Exploring lipid digestion discrepancies between preterm formula and human milk: Insights from in vitro gastrointestinal digestion and the impact of added milk fat.

Lipids play a pivotal role in the nutrition of preterm infants, acting as a primary energy source. Due to their underdeveloped gastrointestinal systems, lipid malabsorption is common, leading to insufficient energy intake and slowed growth. Therefore, it is critical to explore the reasons behind the low lipid absorption rate in formulas for preterm infants. This study utilized a simulated in intro gastrointestinal digestion model to assess the differences in lipid digestion between preterm human milk and various infant formulas. Results showed that the fatty acid release rates for formulas IF3, IF5, and IF7 were 58.90 %, 56.58 %, and 66.71 %, respectively, lower than human milk's 72.31 %. The primary free fatty acids (FFA) and 2-monoacylglycerol (2-MAG) released during digestion were C14:0, C16:0, C18:0, C18:1n-9, and C18:2n-6, in both human milk and formulas. Notably, the higher release of C16:0 in formulas may disrupt fatty acid balance, impacting lipid absorption. Further investigations are necessary to elucidate lipid absorption differences, which will inform the optimization of lipid content in preterm infant formulas.

Human gut-associated Bifidobacterium species salvage exogenous indole, a uremic toxin precursor, to synthesize indole-3-lactic acid via tryptophan.

Indole in the gut is formed from dietary tryptophan by a bacterial tryptophan-indole lyase. Indole not only triggers biofilm formation and antibiotic resistance in gut microbes but also contributes to the progression of kidney dysfunction after absorption by the intestine and sulfation in the liver. As tryptophan is an essential amino acid for humans, these events seem inevitable. Despite this, we show in a proof-of-concept study that exogenous indole can be converted to an immunomodulatory tryptophan metabolite, indole-3-lactic acid (ILA), by a previously unknown microbial metabolic pathway that involves tryptophan synthase ß subunit and aromatic lactate dehydrogenase. Selected bifidobacterial strains converted exogenous indole to ILA via tryptophan (Trp), which was demonstrated by incubating the bacterial cells in the presence of (2-13C)-labeled indole and l-serine. Disruption of the responsible genes variedly affected the efficiency of indole bioconversion to Trp and ILA, depending on the strains. Database searches against 11,943 bacterial genomes representing 960 human-associated species revealed that the co-occurrence of tryptophan synthase ß subunit and aromatic lactate dehydrogenase is a specific feature of human gut-associated Bifidobacterium species, thus unveiling a new facet of bifidobacteria as probiotics. Indole, which has been assumed to be an end-product of tryptophan metabolism, may thus act as a precursor for the synthesis of a host-interacting metabolite with possible beneficial activities in the complex gut microbial ecosystem.

New methods to unveil host-microbe interaction mechanisms along the microbiota-gut-brain-axis.

Links between the gut microbiota and human health have been supported throughout numerous studies, such as the development of neurological disease disorders. This link is referred to as the "microbiota-gut-brain axis" and is the focus of an emerging field of research. Microbial-derived metabolites and gut and neuro-immunological metabolites regulate this axis in health and many diseases. Indeed, assessing these signals, whether induced by microbial metabolites or neuro-immune mediators, could significantly increase our knowledge of the microbiota-gut-brain axis. However, this will require the development of appropriate techniques and potential models. Methods for studying the induced signals originating from the microbiota remain crucial in this field. This review discusses the methods and techniques available for studies of microbiota-gut-brain interactions. We highlight several much-debated elements of these methodologies, including the widely used in vivo and in vitro models, their implications, and perspectives in the field based on a systematic review of PubMed. Applications of various animal models (zebrafish, mouse, canine, rat, rabbit) to microbiota-gut-brain axis research with practical examples of in vitro methods and innovative approaches to studying gut-brain communications are highlighted. In particular, we extensively discuss the potential of "organ-on-a-chip" devices and their applications in this field. Overall, this review sheds light on the most widely used models and methods, guiding researchers in the rational choice of strategies for studies of microbiota-gut-brain interactions.

Diet-microbiota associations in gastrointestinal research: a systematic review.

Interactions between diet and gastrointestinal microbiota influence health status and outcomes. Evaluating these relationships requires accurate quantification of dietary variables relevant to microbial metabolism, however current dietary assessment methods focus on dietary components relevant to human digestion only. The aim of this study was to synthesize research on foods and nutrients that influence human gut microbiota and thereby identify knowledge gaps to inform dietary assessment advancements toward better understanding of diet-microbiota interactions. Thirty-eight systematic reviews and 106 primary studies reported on human diet-microbiota associations. Dietary factors altering colonic microbiota included dietary patterns, macronutrients, micronutrients, bioactive compounds, and food additives. Reported diet-microbiota associations were dominated by routinely analyzed nutrients, which are absorbed from the small intestine but analyzed for correlation to stool microbiota. Dietary derived microbiota-relevant nutrients are more challenging to quantify and underrepresented in included studies. This evidence synthesis highlights advancements needed, including opportunities for expansion of food composition databases to include microbiota-relevant data, particularly for human intervention studies. These advances in dietary assessment methodology will facilitate translation of microbiota-specific nutrition therapy to practice.

Gut dopamine kick: How gut microbes turn on host receptors to fight pathogens.

Common nutrients in our diet often affect our health through unexpected mechanisms. In a recent issue of Nature, Scott et al. show gut microbes convert dietary tryptophan into metabolites activating intestinal dopamine receptors, which can block attachment of bacterial pathogens to host cells.

DNA flow cytometry for detection of genomic instability as a cancer precursor in the gastrointestinal tract.

One of the earliest applications of flow cytometry was the measurement of DNA content in cells. This method is based on the ability to stain DNA in a stoichiometric manner (i.e., the amount of stain is directly proportional to the amount of DNA within the cell). For more than 40years, a number of studies have consistently demonstrated the utility of DNA flow cytometry as a potential diagnostic and/or prognostic tool in patients with most epithelial tumors, including pre-invasive lesions (such as dysplasia) in the gastrointestinal tract. However, its availability as a clinical test has been limited to few medical centers due to the requirement for fresh tissue in earlier studies and perceived technical demands. However, more recent studies have successfully utilized formalin-fixed paraffin-embedded (FFPE) tissue to generate high-quality DNA content histograms, demonstrating the feasibility of this methodology. This review summarizes step-by-step methods on how to perform DNA flow cytometry using FFPE tissue and analyze DNA content histograms based on the published consensus guidelines in order to assist in the diagnosis and/or risk stratification of many different epithelial tumors, with particular emphasis on dysplasia associated with Barrett's esophagus and inflammatory bowel disease.

Dietary lipid sensing through fatty acid oxidation and chylomicron formation in the gastrointestinal tract of rainbow trout.

In mammals, physiological processes related to lipid metabolism, such as chylomicron synthesis or fatty acid oxidation (FAO), modulate eating, highlighting the importance of energostatic mechanisms in feeding control. This study, using rainbow trout (Oncorhynchus mykiss) as model, aimed to characterize the role of FAO and chylomicron formation as peripheral lipid sensors potentially able to modulate feeding in fish. Fish fed with either a normal- (24%) or high- (32%) fat diet were intraperitoneally injected with water alone or containing etomoxir (inhibitor of FAO rate-limiting enzyme carnitine palmitoyl-transferase 1). First, feed intake levels were recorded. We observed an etomoxir-derived decrease in feeding at short times, but a significant increase at 48 h after treatment in fish fed normal-fat diet. Then, we evaluated putative etomoxir effects on the mRNA abundance of genes related to lipid metabolism, chylomicron synthesis and appetite-regulating peptides. Etomoxir treatment upregulated mRNA levels of genes related to chylomicron assembly in proximal intestine, while opposite effects occurred in distal intestine, indicating a clear regionalization in response. Etomoxir also modulated gastrointestinal hormone mRNAs in proximal intestine, upregulating ghrl in fish fed normal-fat diet and pyy and gcg in fish fed high-fat diet. These results provide evidence for an energostatic control of feeding related to FAO and chylomicron formation at the peripheral level in fish.

Coarse-grained modeling and dynamics tracking of nanoparticles diffusion in human gut mucus.

The gastrointestinal (GI) tract's mucus layer serves as a critical barrier and a mediator in drug nanoparticle delivery. The mucus layer's diverse molecular structures and spatial complexity complicates the mechanistic study of the diffusion dynamics of particulate materials. In response, we developed a bi-component coarse-grained mucus model, specifically tailored for the colorectal cancer environment, that contained the two most abundant glycoproteins in GI mucus: Muc2 and Muc5AC. This model demonstrated the effects of molecular composition and concentration on mucus pore size, a key determinant in the permeability of nanoparticles. Using this computational model, we investigated the diffusion rate of polyethylene glycol (PEG) coated nanoparticles, a widely used muco-penetrating nanoparticle. We validated our model with experimentally characterized mucus pore sizes and the diffusional coefficients of PEG-coated nanoparticles in the mucus collected from cultured human colorectal goblet cells. Machine learning fingerprints were then employed to provide a mechanistic understanding of nanoparticle diffusional behavior. We found that larger nanoparticles tended to be trapped in mucus over longer durations but exhibited more ballistic diffusion over shorter time spans. Through these discoveries, our model provides a promising platform to study pharmacokinetics in the GI mucus layer.

Imaging gastrointestinal damage due to acute mercury poisoning using a mitochondria-targeted dual near-infrared fluorescent probe.

Mercury (Hg) is one of the most widespread pollutants that pose serious threats to public health and the environment. People are inevitably exposed to Hg via different routes, such as respiration, dermal contact, drinking or diet. Hg poisoning could cause gingivitis, inflammation, vomiting and diarrhea, respiratory distress or even death. Especially during the developmental stage, there is considerable harm to the brain development of young children, causing serious symptoms such as intellectual disability and motor impairments, and delayed neural development. Therefore, it's of great significance to develop a specific, quick, practical and labor-saving assay for monitoring Hg2+. Herein, a mitochondria-targeted dual (excitation 700 nm and emission 728 nm) near-infrared (NIR) fluorescent probe JZ-1 was synthesized to detect Hg2+, which is a turn-on fluorescent probe designed based on the rhodamine fluorophore thiolactone, with advantages of swift response, great selectivity, and robust anti-interference capability. Cell fluorescence imaging results showed that JZ-1 could selectively target mitochondria in HeLa cells and monitor exogenous Hg2+. More importantly, JZ-1 has been successfully used to monitor gastrointestinal damage of acute mercury poisoning in a drug-induced mouse model, which provided a great method for sensing Hg species in living subjects, as well as for prenatal diagnosis.

p53 promotes revival stem cells in the regenerating intestine after severe radiation injury.

Ionizing radiation induces cell death in the gastrointestinal (GI) epithelium by activating p53. However, p53 also prevents animal lethality caused by radiation-induced acute GI syndrome. Through single-cell RNA-sequencing of the irradiated mouse small intestine, we find that p53 target genes are specifically enriched in regenerating epithelial cells that undergo fetal-like reversion, including revival stem cells (revSCs) that promote animal survival after severe damage of the GI tract. Accordingly, in mice with p53 deleted specifically in the GI epithelium, ionizing radiation fails to induce fetal-like revSCs. Using intestinal organoids, we show that transient p53 expression is required for the induction of revival stem cells and is controlled by an Mdm2-mediated negative feedback loop. Together, our findings reveal that p53 suppresses severe radiation-induced GI injury by promoting fetal-like reprogramming of irradiated intestinal epithelial cells.

Charting the cellular biogeography in colitis reveals fibroblast trajectories and coordinated spatial remodeling.

Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used multiplexed error-robust fluorescence in situ hybridization (MERFISH) to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations, charted their spatial organization, and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.

Gut microbiota plays a significant role in gout.

With the development of social economy, the incidence of gout is increasing, which is closely related to people's increasingly rich diet. Eating a diet high in purine, fat, sugar and low-fibre for a long time further aggravates gout by affecting uric acid metabolism. The renal metabolism mechanism of uric acid has been thoroughly studied. To find a new treatment method for gout, increasing studies have recently been conducted on the mechanism of intestinal excretion, metabolism and absorption of uric acid. The most important research is the relationship between intestinal microbiota and the risk of gout. Gut microbiota represent bacteria that reside in a host's gastrointestinal tract. The composition of the gut microbiota is associated with protection against pathogen colonization and disease occurrence. This review focuses on how gut microbiota affects gout through uric acid and discusses the types of bacteria that may be involved in the occurrence and progression of gout. We also describe potential therapy for gout by restoring gut microbiota homeostasis and reducing uric acid levels. We hold the perspective that changing intestinal microbiota may become a vital method for effectively preventing or treating gout.

Analysis of Protein Digestion and Absorption Using a Ussing Chamber to Simulate the Environment in the Digestive Tract.

The Ussing chamber is a tool for analyzing drug absorption. We investigated whether the Ussing chamber can be used to analyze the process from digestion to absorption of protein in the gastrointestinal tract. Mixtures containing infant formula, whole cow's milk, processed soy milk, enteral nutrition, or human breast milk, were placed in the apical membrane side equipped with Caco-2 cells. After the addition of first pepsin then pancreatin, samples from the apical and basal membranes were collected. Infant formula showed the highest digestibility and absorption rate. This may be attributed to the presence of whey protein, which is rapidly digested and absorbed. The digestion and absorption of human breast milk showed different results in each donor, suggesting that digestion and absorption may vary among individuals. We concluded that the Ussing chamber can continuously analyze the process from digestion to absorption of proteins in the gastrointestinal tract.

Encapsulation of short-chain bioactive peptides (BAPs) for gastrointestinal delivery: a review.

The majority of known peptides with high bioactivity (BAPs) such as antihypertensive, antidiabetic, antioxidant, hypocholesterolemic, anti-inflammatory and antimicrobial actions, are short-chain sequences of less than ten amino acids. These short-chain BAPs of varying natural and synthetic origin must be bioaccessible to be capable of being adsorbed systemically upon oral administration to show their full range of bioactivity. However, in general, in vitro and in vivo studies have shown that gastrointestinal digestion reduces BAPs bioactivity unless they are protected from degradation by encapsulation. This review gives a critical analysis of short-chain BAP encapsulation and performance with regard to the oral delivery route. In particular, it focuses on short-chain BAPs with antihypertensive and antidiabetic activity and encapsulation methods via nanoparticles and microparticles. Also addressed are the different wall materials used to form these particles and their associated payloads and release kinetics, along with the current challenges and a perspective of the future applications of these systems.

Malondialdehyde levels and bioaccessibility in healthy diet bars: A gastrointestinal digestion model.

Malondialdehyde (MDA), which is composed when n-6 and n-3 PUFAs are peroxidized, has been utilized as an indicator of lipid peroxidation and has been considered neurotoxic, cytotoxic, and mutagenic. This study aimed to determine the bioaccessibility level of MDA in diet bars sold as healthy snacks in the market using in vitro gastrointestinal digestive model. In our study, 28 different diet bar samples were bought from markets in Istanbul. MDA contents of the products were determined by the HPLC-FLD method. The investigation showed that diet bars had an average MDA concentration of 116.25 µg/100 g before digestion, while the average MDA concentration was 90.50 µg/100 g after in vitro digestion. In line with these data, the average MDA bioaccessibility of 28 diet bar samples was calculated as 77.3%. For this reason, more studies are needed to understand the relationship between both the MDA content and the reaction and nutritional components.

In vitro gastrointestinal simulated digestion of three plant proteins: determination of digestion rate, free amino acids and peptide contents.

Cassava protein (CP), barley protein (BP) and yellow pea protein (YPP) are important nutrient and integral constituent of staple in pet foods. It is known that the digestion of proteins directly influences their absorption and utilisation. In the present work, we performed in vitro simulated gastrointestinal digestion of three plant proteins as a staple for dog and cat food. The digestion rate of CP, BP and YPP in dog food was 56.33 ± 0.90%, 48.53 ± 0.91%, and 66.96 ± 0.37%, respectively, whereas the digestion rate of CP, BP, and YPP in cat food was 66.25 ± 0.72%, 43.42 ± 0.83%, and 58.05 ± 0.85%, respectively. Using SDS-polyacrylamide gel electrophoresis to determine the molecular weight (MW) of each protein and the products of their digestion, it was revealed that MW of digestion samples decreased, and MW during the small intestine phase was lower than that during the gastric phase. Peptide sequences of digested products were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and it was found that the total number of peptides in the small intestine digestion samples was higher than that in the gastric phase samples. The MW of peptides obtained from CP was within the range of 1000-1500 Da, while MW of peptides derived from BP and YPP was within the range of 400-2000 Da. In addition, free amino acids were mainly produced in the small intestine phase. Furthermore, the percentage of essential amino acids in the small intestine phase (63 ~ 82%) was higher than that in the gastric phase (37 ~ 63%). Taken together, these findings contribute to the current understanding of the utilisation of plant proteins in dog and cat foods and provide important insights into the selection and application of plant proteins as a staple in dog and cat foods.

Thermal stability and in vitro biological fate of lactoferrin-polysaccharide complexes.

Lactoferrin (LF) is a thermally sensitive iron-binding globular glycoprotein. Heat treatment can induce its denaturation and aggregation and thus affect its functional activity. In this study, carrageenan (CG), xanthan gum (XG) and locust bean gum (LBG), allowed to apply in infant food, were used to form protein-polysaccharide complexes to improve the thermal stability of LF. Meanwhile, in vitro simulated infant digestion and absorption properties of LF were also estimated. The results showed that the complexes formed by CG and XG with LF (LF-CG and LF-XG) could significantly inhibit the loss of α-helix structure of LF against heating. LF-CG and LF-LBG could protect LF from digestion in simulated infant gastric fluid and slow down the degradation of LF under the simulated intestinal conditions. Besides, LF, LF-CG and LF-XG showed no adverse effects on the growth of Caco-2 cells in the LF concentration range of 10-300 µg/mL, and LF-XG exhibited better beneficial to improve the cell uptake of the digestive product than the other protein-polysaccharides at the LF concentration of 100 µg/mL. This study may provide a reference for the enhancement of thermal processing stability of LF and development infant food ingredient with high nutrients absorption efficiency in the gastrointestinal environment in the future.

A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche.

Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.

Clinical Analysis and in Vitro Correlation of BCRP-Mediated Drug-Drug Interaction in the Gastrointestinal Tract.

Breast cancer resistance protein (BCRP) is a drug efflux transporter expressed on the epithelial cells of the small intestine and on the lateral membrane of the bile duct in the liver; and is involved in the efflux of substrate drugs into the gastrointestinal lumen and secretion into bile. Recently, the area under the plasma concentration-time curve (AUC) of rosuvastatin (ROS), a BCRP substrate drug, has been reported to be increased by BCRP inhibitors, and BCRP-mediated drug-drug interaction (DDI) has attracted attention. In this study, we performed a ROS uptake study using human colon cancer-derived Caco-2 cells and confirmed that BCRP inhibitors significantly increased the intracellular accumulation of ROS. The correlation between the cell to medium (C/M) ratio of ROS obtained by the in vitro study and the absorption rate constant (ka) ratio obtained by clinical analysis was examined, and a significant positive correlation was observed. Therefore, it is suggested that the in vitro study using Caco-2 cells could be used to quantitatively estimate BCRP-mediated DDI with ROS in the gastrointestinal tract.

Coordinated ASBT and EGFR Mechanisms for Optimized Liraglutide Nanoformulation Absorption in the GI Tract.

Background: For maintenance therapy in type 2 diabetes, glucagon-like peptide-1 agonist (GLP-1A), which exhibits low cardiovascular risk and high efficacy, is a promising peptide therapeutic. However, developing an oral GLP-1A presents challenges due to the analog's poor cellular permeability and gastrointestinal (GI) stability. Methods: To mitigate such limitations, an oral nanoformulation of liraglutide (LG) was designed and achieved by combining LG with bile acid derivatives using the nanoprecipitation method. This strategy allowed the bile acid moieties to localize at the nanoparticle surface, enhancing the binding affinity for apical sodium-dependent bile acid transporter (ASBT) and improving GI stability. The in vitro characteristics, cellular permeability, and absorption mechanisms of the LG nanoformulation (LG/TD-NF) were thoroughly investigated. Furthermore, the in vivo oral absorption in rats and the glucose-lowering effects in a diabetic (db/db) mouse model were evaluated. Results: The LG/TD-NF produced neutral nanoparticles with a diameter of 58.7 ± 4.3 nm and a zeta potential of 4.9 ± 0.4 mV. Notably, when exposed to simulated gastric fluid, 65.7 ± 3.6% of the LG/TD-NF remained stable over 120 min, while free LG was fully degraded. Relative to unformulated LG, the Caco-2 cellular permeability of the nanoformulation improved, measuring 10.9 ± 2.1 (× 10-6 cm/s). The absorption mechanism prominently featured endocytosis simultaneously mediated by both ASBT and epidermal growth factor receptor (EGFR). The oral bioavailability of the LG/TD-NF was determined to be 3.62% at a dosage of 10 mg/kg, which is 45.3 times greater than that of free LG. In a diabetes model, LG/TD-NF at 10 mg/kg/day exhibited commendable glucose sensitivity and reduced HbA1c levels by 4.13% within 28 days, similar to that of subcutaneously administered LG at a dosage of 0.1 mg/kg/day. Conclusion: The oral LG/TD-NF promotes ASBT/EGFR-mediated transcytosis and assures cellular permeability within the GI tract. This method holds promise for the development of oral GLP-1A peptides as an alternative to injections, potentially enhancing patient adherence to maintenance therapy.