Protein Abundance Research Articles

Expression of hepatic genes involved in bile acid metabolism in dairy cows with fatty liver

Bile acids are cholesterol-derived molecules that are primarily produced in the liver. In nonruminants with fatty liver, overproduction of bile acids is associated with liver injury. During the transition period, fatty liver is a metabolic disorder that can affect up to 50% of high-producing dairy cows. The purpose of this study was to provide a comprehensive evaluation on hepatic bile acid metabolism in dairy cows with fatty liver by assessing expression changes of genes involved in bile acid synthesis, export and uptake. The serum activities of aspartate aminotransferase, alanine aminotransferase and glutamate dehydrogenase and concentration of total bile acids were all greater, whereas serum concentration of total cholesterol was lower in cows with fatty liver than in healthy cows. Content of total bile acids was higher but total cholesterol was slightly lower in liver tissues from fatty liver cows than from healthy cows. The hepatic mRNA abundance of cholesterol 7a-hydroxylase (CYP7A1), hydroxy-delta-5-steroid dehydrogenase, 3 β- and steroid delta-isomerase 7 (HSD3B7) and sterol 12α-hydroxylase (CYP8B1), enzymes involved in the classic pathway of bile acid synthesis, was higher in fatty liver cows than in healthy cows. Compared with healthy cows, the hepatic mRNA abundance of alternative bile acid synthesis pathway-related genes sterol 27-hydroxylase (CYP27A1) and oxysterol 7α-hydroxylase (CYP7B1) did not differ in cows with fatty liver. The protein and mRNA abundance of bile acid transporter bile salt efflux pump (BSEP) were lower in the liver of dairy cow with fatty liver. Compared with healthy cows, the hepatic mRNA abundance of bile acid transporters solute carrier family 51 subunit α (SLC51A), ATP binding cassette subfamily C member 1 (ABCC1) and 3 (ABCC3) was greater in cows with fatty liver, whereas the solute carrier family 51 subunit β (SLC51B) did not differ. The expression of genes involved in bile acid uptake, including solute carrier family 10 member 1 (NTCP), solute carrier organic anion transporter family member 1A2 (SLCO1A2) and 2B1 (SLCO2B1) was upregulated in dairy cows with fatty liver. Furthermore, the hepatic protein and mRNA abundance of bile acid metabolism regulators farnesoid X receptor (FXR) and small heterodimer partner (SHP) were lower in cows with fatty liver than in healthy cows. Overall, these data suggest that inhibition of FXR signaling pathway may lead to the increased bile acid synthesis and uptake and decreased secretion of bile acids from hepatocytes to the bile, which elevates hepatic bile acids content in dairy cows with fatty liver. As the hepatotoxicity of bile acids has been demonstrated on nonruminant hepatocytes, it is likely that the liver injury is induced by increased hepatic bile acids content in dairy cows with fatty liver.

Comprehensive Overview of Treponema pallidum Outer Membrane Proteins.

Treponema pallidum, the causative agent of syphilis, is a sexually transmitted microorganism that exhibits remarkable motility capabilities, allowing it to affect various systems. Despite its structural resemblance to gram-negative bacteria due to its dual-membrane, T. pallidum possesses a lower abundance of outer membrane proteins (OMPs), which enables it to effectively conceal itself. This review presents a comprehensive analysis of the clinical diagnostic potential associated with the OMPs of T. pallidum. Furthermore, the known OMPs in T. pallidum that are responsible for mediating host interactions have been progressively elucidated. This review aims to shed light on the pathogenesis of syphilis, encompassing aspects such as vascular inflammation, chancre self-healing, neuroinvasion, and reinfection. Additionally, this review offers a detailed overview of the current state and prospects of development in the field of syphilis vaccines, with the ultimate goal of establishing a foundation for understanding the pathogenesis and implementing effective prevention strategies against syphilis.

Surface tension–driven sorting of human perilipins on lipid droplets

Perilipins (PLINs), the most abundant proteins on lipid droplets (LDs), display similar domain organization including amphipathic helices (AH). However, the five human PLINs bind different LDs, suggesting different modes of interaction. We established a minimal system whereby artificial LDs covered with defined polar lipids were transiently deformed to promote surface tension. Binding of purified PLIN3 and PLIN4 AH was strongly facilitated by tension but was poorly sensitive to phospholipid composition and to the presence of diacylglycerol. Accordingly, LD coverage by PLIN3 increased as phospholipid coverage decreased. In contrast, PLIN1 bound readily to LDs fully covered by phospholipids; PLIN2 showed an intermediate behavior between PLIN1 and PLIN3. In human adipocytes, PLIN3/4 were found in a soluble pool and relocated to LDs upon stimulation of fast triglyceride synthesis, whereas PLIN1 and PLIN2 localized to pre-existing LDs, consistent with the large difference in LD avidity observed in vitro. We conclude that the PLIN repertoire is adapted to handling LDs with different surface properties.

Response of Staphylococcus aureus physiology and Agr quorum sensing to low-shear modeled microgravity.

Staphylococcus aureus is commonly isolated from astronauts returning from spaceflight. Previous analysis of omics data from S. aureus low Earth orbit cultures indicated significantly increased expression of the Agr quorum sensing system and its downstream targets in spaceflight samples compared to ground controls. In this current study, the rotary cell culture system (RCCS) was used to investigate the effect of low-shear modeled microgravity (LSMMG) on S. aureus physiology and Agr activity. When cultured in the same growth medium and temperature as the previous spaceflight experiment, S. aureus LSMMG cultures exhibited decreased agr expression and altered growth compared to normal gravity control cultures, which are typically oriented with oxygenation membrane on the bottom of the high aspect rotating vessel (HARV). When S. aureus was grown in an inverted gravity control orientation (oxygenation membrane on top of the HARV), reduced Agr activity was observed relative to both traditional control and LSMMG cultures, signifying that oxygen availability may affect the observed differences in Agr activity. Metabolite assays revealed increased lactate and decreased acetate excretion in both LSMMG and inverted control cultures. Secretomics analysis of LSMMG, control, and inverted control HARV culture supernatants corroborated these results, with inverted and LSMMG cultures exhibiting a decreased abundance of Agr-regulated virulence factors and an increased abundance of proteins expressed in low-oxygen conditions. Collectively, these studies suggest that the orientation of the HARV oxygenation membrane can affect S. aureus physiology and Agr quorum sensing in the RCCS, a variable that should be considered when interpreting data using this ground-based microgravity model.IMPORTANCES. aureus is commonly isolated from astronauts returning from spaceflight and from surfaces within human-inhabited closed environments such as spacecraft. Astronaut health and immune function are significantly altered in spaceflight. Therefore, elucidating the effects of microgravity on S. aureus physiology is critical for assessing its pathogenic potential during long-term human space habitation. These results also highlight the necessity of eliminating potential confounding factors when comparing simulated microgravity model data with actual spaceflight experiments.

Ca2+ channel and active zone protein abundance intersects with input-specific synapse organization to shape functional synaptic diversity

Synaptic heterogeneity is a hallmark of nervous systems that enables complex and adaptable communication in neural circuits. To understand circuit function, it is thus critical to determine the factors that contribute to the functional diversity of synapses. We investigated the contributions of voltage-gated calcium channel (VGCC) abundance, spatial organization, and subunit composition to synapse diversity among and between synapses formed by two closely related Drosophila glutamatergic motor neurons with distinct neurotransmitter release probabilities (Pr). Surprisingly, VGCC levels are highly predictive of heterogeneous Pr among individual synapses of either low- or high-Pr inputs, but not between inputs. We find that the same number of VGCCs are more densely organized at high-Pr synapses, consistent with tighter VGCC-synaptic vesicle coupling. We generated endogenously tagged lines to investigate VGCC subunits in vivo and found that the α2δ–3 subunit Straightjacket along with the CAST/ELKS active zone (AZ) protein Bruchpilot, both key regulators of VGCCs, are less abundant at high-Pr inputs, yet positively correlate with Pr among synapses formed by either input. Consistently, both Straightjacket and Bruchpilot levels are dynamically increased across AZs of both inputs when neurotransmitter release is potentiated to maintain stable communication following glutamate receptor inhibition. Together, these findings suggest a model in which VGCC and AZ protein abundance intersects with input-specific spatial and molecular organization to shape the functional diversity of synapses.

Ca2+ channel and active zone protein abundance intersects with input-specific synapse organization to shape functional synaptic diversity.

Synaptic heterogeneity is a hallmark of nervous systems that enables complex and adaptable communication in neural circuits. To understand circuit function, it is thus critical to determine the factors that contribute to the functional diversity of synapses. We investigated the contributions of voltage-gated calcium channel (VGCC) abundance, spatial organization, and subunit composition to synapse diversity among and between synapses formed by two closely related Drosophila glutamatergic motor neurons with distinct neurotransmitter release probabilities (Pr). Surprisingly, VGCC levels are highly predictive of heterogeneous Pr among individual synapses of either low- or high-Pr inputs, but not between inputs. We find that the same number of VGCCs are more densely organized at high-Pr synapses, consistent with tighter VGCC-synaptic vesicle coupling. We generated endogenously tagged lines to investigate VGCC subunits in vivo and found that the α2δ-3 subunit Straightjacket along with the CAST/ELKS active zone (AZ) protein Bruchpilot, both key regulators of VGCCs, are less abundant at high-Pr inputs, yet positively correlate with Pr among synapses formed by either input. Consistently, both Straightjacket and Bruchpilot levels are dynamically increased across AZs of both inputs when neurotransmitter release is potentiated to maintain stable communication following glutamate receptor inhibition. Together, these findings suggest a model in which VGCC and AZ protein abundance intersects with input-specific spatial and molecular organization to shape the functional diversity of synapses.

A Multiplexed Quantitative Proteomics Approach to the Human Plasma Protein Signature

Despite the plasma proteome being able to provide a unique insight into the health and disease status of individuals, holding singular promise as a source of protein biomarkers that could be pivotal in the context of personalized medicine, only around 100 proteins covering a few human conditions have been approved as biomarkers by the US Food and Drug Administration (FDA) so far. Mass spectrometry (MS) currently has enormous potential for high-throughput analysis in clinical research; however, plasma proteomics remains challenging mainly due to the wide dynamic range of plasma protein abundances and the time-consuming procedures required. We applied a new MS-based multiplexed proteomics workflow to quantitate proteins, encompassing 67 FDA-approved biomarkers, in >1300 human plasma samples from a clinical cohort. Our results indicate that this workflow is suitable for large-scale clinical studies, showing good accuracy and reproducibility (coefficient of variation (CV) < 20 for 90% of the proteins). Furthermore, we identified plasma signature proteins (stable in time on an individual basis), stable proteins (exhibiting low biological variability and high temporal stability), and highly variable proteins (with low temporal stability) that can be used for personalized health monitoring and medicine.

Charting the Dynamic Trophoblast Plasma Membrane Identifies LYN As a Functional Regulator of Syncytialization.

The differentiation of placental cytotrophoblasts (CTBs) into the syncytiotrophoblast (STB) layer results in a significant remodeling of the plasma membrane proteome. Here, we use a peroxidase-catalyzed proximity labeling strategy to map the dynamic plasma membrane proteomes of CTBs and STBs. Coupled with mass-spectrometry-based proteomics, we identify hundreds of plasma membrane proteins and observe relative changes in protein abundance throughout differentiation, including the upregulation of the plasma-membrane-localized nonreceptor tyrosine kinase LYN. We show that both siRNA-mediated knockdown and small molecule inhibition of LYN kinase function impairs CTB fusion and reduces the expression of syncytialization markers, presenting a function for LYN outside of its canonical role in immunological signaling. Our results demonstrate the use of the proximity labeling platform to discover functional regulators within the plasma membrane and provide new avenues to regulate trophoblast differentiation.

Vgll2 as an integrative regulator of mitochondrial function and contractility specific to skeletal muscle.

During skeletal muscle adaptation to physiological or pathophysiological signals, contractile apparatus and mitochondrial function are coordinated to alter muscle fiber type. Although recent studies have identified various factors involved in modifying contractile proteins and mitochondrial function, the molecular mechanisms coordinating contractile and metabolic functions during muscle fiber transition are not fully understood. Using a gene-deficient mouse approach, our previous studies uncovered that vestigial-like family member 2 (Vgll2), a skeletal muscle-specific transcription cofactor activated by exercise, is essential for fast-to-slow adaptation of skeletal muscle. The current study provides evidence that Vgll2 plays a role in increasing muscle mitochondrial mass and oxidative capacity. Transgenic Vgll2 overexpression in mice altered muscle fiber composition toward the slow type and enhanced exercise endurance, which contradicted the outcomes observed with Vgll2 deficiency. Vgll2 expression was positively correlated with the expression of genes related to mitochondrial function in skeletal muscle, mitochondrial DNA content, and protein abundance of oxidative phosphorylation complexes. Additionally, Vgll2 overexpression significantly increased the maximal respiration of isolated muscle fibers and enhanced the suppressive effects of endurance training on weight gain. Notably, no additional alteration in expression of myosin heavy chain genes was observed after exercise, suggesting that Vgll2 plays a direct role in regulating mitochondrial function, independent of its effect on contractile components. The observed increase in exercise endurance and metabolic efficiency may be attributed to the acute upregulation of genes promoting fatty acid utilization as a direct consequence of Vgll2 activation facilitated by endurance exercise. Thus, the current study establishes that Vgll2 is an integrative regulator of mitochondrial function and contractility in skeletal muscle.

Age-dependent changes in cytochrome P450 abundance and composition in human liver.

Cytochrome P450 (CYP) superfamily represents the major drug metabolizing enzymes responsible for metabolizing over 65% of therapeutic drugs, including those for pediatric use. CYP-ontogeny based physiologically-based pharmacokinetic (PBPK) models have emerged as useful tools to mechanistically extrapolate adult pharmacokinetic data to children. However, these models integrate physiological differences in pediatric population including age-dependent differences in the abundances of CYP enzymes. Conventionally, developmental changes in CYP enzymes have been reported using protein abundance and activity data from subcellular fractions such as microsomes, which is prone to high technical variability. Similarly, the available pediatric pharmacokinetic data suffer from the lack of specific CYP substrates, especially in younger children. In the present study, we utilized viable hepatocytes from 50 pediatric (age, day 1- 18 yr) and 8 adult human donors and carried out global proteomics-based quantification of all major hepatic CYP enzymes, including orphan enzymes that have not been studied previously. While CYPs 2B6, 3A5, 4A11, 4F3, and 4V2 did not show significant association with age, all other quantified isoforms either increased or decreased with age. CYPs 1A2, 2C8, 2C18, and 2C19 were absent or barely detected in the neonatal group, while CYP3A7 was the highest in this group. The >1-2 yr age-group showed the highest total abundance of all CYP enzymes. The age-dependent differences in CYP enzymes reported in this study can be used to develop ontogeny-based PBPK models, which in turn can help improve pediatric dose-prediction based on adult dosing, leading to safer drug pharmacology in children. Significance Statement We quantified age-dependent differences in the abundances of hepatic CYP enzymes using a large set of viable pediatric and adult hepatocytes using quantitative global proteomics. We report for the first time the ontogeny in the abundance of CYP enzymes in human hepatocytes, especially, orphan CYPs 20A1, 27A1, 51A1, 7B1, and 8B1 and the CYP4 subfamily of enzymes. Our study provides important data about CYP ontogeny that can be used for the better prediction of pediatric pharmacokinetics using physiologically-based pharmacokinetic modeling.

In silico analysis of secreted effectorome of the rubber tree pathogen Rigidoporus microporus highlights its potential virulence proteins

Rigidoporus microporus, the causative agent of the white root rot disease of rubber trees, poses a significant threat to natural rubber production worldwide. Understanding the molecular mechanisms facilitating its pathogenicity would be crucial for developing effective disease management strategies. The pathogen secretes effector proteins, which play pivotal roles in modulating host immune responses and infection. In this study, in silico analyses identified 357 putative secreted effector proteins from the R. microporus genome. These were then integrated into previous RNA-seq data obtained in response to rubber tree latex exposure. Annotation of putative effectors suggested the abundance of proteins in several families associated with the virulence of R. microporus, especially hydrophobin proteins and glycoside hydrolase (GH) proteins. The contribution of secreted effectors to fungal pathogenicity was discussed, particularly in response to rubber tree latex exposure. Some unknown highly expressed effectors were predicted for the protein structures, revealing their similarity to aminopeptidase, ubiquitin ligase, spherulin, and thaumatin protein. This integrative study further elucidates the molecular mechanism of R. microporus pathogenesis and offers alternative targets for developing control strategies for managing white root rot disease in rubber plantations.

Chemoenzymatic Labeling, Detection and Profiling of Core Fucosylation in Live Cells.

Core fucosylation, the attachment of an α-1,6-linked-fucose to the N-glycan core pentasaccharide, is an abundant protein modification that plays critical roles in various biological processes such as cell signaling, B cell development, antibody-dependent cellular cytotoxicity, and oncogenesis. However, the tools currently used to detect core fucosylation suffer from poor specificity, exhibiting cross-reactivity against all types of fucosylation. Herein we report the development of a new chemoenzymatic strategy for the rapid and selective detection of core fucosylated glycans. This approach employs a galactosyltransferase enzyme identified fromCaenorhabditis elegansthat specifically transfers an azido-appended galactose residue onto core fucose via a β-1,4 glycosidic linkage. We demonstrate that the approach exhibits superior specificity toward core fucose on a variety of complex N-glycans. The method enables detection of core fucosylated glycoproteins from complex cell lysates, as well as on live cell surfaces, and it can be integrated into a diagnostic platform to profile protein-specific core fucosylation levels. This chemoenzymatic labeling approach offers a new strategy for the identification of disease biomarkers and will allow researchers to further characterize the fundamental role of this important glycan in normal and disease physiology.

Proteomic profiles revealed enzymatic activities associated with the flavor formation of salted shrimp paste influenced by Bacillus subtilis K-C3 inoculation.

Enzymatic proteomic profiles were examined to comprehend the predominant enzymes involved in the flavor development of salted shrimp paste influenced by Bacillus subtilis K-C3 inoculation (Inoc), compared to those without inoculation (CON). Inoc showed greater proteolytic, lipolytic, and chitinolytic activities than CON (P < 0.05) throughout 30 days of fermentation, indicating B. subtilis's ability to accelerate the fermentation rate and render distinctive flavor profiles to shrimp paste. Among 50 differential abundance proteins (DAPs), 24 DAPs were identified as potential key regulating enzymes, with a P-value < 0.05 and |FC| > 0.50, indicating their significance and regulating capacity within specific metabolic pathways. Notably, 27 and 23 DAPs were up-regulated in Inoc and CON, respectively. Moreover, gene ontology (GO) enrichment analysis revealed that hydrolases, involved in carbohydrate metabolic processes and proteolysis, were the most differentiating pathways between Inoc and CON. Both samples exhibited different flavor profiles. A greater abundance of N-containing volatile compounds with a lower total abundance of aldehydes, ketones, alcohols, and acids could suggest a more favorable flavor in Inoc, compared to CON. Principal component analysis (PCA) revealed a positive correlation between L-ascorbate peroxidase, carboxypeptidase, and tripeptidyl peptidase sed2, with proteolytic and lipolytic activities in Inoc (P < 0.05). Meanwhile, acids and alcohols were positively correlated with CON. Therefore, B. subtilis inoculation could produce a distinctive flavor with a desirable sensory perception of shrimp paste regarding its ability to release extracellular enzymes/proteins. B. subtilis K-C3 inoculation could be suggested in the production of shrimp paste to improve its flavor characteristics.

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 IA-04: Targeting the oncogenic state of RAS with tri-complex inhibitors

Abstract KRAS oncogenic driver mutations occur in approximately 30% of all human cancers, and in particular, in over 90% of patients with pancreatic ductal adenocarcinoma (PDAC). Small molecule inhibitors that selectively target the inactive, GDP-bound state of KRAS G12C mutant proteins have demonstrated clinical efficacy in non-small cell lung cancer (NSCLC) as monotherapies, and in the small subset of PDAC patients that harbor this mutation. However, there are no currently approved RAS-targeted therapy options for the majority of patients with non-G12C RAS mutant PDAC. We have designed a series of tri-complex inhibitors that specifically target the GTP-bound, active state of RAS (RAS(ON)). These small molecule inhibitors bind non-covalently to an abundant intracellular protein, cyclophilin A (CypA). The resulting binary complex selectively engages RAS(ON), forming a tri-complex that sterically inhibits RAS interaction with downstream effectors. We have generated mutant-selective inhibitors that covalently engage RAS(ON) G12C (RMC-6291) or RAS(ON) G12D (RMC-9805). In addition, RMC-6236 is a RAS(ON) multi-selective inhibitor that non-covalently inhibits the active, GTP-bound state of mutant and wild-type variants of the canonical RAS isoforms (KRAS, NRAS, and HRAS). Here we will describe the mechanism of action of these investigational agents and the preclinical profiles that support their clinical evaluation in patients with PDAC as monotherapies and in combination therapy regimens with standard-of-care agents or as novel RAS(ON) inhibitor doublets. Citation Format: Mallika Singh. Targeting the oncogenic state of RAS with tri-complex inhibitors [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 IA-04.

Chlorogenic Acid Enhances the Intestinal Health of Weaned Piglets by Inhibiting the TLR4/NF-κB Pathway and Activating the Nrf2 Pathway

Chlorogenic acid (CGA) is a natural polyphenol with potent antioxidant and anti-inflammatory activities. However, the exact role of it in regulating intestinal health under oxidative stress is not fully understood. This study aims to investigate the effects of dietary CGA supplementation on the intestinal health of weaned piglets under oxidative stress, and to explore its regulatory mechanism. Twenty-four piglets were randomly divided into two groups and fed either a basal diet (CON) or a basal diet supplemented with 200 mg/kg CGA (CGA). CGA reduced the diarrhea rate, increased the villus height in the jejunum, and decreased the crypt depth in the duodenum, jejunum, and ileum of the weaned piglets (p &lt; 0.05). Moreover, CGA increased the protein abundance of Claudin-1, Occludin, and zonula occludens (ZO)-1 in the jejunum and ileum (p &lt; 0.05). In addition, CGA increased the mRNA expression of pBD2 in the jejunum, and pBD1 and pBD2 in the ileum (p &lt; 0.05). The results of 16S rRNA sequencing showed that CGA altered the ileal microbiota composition and increased the relative abundance of Lactobacillus reuteri and Lactobacillus pontis (p &lt; 0.05). Consistently, the findings suggested that the enhancement of the intestinal barrier in piglets was associated with increased concentrations of T-AOC, IL-22, and sIgA in the serum and T-AOC, T-SOD, and sIgA in the jejunum, as well as T-AOC and CAT in the ileum caused by CGA (p &lt; 0.05). Meanwhile, CGA decreased the concentrations of MDA, IL-1β, IL-6, and TNF-α in the serum and jejunum and IL-1β and IL-6 in the ileum (p &lt; 0.05). Importantly, this study found that CGA alleviated intestinal inflammation and oxidative stress in the piglets by inhibiting the TLR4/NF-κB signaling pathway and activating the Nrf2 signaling pathway. These findings showed that CGA enhances the intestinal health of weaned piglets by inhibiting the TLR4/NF-κB pathway and activating the Nrf2 pathway.

PSI-28 Effect of supplementing a Bacillus spp.-based probiotic on intestinal tight junction proteins expression and abundance of selected microorganisms in heat stressed pigs

Abstract The maintenance of small intestine epithelial barrier function, depending in part on tight junction proteins (TJP), prevents the system from invasion of undesired materials but may become altered in heat stressed pigs. Heat stress (HS) also appears to modify the composition of intestinal microbiota that, combined with a weak barrier, may cause health deterioration of pigs. Probiotics, in contrast, can help animals to maintain a healthy intestinal environment. A study, conducted with pigs [n = 80; body weight (BW) = 21.6 ± 3.4 kg], analyzed the effect of HS and the supplementation of a Bacillus spp-based probiotic on gene expression of the TJP occludin, claudins, and TJP1 in jejunum and ileum. Abundance of Lactobacillus sp., Bifidobacterium sp., Bacillus subtilis and E. coli in ileal content was also analyzed. Treatments were thermal neutral (TN) pigs fed a control diet without probiotic (TN-C) or with 0.05% probiotic (TN-P), and HS pigs fed a control without probiotic (HS-C) or with 0.05% probiotic (HS-P). The control diet was based on wheat and soybean meal, added with free Lys, Thr, Met, and Val. Feed and water were freely available during the 21-d study. At termination, six pigs per treatment were sacrificed and samples from jejunum and ileum tissue were collected to analyze TJP gene expression; ileal content was collected to analyze abundance of intestinal microorganisms. Ambient temperature inside TN and HS rooms ranged from 19 to 25°C and 30 to 38.5°C, respectively. In jejunum, claudin2 expression was greater in TN-P than in TN-C (P = 0.05) but there was no effect of HS. In ileum, HS increased claudin3 but decreased occludin gene expression (P &amp;lt; 0.05). HS did not affect TJP1gene expression (P &amp;gt; 0.10). Probiotic supplementation did not affect gene expression of TJP (P &amp;gt; 0.10). Relative to TN-C pigs, abundance of Lactobacillus sp. and Bifidobacterium sp. tended to decrease in HS-C pigs (P &amp;lt; 0.10), and that of Bifidobacterium sp. decreased in HS-P pigs (P &amp;lt; 0.05). Abundance of E. coli tended to be greater (P &amp;lt; 0.10) in both HS-C and HS-P pigs, than in the TN-C pigs. Abundance of Bacillus sp. was greater (P &amp;lt; 0.01) in HS pigs fed the diet supplemented with the probiotic, compared with the TN-C (P &amp;lt; 0.01) and HS-C (P &amp;lt; 0.05) pigs. In conclusion, these data indicate that HS affects TJP gene expression and composition of intestinal microbiota, but supplementing 0.05% of a probiotic based on Bacillus spp. did not affect the abundance of measured microbiota except for Bacillus sp. under HS conditions.

PSII-18 Prematurity alters nutrient signaling and protein synthesis in skeletal muscle of neonatal piglets

Abstract Extrauterine growth restriction is common in infants born preterm, and negatively affects lean mass accretion and health. Prematurity has been shown to inhibit the feeding-induced stimulation of protein synthesis in skeletal muscle. We hypothesized that nutrient signaling, activation of the mechanistic target of rapamycin complex 1 (mTORC1), and protein synthesis in the muscle are limited in preterm infants which negatively impacts muscle growth. The objective of this study was to examine how prematurity influences the mechanisms involved in insulin- and amino acid-induced signaling and protein synthesis in skeletal muscle of piglets delivered either 9 d preterm (104 d, n = 25) or at term (112 d, n = 26) by cesarean section. After resuscitation, they were surgically implanted with jugular vein and carotid artery catheters and placed in individual incubators. They were randomly allotted to one of three treatments within each preterm and term birth groups: euaminoacidemic-euglycemic (FAST), hyperinsulinemic-euaminoacidemic-euglycemic (INS), or euinsulinemic-hyperaminoacidemic-euglycemic (AA) clamps on 4 d after birth. After the clamp procedure, the piglets were euthanized to collect longissimus dorsi muscle for estimating in vivo fractional protein synthesis rates and the abundance and activation of components related to insulin and amino acid signaling. Data were analyzed using the MIXED procedure of SAS. The leucine sensor, Sestrin1 bound to GATOR2 (P &amp;lt; 0.05) was reduced in response to AA whereas the abundance of the mTOR-RagA and mTOR-RagC complexes increased (P &amp;lt; 0.05). The phosphorylation of Akt was increased (P &amp;lt; 0.05) in response to INS and the phosphorylation of mTORC1 and protein synthesis increased (P &amp;lt; 0.05) in response to both AA and INS. Prematurity did not affect the protein abundances of the AA transporters for glutamine (SLC38A2), leucine (SLC7A5), and arginine (SLC38A9). However, prematurity reduced (P &amp;lt; 0.05) the abundances of the leucine sensors SAR1B, the Sestrin1-GATOR complex, the AA sensor, RAB1A, and the threonine sensor, TARS2 (P &amp;lt; 0.05). The abundances of the glutamine sensor, ARF1, the arginine sensor, CASTOR, and the methionine-SAMTOR GATOR complex were similar. Prematurity reduced (P &amp;lt; 0.05) the abundance of the mTOR-RagA and mTOR-RagC complexes, the phosphorylation of mTORC1 and muscle protein synthesis (P &amp;lt; 0.05). In conclusion, prematurity negatively affects protein synthesis in skeletal muscle of neonatal preterm piglets by blunting anabolic pathways responsible for nutrient-sensing and mTORC1 activation. The reduced anabolic response likely contributes to reduced lean growth and extrauterine growth restriction following preterm birth.

Decoupled transcript and protein concentrations ensure histone homeostasis in different nutrients.

To maintain protein homeostasis in changing nutrient environments, cells must precisely control the amount of their proteins, despite the accompanying changes in cell growth and biosynthetic capacity. As nutrients are major regulators of cell cycle length and progression, a particular challenge arises for the nutrient-dependent regulation of 'cell cycle genes', which are periodically expressed during the cell cycle. One important example are histones, which are needed at a constant histone-to-DNA stoichiometry. Here we show that budding yeast achieves histone homeostasis in different nutrients through a decoupling of transcript and protein abundance. We find that cells downregulate histone transcripts in poor nutrients to avoid toxic histone overexpression, but produce constant amounts of histone proteins through nutrient-specific regulation of translation efficiency. Our findings suggest that this allows cells to balance the need for rapid histone production under fast growth conditions with the tight regulation required to avoid toxic overexpression in poor nutrients.

189 Association of the liver phosphoproteome with divergent dry matter intake in finishing beef steers

Abstract As the need for improving the sustainability of the beef industry increases, optimizing feed efficiency is of the utmost importance. The liver has a central role in nutrient metabolism, and for ruminants particularly, it is essential for providing glucose through gluconeogenesis via substrates such as propionate, lactate, and glycerol. Further, through cell signaling and vagal afferent signals, the liver acts to stimulate and inhibit dry matter intake (DMI). Thus, the objective of this study was to investigate alterations in the phosphoproteome of finishing beef steers in relation to divergent DMI to identify changes in the abundance of signaling proteins. Angus steers [n = 57; initial body weight (BW) = 518 ± 27 kg] underwent a 63-d intake trial utilizing an Insentec Roughage Intake Control System (Hokofarm Group, Emmeloord, The Netherlands). Prior to beginning the trial, liver biopsy samples were collected from all steers. Samples from steers with the greatest and least DMI (n = 8 each) were utilized for phosphoproteomic analysis. Liver protein was isolated and labeled with isobaric tandem mass tagging reagents. Samples were enriched for phosphopeptides using immobilized metal affinity chromatography and subsequently analyzed using high-performance liquid chromatography tandem mass spectrometry. Spectral data were searched in the National Center for Biotechnology Information Bos taurus protein database using MaxQuant (version 2.2.0.0; Max Planck Inst., Munich, Germany), and the data were analyzed using Perseus (version 2.0.10.0; Max Planck Inst.). Two sample t-tests were conducted to determine the difference in phosphopeptide abundance between steers with high and low DMI. A total of 16,365 phosphopeptides were identified, and 287 were differentially expressed (False Discovery Rate &amp;lt; 0.05). In addition, 2,777 phosphopeptides were differentially expressed based on an unadjusted P &amp;lt; 0.05 which were used for enrichment analysis of Gene Ontology processes and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the Database for Annotation, Visualization, and Integrated Discovery. The background for this analysis was all identified phosphopeptides. Gene ontology terms enriched included processes related to phosphorylation activity, intracellular signal transduction, response to insulin, kinase activity, and signaling protein binding. Enriched KEGG pathways included pathways related to insulin signaling, such as AMPK signaling, mTOR signaling, and MAPK signaling. Further, pathways related to glycolysis and gluconeogenesis, glucagon signaling, insulin resistance, carbon metabolism, and various other signaling pathways were also enriched. These results suggest a link between the liver phosphoproteome and DMI, indicating that divergent DMI is associated with alterations in signaling pathways related to nutrient uptake and metabolic processes.