This study evaluated pure ferulic acid (FA) or a FA-rich corn pericarp extract (CPE) dietary supplementation on broiler growth performance and associated physiological and microbial responses. Ross 708 d-old male chicks (n = 300) were randomly assigned to three corn-soybean meal diets: no additive (Control), 100 mg/kg FA, or 600 mg/kg CPE, standardized to provide 100 mg/kg FA equivalents. From 0-21 d, birds were reared in battery cages (5 birds/pen; 20 pens/treatment) with a subset transferred to floor pens (10 birds/pen; 3 pens/treatment) from 22 to 42 d. No overall differences in growth performance were observed from 0 to 21 d; however, differences within wk were detected. At 7 d, CPE increased BW, body weight gain (BWG), and FI compared to FA, while BWG was reduced at 14 d. FA increased BW at 35 and 42 d along with BWG from 22 to 42 d compared to CPE. CPE increased intestinal weights at 42 d and altered duodenal morphology at 21 d, including decreased villus height (VH), increased crypt depth (CD), and reduced VH/CD ratio. At 42 d, both FA and CPE upregulated hepatic GPX1 expression and modulated cytokine gene expression. Age primarily influenced microbial diversity; however, FA and CPE promoted early enrichment of beneficial taxa at 7 d. Overall, age primarily drove physiological and microbial changes; however, FA and CPE differentially influenced growth, intestinal development, hepatic function, and gut microbiota in an age-dependent manner. These findings support potential for FA-based additives to impact broiler performance, with associated effects on gut health and physiology.

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects approximately one-third of the global population and can progress to metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis, increasing the risk of cirrhosis, hepatocellular carcinoma, and mortality. Gut microbes driven by diets high in saturated fat, simple sugar, and cholesterol contribute to disease progression, yet the underlying mechanisms remain undefined. We explored the independent and synergistic effects of dietary saturated fat and cholesterol on MASH development using specific pathogen-free (SPF) and germ-free (GF) mice. We demonstrate that (1) both dietary cholesterol and saturated fat are required to induce fibrosing MASH in SPF mice, whereas GF mice are protected, (2) saturated fat and cholesterol individually alter gut microbial membership, potentially via altered bile acid metabolism, while their combination promotes a distinct composition, including an increase in Parasutterella spp. which correlates with hepatic fibrosis, and (3) diluted cecal contents from SPF, but not GF, mice fed high-fat, high-cholesterol diets are enriched in deoxycholic acid and activate human hepatic stellate cells in vitro, suggesting a mechanistic link between dietary lipid-induced microbiota and liver fibrogenesis. These findings reveal how specific Western dietary components shape the gut microbiota and contribute to hepatic fibrosis via stellate cell activation, offering potential targets for therapeutic interventions against MASLD/MASH.

Many cellular processes are governed by protein-protein interactions that require tight spatial and temporal regulation. Accordingly, it is necessary to understand the dynamics of these interactions to fully comprehend and elucidate cellular processes and pathological disease states. To map de novo protein-protein interactions with time resolution at an organelle-wide scale, we developed a quantitative mass spectrometry method, time-resolved interactome profiling (TRIP). We apply TRIP to elucidate aberrant protein interaction dynamics that lead to the protein misfolding disease congenital hypothyroidism. We deconvolute altered temporal interactions of the thyroid hormone precursor thyroglobulin with pathways implicated in hypothyroidism pathophysiology, such as Hsp70-/90-assisted folding, disulfide/redox processing, and N-glycosylation. Functional siRNA screening identified VCP and TEX264 as key protein degradation components whose inhibition selectively rescues mutant prohormone secretion. Ultimately, our results provide novel insight into the temporal coordination of protein homeostasis, and our TRIP method should find broad applications in investigating protein-folding diseases and cellular processes.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is rapidly increasing in prevalence, impacting over a third of the global population. The advanced form of MASLD, Metabolic dysfunction-associated steatohepatitis (MASH), is on track to become the number one indication for liver transplant. FDA-approved pharmacological agents are limited for MASH, despite over 400 ongoing clinical trials, with only a single drug (resmetirom) currently on the market. This is likely due to the heterogeneous nature of disease pathophysiology, which involves interactions between highly individualized genetic and environmental factors. To apply precision medicine approaches that overcome interpersonal variability, in-depth insights into interactions between genetics, nutrition, and the gut microbiome are needed, given that each have emerged as dynamic contributors to MASLD and MASH pathogenesis. Here, we discuss the associations and molecular underpinnings of several of these factors individually and outline their interactions in the context of both patient-based studies and preclinical animal model systems. Finally, we highlight gaps in knowledge that will require further investigation to aid in successfully implementing precision medicine to prevent and alleviate MASLD and MASH.

SummaryNonalcoholic fatty liver disease (NAFLD) is multifactorial in nature, affecting over a billion people worldwide. The gut microbiome has emerged as an associative factor in NAFLD, yet mechanistic contributions are unclear. Here, we show fast food (FF) diets containing high fat, added cholesterol, and fructose/glucose drinking water differentially impact short- vs. long-term NAFLD severity and progression in conventionally-raised, but not germ-free mice. Correlation and machine learning analyses independently demonstrate FF diets induce early and specific gut microbiota changes that are predictive of NAFLD indicators, with corresponding microbial community instability relative to control-fed mice. Shotgun metagenomics showed FF diets containing high cholesterol elevate fecal pro-inflammatory effectors over time, relating to a reshaping of host hepatic metabolic and inflammatory transcriptomes. FF diet-induced gut dysbiosis precedes onset and is highly predictive of NAFLD outcomes, providing potential insights into microbially-based pathogenesis and therapeutics.HighlightsGerm-free mice are protected from fast-food diet-induced NAFLD.Fast-food diets rapidly shift gut microbiota composition and function.Increasing dietary cholesterol exacerbates hepatic inflammation only in SPF mice.Fast-food diet-induced gut dysbiosis precedes and predicts late-stage NAFLD severity.

Skeletal muscle size is controlled by the balance between protein synthesis and protein degradation. Given the essential role of skeletal muscle in maintaining a high quality of life, understanding the mechanisms that modulate this balance are of critical importance. Previously, we demonstrated that muscle-specific knockout of TRIM28 reduces muscle size and function and in the current study, we discovered that this effect is associated with an increase in protein degradation and a dramatic reduction in the expression of Mettl21c. Importantly, we also determined that overexpression of Mettl21c is sufficient to induce hypertrophy in both control and TRIM28 knockout muscles. Moreover, we developed a simple pulse-chase biorthogonal non-canonical amino acid tagging technique that enabled us to visualize the in vivo rate of protein degradation, and with this technique were able to conclude that the hypertrophic effect of Mettl21c is due, at least in part, to an inhibition of protein degradation.

Summary Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism that ensure optimal energy balance. Yet, the interplay between diet, microbes, and host factors that sustain intestinal oscillations is complex and poorly understood. Here, we report the host C-type lectin antimicrobial peptide Reg3γ works with key ileal microbes to orchestrate these interactions in a bi-directional manner, independent from the intestinal core circadian clock. High fat diet diminishes physiologically relevant microbial oscillators essential for host metabolic homeostasis, resulting in arrhythmic host Reg3γ expression and increased abundance and oscillation of Reg3γ -independent gut microbes. This illustrates a transkingdom co-evolved biological rhythm involving reciprocating, sensor-effector signals between key host and microbial components that ultimately drive metabolism, but are also heavily influenced by diet. Restoring the gut microbiota’s capacity to sense and transduce dietary signals mediated by specific host factors such as Reg3γ could be harnessed to improve metabolic dysfunction.

Protein homeostasis plays a critical role in the regulation of skeletal muscle size, and the maintenance of skeletal muscle size contributes significantly to disease prevention and quality of life. Over the past few decades, it has become widely accepted that skeletal muscle size is controlled by the net balance between the rates of protein synthesis and protein degradation. Despite this assertion, the mechanisms that modulate this balance and lead to changes in muscle size remain incompletely defined. Nevertheless, advancements have been made. For instance, a recent study from our lab revealed that the myofiber-specific loss of a transcriptional intermediary factor named TRIM28 led to a significant reduction in basal myofiber size and attenuated the increase in myofiber size that occurs in response to mechanical overload. Moreover, rigorous follow-up studies indicate that the TRIM28 knockout-induced deficits in myofiber size are not driven by a decrease in the rate of protein synthesis, but that this phenotype is instead associated with elevated levels of canonical markers of protein degradation. Together, these observations led to our central hypothesis that TRIM28 confers its effects on myofiber size via the regulation of protein degradation. Thus, in an effort to gain further insight into this possibility, we performed a deep RNA-sequencing analysis, the results of which led to the identification of Mettl21c and Mettl21e as two genes whose expression were significantly downregulated by the loss of TRIM28. We were intrigued by this discovery because recent reports have implicated Mettl21c and Mettl21e in the regulation of protein degradation, and it has been shown that the loss of these proteins in muscle promotes aberrant protein degradation and reduces muscle size. Combined with our initial observations, it became apparent that Mettl21c and Mettl21e might be important parts of the pathway via which TRIM28 regulates protein degradation to control myofiber size. To test this theory, we generated expression plasmids encoding HA-Mettl21c and HA-Mettl21e, and then used electroporation to demonstrate that the expression of Mettl21c and Mettl21e in myofibers is sufficient to induce a robust hypertrophic response in control animals, and that the hypertrophic effect of Mettl21c, but not Mettl21e, is conserved in muscles lacking TRIM28. Collectively, these findings provide evidence that TRIM28 regulates myofiber size via the regulation of protein degradation, and that Mettl21c, but not Mettl21e, might play an important role in the pathway via which TRIM28 confers this effect.

Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism ensuring optimal energy balance. However, the interplay between diet, microbes, and host factors sustaining intestinal oscillations is complex and poorly understood. Here, using a mouse model, we report the host C-type lectin antimicrobial peptide Reg3γ works with key ileal microbes to orchestrate these interactions in a bidirectional manner and does not correlate with the intestinal core circadian clock. High-fat diet is the primary driver of microbial oscillators that impair host metabolic homeostasis, resulting in arrhythmic host Reg3γ expression that secondarily drives abundance and oscillation of key gut microbes. This illustrates transkingdom coordination of biological rhythms primarily influenced by diet and reciprocal sensor-effector signals between host and microbial components, ultimately driving metabolism. Restoring the gut microbiota’s capacity to sense dietary signals mediated by specific host factors such as Reg3γ could be harnessed to improve metabolic dysfunction.

Genetic missense tolerance ratio (MTR) analysis systematically evaluates all possible segments in a given protein‐encoding transcript found in the human population. This method scores each segment for the number of observed missense variants versus the number of silent mutations in that same segment. An MTR score of 0 indicates that no missense mutations are observed within a given segment. This is indicative of evolutionary purifying selection, which excludes mutations in that segment from the general human population. Here, we conducted MTR analysis on each of the roughly 20,000 protein‐encoding human genes. It was seen that there are 257 genes with at least one 31‐residue encoding segment with MTR = 0 (1.3% of all human genes). The proteins encoded by these 257 genes were tabulated along with information regarding the sequence location of each intolerant segment, the likely function of the protein, and so forth. The most functionally‐enriched family among these proteins is a collection of several dozen proteins that are directly involved in RNA splicing. Some of the other proteins with zero‐tolerance segments have thus far escaped significant characterization. Indeed, while a number of these proteins have previously been genetically linked to human disorders, many have not. We hypothesize that this compendium of human proteins with zero‐tolerance segments can be used to complement disease mutation data as a pointer to genes and proteins that are associated with interesting and underexplored human biology.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are hepatic manifestations of metabolic syndrome and major indications for liver transplantation. Western diet contributes to disease pathogenesis, partially mediated through the gut microbiome, yet mechanisms remain elusive. Human epidemiological studies identified high dietary cholesterol intake as a NAFLD risk factor and it is essential to drive disease in murine models, yet little is known about its role in reshaping gut microbiota. Using the fast food (FF) diet murine model in germ‐free (GF) mice completely devoid of all microbes and their conventionally‐raised (control) counterparts harboring complex microbiomes, we hypothesized high dietary cholesterol‐induced gut microbiota impact NAFLD onset, progression, and severity.

Mechanical signals, such as those evoked by maximal-intensity contractions (MICs), can induce an increase in muscle mass. Rapamycin-sensitive signaling events are widely implicated in the regulation of this process; however, recent studies indicate that rapamycin-insensitive signaling events are also involved. Thus, to identify these events, we generate a map of the MIC-regulated and rapamycin-sensitive phosphoproteome. In total, we quantify more than 10,000 unique phosphorylation sites and find that more than 2,000 of these sites are significantly affected by MICs, but remarkably, only 38 of the MIC-regulated events are mediated through a rapamycin-sensitive mechanism. Further interrogation of the rapamycin-insensitive phosphorylation events identifies the S473 residue on Tripartite Motif-Containing 28 (TRIM28) as one of the most robust MIC-regulated phosphorylation sites, and extensive follow-up studies suggest that TRIM28 significantly contributes to the homeostatic regulation of muscle size and function as well as the hypertrophy that occurs in response to increased mechanical loading.

Many biologically active natural products contain phenol groups that are prone to rapid phase‐II metabolism. In order to better understand the structural basis for these metabolic transformations, we have investigated the in vitro metabolism of rooperol, a bis‐catechol with anti‐cancer activity, and caffeic acid phenethyl amide (CAPA), a synthetic analog of a natural product with antioxidant and cytoprotective activities. In the presence of pig liver microsomes supplemented with UDP‐glucuronic acid, both of these catechols undergo rapid phase‐II metabolism to form mono‐ (CAPA and rooperol) and bis‐ (rooperol) glucuronide metabolites. The identity of these metabolites was determined by HPLC‐MS. We followed the kinetics of these in vitro metabolism reactions using HPLC. In both cases, the disappearance of the compound over time corresponded to a first‐order process with half‐life of 4.0 ± 0.5 min for rooperol and 5.1 ± 0.8 min for CAPA. In the case of rooperol, this data is in agreement with previously published pharmacokinetic (PK) studies in humans, in which only phase‐II metabolites were detected after oral administration, as well as PK studies in baboons which also report rapid phase‐II metabolism following IV administration. While human or non‐human primate PK data for CAPA have not been reported, our data suggest that this compound will also suffer from rapid phase‐II metabolism, and thus more metabolically stable analogs of both rooperol and CAPA are required.Support or Funding InformationThis research was supported by the U.S. Department of Education HSI STEM program (841.031c), Award #P021C160036The Robert and Ella Owens Medical Research Foundation