis are discussed, aiming to open new pathways for therapeutic intervention.

Zinc oxide nanoparticles reduce cadmium accumulation in hydroponic lettuce (Lactuca sativa L.) by increasing photosynthetic capacity and regulating phenylpropane metabolism

Due to the continuous production of industrial wastes and the excessive use of chemical fertilizers and pesticides, severe cadmium (Cd) pollution in soil has occurred globally. This study investigated the impacts of incorporating zinc oxide nanoparticles (ZnONPs) into hydroponically grown lettuce (Lactuca sativa) under cadmium stress conditions, to seek effective methods to minimize Cd buildup in green leafy vegetables. The results showed that 1 mg/L of Cd significantly inhibited lettuce growth, decreasing in leaves (29 %) and roots (33 %) biomass. However, when lettuce was exposed to 2.5 mg/L ZnONPs under cadmium stress, the growth, chlorophyll content, net photosynthetic rate (Pn), stomatal conductance (Gs), actual photochemical efficiency of PSII (φPSII), and activity of key enzymes in photosynthesis were all significantly enhanced. Furthermore, ZnONPs significantly decreased the accumulation of Cd in lettuce leaves (36 %) and roots (13 %). They altered the subcellular distribution and chemical morphology of Cd in lettuce by modifying the composition of cell walls (such as pectin content) and the levels of phenolic compounds, resulting in a reduction of 27 % in Cd translocation from roots to leaves. RNA sequencing yielded 45.9 × 107 and 53.4 × 107 clean reads from plant leaves and roots in control (T0), Cd (T1), Cd+ZnONPs (T2), and ZnONPs (T3) treatment groups respectively, and 3614 and 1873 differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified photosynthesis, carbon fixation, and phenylpropanoid metabolism as the main causes of ZnONPs-mediated alleviation of Cd stress in lettuce. Specifically, the DEGs identified included 12 associated with photosystem I, 13 with photosystem II and 23 DEGs with the carbon fixation pathway of photosynthesis. Additionally, DEGs related to phenylalanine ammonia-lyase, caffeoyl CoA 3-O-methyltransferase, peroxidase, 4-coumarate-CoA ligase, hydroxycinnamoyl transferase, and cytochrome P450 proteins were also identified. Therefore, further research is recommended to elucidate the molecular mechanisms by which ZnONPs reduce Cd absorption in lettuce through phenolic acid components in the phenylpropanoid metabolism pathway. Overall, treatments with ZnONPs are recommended to effectively reduce Cd accumulation in the edible portion of lettuce.

A comprehensive review on microbial diversity and anticancer compounds derived from seaweed endophytes: a pharmacokinetic and pharmacodynamic approach.

Seaweed endophytes are a rich source of microbial diversity and bioactive compounds. This review provides a comprehensive analysis of the microbial diversity associated with seaweeds and their interaction between them. These diverse bacteria and fungi have distinct metabolic pathways, which result in the synthesis of bioactive compounds with potential applications in a variety of health fields. We examine many types of seaweed-associated microorganisms, their bioactive metabolites, and their potential role in cancer treatment using a comprehensive literature review. By incorporating recent findings, we hope to highlight the importance of seaweed endophytes as a prospective source of novel anticancer drugs and promote additional studies in this area. We also investigate the pharmacokinetic and pharmacodynamic profiles of these bioactive compounds because understanding their absorption, distribution, metabolism, excretion (ADMET), and toxicity profiles is critical for developing bioactive compounds with anticancer potential into effective cancer drugs. This knowledge ensures the safety and efficacy of proposed medications prior to clinical trials. This study not only provides promise for novel and more effective treatments for cancer with fewer side effects, but it also emphasizes the necessity of sustainable harvesting procedures and ethical considerations for protecting the delicate marine ecology during bioprospecting activities.

Causal effects of genetically determined metabolites and metabolite ratios on esophageal diseases: a two-sample Mendelian randomization study

BackgroundEsophageal diseases (ED) are a kind of common diseases of upper digestive tract. Previous studies have proved that metabolic disorders are closely related to the occurrence and development of ED. However, there is a lack of evidence for causal relationships between metabolites and ED, as well as between metabolite ratios representing enzyme activities and ED. Herein, we explored the causality of genetically determined metabolites (GDMs) on ED through Mendelian Randomization (MR) study.MethodsTwo-sample Mendelian randomization analysis was used to assess the causal effects of genetically determined metabolites and metabolite ratios on ED. A genome-wide association analysis (GWAS) encompassing 850 individual metabolites along with 309 metabolite ratios served as the exposures. Meanwhile, the outcomes were defined by 10 types of ED phenotypes, including Congenital Malformations of Esophagus (CME), Esophageal Varices (EV), Esophageal Obstructions (EO), Esophageal Ulcers (EU), Esophageal Perforations (EP), Gastroesophageal Reflux Disease (GERD), Esophagitis, Barrett's Esophagus (BE), Benign Esophageal Tumors (BETs), and Malignant Esophageal Neoplasms (MENs). The standard inverse variance weighted (IVW) method was applied to estimate the causal relationship between exposure and outcome. Sensitivity analyses were carried out using multiple methods, including MR-Egger, Weighted Median, MR-PRESSO, Cochran's Q test, and leave-one-out analysis. P < 0.05 was conventionally considered statistically significant. After applying the Bonferroni correction for multiple testing, a threshold of P < 4.3E-05 (0.05/1159) was regarded as indicative of a statistically significant causal relationship. Furthermore, metabolic pathway analysis was performed using the web-based MetaboAnalyst 6.0 software.ResultsThe findings revealed that initially, a total of 869 candidate causal association pairs (Pivw\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${P}_{ivw}$$\\end{document} < 0.05) were identified, involving 442 metabolites, 145 metabolite ratios and 10 types of ED. However, upon applying the Bonferroni correction for multiple testing, only 36 pairs remained significant, involving 28 metabolites (predominantly lipids and amino acids), 5 metabolite ratios and 6 types of ED. Sensitivity analyses and reverse MR were performed for these 36 causal association pairs, where the results showed that the pair of EV and 1-(1-enyl-palmitoyl)-2-linoleoyl-GPE (p-16:0/18:2) did not withstand the sensitivity tests, and Hexadecenedioate (C16:1-DC) was found to have a reverse causality with GERD. The final 34 robust causal pairs included 26 metabolites, 5 metabolite ratios and 5 types of ED. The involved 26 metabolites predominantly consisted of methylated nucleotides, glycine derivatives, sex hormones, phospholipids, bile acids, fatty acid dicarboxylic acid derivatives, and N-acetylated amino acids. Furthermore, through metabolic pathway analysis, we uncovered 8 significant pathways that played pivotal roles in five types of ED conditions.ConclusionsThis study integrated genomics with metabolomics to assess causal relationships between ED and both metabolites and metabolite ratios, uncovering several key metabolic features in ED pathogenesis. These findings have potential as novel biomarkers for ED and provide insights into the disease's etiology and progression. However, further clinical and experimental validations are necessary

PSVI-6 Genomic determinants of the rumen microbiome and metabolome in Angus steers

Abstract The rumen microbiome provides approximately 70% of energy required for the ruminant host through fermentation byproducts. Due to microbial impacts on host health and productivity, several studies have associated microbiome and metabolome variation with important sustainability phenotypes, such as feed efficiency and methane emissions. Recent studies have indicated the microbial communities present in rumen fluid are under low-to-moderate host genetic control. However, microorganisms present on the rumen wall have been underrepresented in research, negating their unique role in host-microbiome interactions. Current studies investigating heritability of the rumen planktonic microorganisms are plagued by small sample sizes, high management and breed heterogeneity, and low resolution for classifying microbial communities. To determine the heritability of the rumen fluid and epithelium-associated microbiome and metabolome, Angus steers (n = 350) were enrolled in a 70-d feed efficiency trial, where real-time dry matter intake was measured. Body weights were collected throughout the trial to calculate average daily gain and residual feed intake. Mid-trial, a novel surgery method was used to swab the rumen wall to capture epithelium-associated microorganisms. On d 70, 50 mL of rumen fluid was obtained from steers using orogastric tubing for collection of planktonic microbial communities. Metagenomic DNA extracted from both rumen fluid and epimural samples was used for reduced representation sequencing to predict microbial features such as diversity, composition, and putative function. Genomic DNA isolated from whole blood was used to perform low-pass whole genome sequencing for use in downstream heritability estimates and variant association analyses. Many core microbial abundances are under low-to-moderate host genetic control (h2 &amp;gt; 0.15; P &amp;lt; 0.05); however, many microorganisms had low heritability estimates (h2 &amp;lt; 0.10). Preliminary genome-wide associations studies are vastly underpowered to detect host genetic variants associated with microbial features, but data generated using the total number of animals will better refine these polymorphisms. Several key heritable metabolites were identified in amino acid metabolism pathways in rumen fluid (P &amp;lt; 0.05). The rumen microbiome appears to act as a complex polygenic trait and incorporating microbial features into prediction models may improve feed efficiency estimations. Understanding host genetic control of the rumen microbiome and metabolome may empower downstream applications to enhance genomic and phenotypic predictions for feed efficiency and other sustainability traits in beef cattle.

Efficacy of DHA-enriched phosphatidylserine and its underlying mechanism in alleviating polystyrene nanoplastics-induced hepatotoxicity in mice

ObjectivePlastic pollution has become a global pollution problem that cannot be ignored. As the main destination of human oral intake, the toxic effects of plastic on the digestive system represented by the intestine and liver are the focus of current research. Marine-derived DHA-PS has a variety of biological activities, mainly focusing on improving brain function and regulating lipid metabolism. However, whether it has an improvement effect on PS-NPs-induced hepato-intestinal injury and the underlying mechanism remain unclear. MethodsA murine liver injury model was established by gavage of PS-NPs for six weeks. By integrating approaches from lipidomics, transcriptomics, and gut microbiota analysis, the molecular mechanism by which DHA-PS alleviates PS-NPs-induced murine hepatotoxicity was explored through the “gut-liver axis”. ResultsOur findings reveal that prolonged exposure to PS-NPs results in significant murine liver damage and dysfunction, characterized by increased oxidative stress and inflammation, along with exacerbated hepatic lipid accumulation. Mechanistically, PS-NPs disrupt the hepatic SIRT1-AMPK pathway by suppressing the expression of SIRT1, AMPKα, and PPARα, while enhancing the expression of SREBP-1c, ultimately leading to disordered hepatic lipid metabolism. The sphingolipid and glycerophospholipid metabolic pathways were particularly affected. Additionally, in agreement with transcriptomic analyses, PS-NPs activate the hepatic TLR4/NF-κB pathway. At the same time, exposure to PS-NPs decreases the expression of ZO-1, occludin, and claudin-1, diminishes the relative abundance of beneficial gut bacteria (norank_f_Muribaculaceae, Akkermansia, and norank_f_norank_o_Clostridia_UCG-014), and increases the prevalence of pathogenic gut bacteria (Coriobacteriaceae_UCG-002 and Desulfovibrio), exacerbating liver injury through the gut-liver axis. However, administering DHA-PS (50 mg/kg) effectively alleviated these injuries. ConclusionThis study was the first to employ multi-omics techniques to elucidate the potential mechanisms underlying hepatotoxicity induced by PS-NPs, thereby supporting the use of DHA-PS as a dietary supplement to mitigate the effects of nanoplastic pollutants.

The effects of alfalfa powder combined with health education on patients with dyslipidemia: A randomized controlled trial

BackgroundCardiovascular diseases (CVDs) remain the leading cause of global mortality, with dyslipidemia recognized as a significant risk factor. Dyslipidemia, characterized by elevated levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG), is associated with an increased lifetime risk of CVD. Although alfalfa’s cholesterol-lowering effects have been demonstrated in animal models, clinical trial data supporting its efficacy in human dyslipidemia treatment are limited. MethodsA randomized controlled trial was conducted from September 20, 2021, to January 12, 2023, involving patients diagnosed with dyslipidemia. Approved by the China Ethics Committee of Registering Clinical Trials, the study included multiple sites in China. Eligible participants were adults aged 18 years or older, meeting criteria from the Chinese Guidelines for the Prevention and Treatment of Adult Blood Lipid Disorders. Participants were randomly assigned to either a health education-only group or a combined alfalfa powder supplementation and health education group for a three-month intervention period. ResultsA total of 160 eligible patients were enrolled and randomized into two groups: the Alfalfa group (n = 80) and the Education group (n = 80). The intervention resulted in significant improvements in key lipid parameters in the Alfalfa group, including reductions in TC, LDL-C, and Apo A1 levels. Although changes in HDL-C levels were not significantly different between the groups, the Alfalfa group exhibited a larger increase. Serum lipid metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) indicated that the combined intervention did not significantly alter the overall blood lipid metabolome but identified fifteen regulated differential metabolites, with the glycerophospholipid metabolism pathway significantly affecting lipid metabolism levels. ConclusionSupplementation with alfalfa powder combined with health education significantly reduced TC, LDL-C, and Apo A1 levels in patients with dyslipidemia. KEGG pathway analysis suggests an association between the glycerophospholipid metabolism pathway and dyslipidemia. Further research is warranted to validate these findings and explore the underlying mechanisms.