The lipopolysaccharide structure affects the detoxifying ability of intestinal alkaline phosphatases.

Lipopolysaccharide (LPS) is one of the most potent mediators of inflammation. In swine husbandry, weaning is associated with LPS-induced intestinal inflammation, resulting in decreased growth rates due to malabsorption of nutrients by the inflamed gut. A potential strategy to treat LPS-mediated disease is administering intestinal alkaline phosphatase (IAP). The latter can detoxify lipid A, the toxic component of LPS, by removal of phosphate groups. Currently, 183 LPS O-serotypes from E. coli have been described, however, comparative experiments to elucidate functional differences between LPS serotypes are scarce. In addition, these functional differences might affect the efficacy of LPS detoxifying enzymes. Here, we evaluated the ability of four LPS serotypes (O26:B6, O55:B5, O111:B4 and O127:B8) derived from Escherichia coli to trigger the secretion of pro-inflammatory cytokines by porcine PBMCs. We also tested the ability of three commercially available IAPs to detoxify these LPS serotypes. The results show that LPS serotypes differ in their ability to trigger cytokine secretion by immune cells, especially at lower concentrations. Moreover, IAPs displayed a different detoxification efficiency of the tested serotypes. Together, this study sheds light on the impact of LPS structure on the detoxification by IAPs. Further research is however needed to elucidate the LPS serotype-specific effects and their implications for the development of novel treatment options to alleviate LPS-induced gut inflammation in weaned piglets.

The effect of various dietary MUFA content and n-6/n-3 ratios on rainbow trout (Oncorhynchus mykiss) fed on a whole plant protein based diet: growth performance, digestive enzymes activity, intestinal morphometry and fatty acids profile of muscle and liver.

The effect of diets with various dietary MUFA content and n-6/n-3 ratios, diets 1 (55.33, 1.00), 2 (25.30, 1.00), 3 (55.13, 2.05), 4 (24.92, 2.03), 5 (54.94, 8.06) and 6 (24.91, 8.06) and a control diet with fish oil (FO), diet 7 (33.60, 5.97), was studied on growth and digestive physiology of rainbow trout, Oncorhynchus mykiss. Juveniles, 14.65 g ± 0.17, were fed the experimental diets for eight weeks. Those fish fed diet 1 (55.33, 1.00) had the lowest growth and nutritional indices, while fish fed diet 6 (24.91, 8.06) possessed the highest indices. However, fish fed D7 (33.60, 5.97) was intermediate in this regard. Proximate body composition results revealed no significant differences among various experimental groups (P > 0.05). Fish fed on diet 1 possessed the highest alkaline protease activity, while fish raised on diet 2 showed the lowest enzyme activity (P < 0.05). Fish fed on diets 1, 3 and 4 had the highest lipase activity (P > 0.05). Amylase activity and intestinal parameters did not differ among groups (P > 0.05). MUFA contents of liver and muscle reflected the MUFA contents of the diets; however, liver PUFA contents was not affected by dietary PUFA contents (P < 0.05). However, 18:3n-3 contents of liver decreased as dietary n-6/n-3 ratio increased (P < 0.05). Furthermore, the liver 20:4n-6 contents considerably increased as dietary n-6/n-3 increased to 8.06 (P < 0.05). Muscle 22:6n-3 content of groups 1 to 6 was lower than group 7 (P < 0.05). In conclusion, rainbow trout was considerably capable of de novo synthesis of 22:6n-3. Meanwhile, increasing dietary n-6/n-3 ratio to 8.06 decreased the muscle 22:6n-3 content which requires further considerations regarding fish immune competence and nutritional quality for human consumption.

Synthesis and In Silico Analysis of New Polyheterocyclic Molecules Derived from [1,4]-Benzoxazin-3-one and Their Inhibitory Effect against Pancreatic α-Amylase and Intestinal α-Glucosidase.

This study focuses on synthesizing a new series of isoxazolinyl-1,2,3-triazolyl-[1,4]-benzoxazin-3-one derivatives 5a-5o. The synthesis method involves a double 1,3-dipolar cycloaddition reaction following a "click chemistry" approach, starting from the respective [1,4]-benzoxazin-3-ones. Additionally, the study aims to evaluate the antidiabetic potential of these newly synthesized compounds through in silico methods. This synthesis approach allows for the combination of three heterocyclic components: [1,4]-benzoxazin-3-one, 1,2,3-triazole, and isoxazoline, known for their diverse biological activities. The synthesis procedure involved a two-step process. Firstly, a 1,3-dipolar cycloaddition reaction was performed involving the propargylic moiety linked to the [1,4]-benzoxazin-3-one and the allylic azide. Secondly, a second cycloaddition reaction was conducted using the product from the first step, containing the allylic part and an oxime. The synthesized compounds were thoroughly characterized using spectroscopic methods, including 1H NMR, 13C NMR, DEPT-135, and IR. This molecular docking method revealed a promising antidiabetic potential of the synthesized compounds, particularly against two key diabetes-related enzymes: pancreatic α-amylase, with the two synthetic molecules 5a and 5o showing the highest affinity values of 9.2 and 9.1 kcal/mol, respectively, and intestinal α-glucosidase, with the two synthetic molecules 5n and 5e showing the highest affinity values of -9.9 and -9.6 kcal/mol, respectively. Indeed, the synthesized compounds have shown significant potential as antidiabetic agents, as indicated by molecular docking studies against the enzymes α-amylase and α-glucosidase. Additionally, ADME analyses have revealed that all the synthetic compounds examined in our study demonstrate high intestinal absorption, meet Lipinski's criteria, and fall within the required range for oral bioavailability, indicating their potential suitability for oral drug development.

Fucosylation and galactosylation in N-glycans of bovine intestinal alkaline phosphatase and their role in its enzymatic activity.

Bovine intestinal alkaline phosphatase (biALP), a membrane-bound plasma metalloenzyme, maintains intestinal homeostasis, regulates duodenal surface pH, and protects against infections caused by pathogenic bacteria. The N-glycans of biALP regulate its enzymatic activity, protein folding, and thermostability, but their structures are not fully reported. In this study, the structures and quantities of the N-glycans of biALP were analyzed by liquid chromatography-electrospray ionization-high energy collision dissociation-tandem mass spectrometry. In total, 48 N-glycans were identified and quantified, comprising high-mannose [6 N-glycans, 33.1 % (sum of relative quantities of each N-glycan)], hybrid (6, 11.9 %), and complex (36, 55.0 %) structures [bi- (13, 26.1 %), tri- (16, 21.5 %), and tetra-antennary (7, 7.4 %)]. These included bisecting N-acetylglucosamine (33, 56.6 %), mono-to tri-fucosylation (32, 53.3 %), mono-to tri-α-galactosylation (16, 20.7 %), and mono-to tetra-ß-galactosylation (36, 58.5 %). No sialylation was identified. N-glycans with non-bisecting GlcNAc (9, 10.3 %), non-fucosylation (10, 13.6 %), non-α-galactosylation (26, 46.2 %), and non-ß-galactosylation (6, 8.4 %) were also identified. The activity (100 %) of biALP was reduced to 37.3 ± 0.2 % (by de-fucosylation), 32.7 ± 2.9 % (by de-α-galactosylation), and 0.2 ± 0.2 % (by de-ß-galactosylation), comparable to inhibition by 10-4 to 101 mM EDTA, a biALP inhibitor. These results indicate that fucosylated and galactosylated N-glycans, especially ß-galactosylation, affected the activity of biALP. This study is the first to identify 48 diverse N-glycan structures and quantities of bovine as well as human intestinal ALP and to demonstrate the importance of the role of fucosylation and galactosylation for maintaining the activity of biALP.

The Role of Intestinal Cytochrome P450s in Vitamin D Metabolism.

Vitamin D hydroxylation in the liver/kidney results in conversion to its physiologically active form of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 controls gene expression through the nuclear vitamin D receptor (VDR) mainly expressed in intestinal epithelial cells. Cytochrome P450 (CYP) 24A1 is a catabolic enzyme expressed in the kidneys. Interestingly, a recently identified mutation in another CYP enzyme, CYP3A4 (gain-of-function), caused type III vitamin D-dependent rickets. CYP3A are also expressed in the intestine, but their hydroxylation activities towards vitamin D substrates are unknown. We evaluated CYP3A or CYP24A1 activities on vitamin D action in cultured cells. In addition, we examined the expression level and regulation of CYP enzymes in intestines from mice. The expression of CYP3A or CYP24A1 significantly reduced 1,25(OH)2D3-VDRE activity. Moreover, in mice, Cyp24a1 mRNA was significantly induced by 1,25(OH)2D3 in the intestine, but a mature form (approximately 55 kDa protein) was also expressed in mitochondria and induced by 1,25(OH)2D3, and this mitochondrial enzyme appears to hydroxylate 25OHD3 to 24,25(OH)2D3. Thus, CYP3A or CYP24A1 could locally attenuate 25OHD3 or 1,25(OH)2D3 action, and we suggest the small intestine is both a vitamin D target tissue, as well as a newly recognized vitamin D-metabolizing tissue.

Use of human Caco-2 cells and HPAE-PAD for α-glucosidase assay.

To measure α-glucosidase activity, rat intestinal acetone powder is commonly used as a source of α-glucosidase, and the mutarotase-glucose oxidase (GOD) methods commonly used to quantitate glucose produced by enzymatic hydrolysis of the substrates. In this study, we compared human Caco-2 cell extracts with rat intestinal acetone powder extracts. We also compared high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with the mutarotase-GOD method. The sensitivity of HPAE-PAD was higher than that of mutarotase-GOD. The glucose concentration quantified by HPAE-PAD was similar to that quantified using the mutarotase-GOD method. In the maltase reaction, 1-deoxynojirimycin (1-DNJ) exerted a more potent inhibitory effect on human enzymes than on rat enzymes. This order was reversed during the sucrase reaction. These results suggested that the combined use of Caco-2 cell extracts and HPAE-PAD is advantageous for use in α-glucosidase-related basic research.

Uniquely preserved gut contents illuminate trilobite palaeophysiology.

Trilobites are among the most iconic of fossils and formed a prominent component of marine ecosystems during most of their 270-million-year-long history from the early Cambrian period to the end Permian period1. More than 20,000 species have been described to date, with presumed lifestyles ranging from infaunal burrowing to a planktonic life in the water column2. Inferred trophic roles range from detritivores to predators, but all are based on indirect evidence such as body and gut morphology, modes of preservation and attributed feeding traces; no trilobite specimen with internal gut contents has been described3,4. Here we present the complete and fully itemized gut contents of an Ordovician trilobite, Bohemolichas incola, preserved three-dimensionally in a siliceous nodule and visualized by synchrotron microtomography. The tightly packed, almost continuous gut fill comprises partly fragmented calcareous shells indicating high feeding intensity. The lack of dissolution of the shells implies a neutral or alkaline environment along the entire length of the intestine supporting digestive enzymes comparable to those in modern crustaceans or chelicerates. Scavengers burrowing into the trilobite carcase targeted soft tissues below the glabella but avoided the gut, suggesting noxious conditions and possibly ongoing enzymatic activity.

Inhibition of Transglutaminase 2 as a Therapeutic Strategy in Celiac Disease-In Vitro Studies in Intestinal Cells and Duodenal Biopsies.

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 µM, 15 ± 3 vs. 48 ± 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 µM; 12 ± 7% vs. 45 ± 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 µM, 25 ± 13% vs. 22 ± 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 µM. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD.

Effects of Various Marine Toxins on the Mouse Intestine Organoid Model.

Because of their trace existence, exquisite structure and unique role, highly toxic marine biotoxins have always led to the development of natural product identification, structure and function research, chemistry and biosynthesis, and there are still many deficiencies in the injury and protection of highly toxic organisms, toxin biosynthesis, rapid detection, poisoning and diagnosis and treatment. In this study, a mouse intestine organoid (MIO) model was constructed to explore the effects of the marine toxins okadaic acid (OA) and conotoxin (CgTx) on MIO. The results showed that the cell mortality caused by the two toxins at middle and high concentrations was significantly higher than the cell mortality of the control group, the ATPase activity in each group exposed to OA was significantly lower than the ATPase activity of the control group, all the CgTx groups were significantly higher than that of the control group, and the number of apoptotic cells was not significantly higher than the number of apoptotic cells of the control group. Through RNA-Seq differential genes, Gene Ontology (GO) and pathway analysis, and Gene Set Enrichment Analysis (GSEA) experimental results, it was demonstrated that OA reduced cell metabolism and energy production by affecting cell transcription in MIO. Ultimately, cell death resulted. In contrast, CgTx upregulated the intracellular hormone metabolism pathway by affecting the nuclear receptor pathway of MIO, which resulted in cell death and the generation of energy in large amounts.

Different Effects of Lard and Vegetable Blend Oil on Intestinal Microorganisms, Enzyme Activity and Blood Routine in Mice.

The intake of moderate oils and fats is necessary to maintain the body's energy balance, and the fatty acid composition of different oils and fats varies in their nutrition and function. The study aimed to investigate the effects of lard and vegetable blend oil on gut microbiota, intestinal enzyme activities, and blood routine. Kunming mice were assigned to the three groups: (1) Control group (CK) was gavage administration with distilled water, (2) Plant oil group (ZWY) was gavage administration with edible vegetable blend oil, (3) Lard group (DWY) was gavage administration with lard. After 42 days, microbiological, digestive enzymes, and blood routine were performed. Compared with the CK group, Escherichia coli, Lactobacilli, and Bifidobacteria were significantly decreased (p < 0.05), the activities of protease, cellulase, amylase, and xylanase were markedly reduced (p < 0.05), the hemoglobin was significantly increased (p < 0.05) in the ZWY group and DWY groups, and the hematocrit was increased in the ZWY group (p < 0.05), while other routine blood indices were increased (p > 0.05). Compared to the ZWY group, the activity of cellulase and amylase were significantly increased (p < 0.05), the intestinal microorganism and the routine blood indexes had no significant difference in the DWY group. Lard and vegetable blend oil diet affected the composition of the intestinal microorganisms, and the functions of digestive enzymes. Meanwhile, the levels of digestive enzymes may be correlated with the intestinal microbiota.

Microcystin-leucine arginine exposure induced intestinal lipid accumulation and MC-LR efflux disorder in Lithobates catesbeianus tadpoles.

Few studies exist on the toxic effects of chronic exposure to microcystins (MCs) on amphibian intestines, and the toxicity mechanisms are unclear. Here, we evaluated the impact of subchronic exposure (30 days) to environmentally realistic microcystin-leucine arginine (MC-LR) concentrations (0 µg/L, 0.5 µg/L and 2 µg/L) on tadpole (Lithobates catesbeianus) intestines by analyzing the histopathological and subcellular microstructural damage, the antioxidative and oxidative enzyme activities, and the transcriptome levels. Histopathological results showed severe damage accompanied by inflammation to the intestinal tissues as the MC-LR exposure concentration increased from 0.5 µg/L to 2 µg/L. RNA-sequencing analysis identified 634 and 1,147 differentially expressed genes (DEGs) after exposure to 0.5 µg/L and 2 µg/L MC-LR, respectively, compared with those of the control group (0 µg/L). Biosynthesis of unsaturated fatty acids and the peroxisome proliferator-activated receptor (PPAR) signaling pathway were upregulated in the intestinal tissues of the exposed groups, with many lipid droplets being observed on transmission electron microscopy, implying that MC-LR may induce lipid accumulation in frog intestines. Moreover, 2 µg/L of MC-LR exposure inhibited the xenobiotic and toxicant biodegradation related to detoxification, implying that the tadpoles' intestinal detoxification ability was weakened after exposure to 2 µg/L MC-LR, which may aggravate intestinal toxicity. Lipid accumulation and toxin efflux disorder may be caused by MC-LR-induced endoplasmic reticular stress. This study presents new evidence that MC-LR harms amphibians by impairing intestinal lipid metabolism and toxin efflux, providing a theoretical basis for evaluating the health risks of MC-LR to amphibians.

Intestinal UDP-Glucuronosyltransferase 1A1 and Protection against Irinotecan-Induced Toxicity in a Novel UDP-Glucuronosyltransferase 1A1 Tissue-Specific Humanized Mouse Model.

The human UDP-glucuronosyltransferases (UGTs) represent an important family of drug-metabolizing enzymes, with UGT1A1 targeting the conjugation and detoxification of many exogenous substances, including pharmaceutical drugs. In this study we generated humanized UGT1A1 mice expressing the human UGT1A1 gene in either liver (hUGT1A1HEP ) or intestine (hUGT1A1GI ), enabling experiments to examine tissue-specific properties of UGT1A1-specific glucuronidation. Hepatic and intestinal tissue-specific expression and function of UGT1A1 were demonstrated. Although the liver is considered a major organ for detoxification, intestinal UGT1A1 is an important contributor for drug clearance. Mice were challenged with irinotecan (CPT-11), a prodrug hydrolyzed by carboxylesterases to form the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) and detoxified by UGT1A1. Humanized UGT1A1HEP mice that have no intestinal UGT1A1 displayed a greater lethality rate when exposed to CPT-11 than hUGT1A1GI mice. When exposed to a low dose of CPT-11 (10 mg/kg), hUGT1A1HEP mice displayed greater intestinal inflammatory (IL-1ß and IL-6) insult in addition to p53-triggered apoptotic responses. In vitro studies with intestinal crypt organoids exposed to CPT-11 confirmed the results observed in vivo and indicated that CPT-11 impacts stemness, apoptosis, and endoplasmic reticulum (ER) stress in organoids deficient in UGT1A1. When we examined the induction of ER stress in organoids with thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase, apoptosis and the caspase surge that occurred in hUGT1A1HEP mice were blocked in hUGT1A1GI organoids. This study reveals the importance of intestinal UGT1A1 in preventing inflammation, apoptosis, and loss of stemness capacity upon systemic challenge with an important chemotherapeutic agent. SIGNIFICANCE STATEMENT: Hepatic and intestinal UGT1A1 play a key role in the metabolism and detoxification of endogenous and exogenous compounds. The use of tissue-specific humanized models expressing UGT1A1 in liver or intestine has confirmed the relevance of the intestinal tract in the detoxification of irinotecan. Mechanistic studies using intestinal organoids highlighted the importance of UGT1A1 in reducing inflammation, apoptosis, and loss of stemness. These new models provide valuable tools for studying tissue-specific glucuronidation of substances that are metabolized by human UGT1A1.

Effects of Dietary Mannan Oligosaccharides on Non-Specific Immunity, Intestinal Health, and Antibiotic Resistance Genes in Pacific White Shrimp Litopenaeus vannamei.

This study was conducted to comprehensively investigate the beneficial effects of a mannan oligosaccharide product (hereinafter called MOS) on Litopenaeus vannamei and optimum level of MOS. Five isonitrogenous and isolipid diets were formulated by adding 0%, 0.02%, 0.04%, 0.08%, and 0.16% MOS in the basal diet. Each diet was randomly fed to one group with four replicates of shrimp in an 8-week feeding trial. The results showed that dietary MOS improved the growth performance and the ability of digestion of shrimp. Dietary MOS significantly increased the activity of total superoxide dismutase, catalase, and glutathione peroxidase and decreased the content of malondialdehyde in plasma of shrimp. Dietary MOS significantly increased the activity of alkaline phosphatase and lysozyme in plasma and the hemocyte counts. Dietary MOS significantly upregulated the expression of Toll, lysozyme, anti-lipopolysaccharide factor, Crustin, and heat shock protein 70 in the hepatopancreas. And dietary MOS significantly upregulated the expression of intestinal mucin-2, mucin-5B, and mucin-19, while it decreased the expression of intestinal mucin-1 and macrophage migration inhibitory factor. Dietary MOS improved the bacterial diversity; increased the abundance of Lactobacillus, Bifidobacterium, Blautia, and Pseudoalteromonas; and decreased the abundance of Vibrio in the intestine. Shrimp fed MOS diets showed lower mortality after being challenged by Vibrio parahaemolyticus. Notably, this study found a decrease in antibiotic resistance genes and mobile genetic elements after MOS supplementation for the first time. The present results showed that diet with MOS supplementation enhanced the organismal antioxidant capacity and immunity, improved intestinal immunity, optimized intestinal microecology, mitigated the degree of antibiotic resistance, and increased the resistance to V. parahaemolyticus in L. vannamei, especially when supplemented at 0.08% and 0.16%.

RNA-Seq unveiled section-specific host response to lack of gut microbiota in mouse intestine.

To identify host responses induced by commensal microbiota in intestine, transcriptomes of four sections of the intestine were compared between germ-free (GF) mice and conventional (CV) controls using RNA-Seq. Cuffdiff revealed that jejunum had the highest number of differentially expressed genes (over 2000) between CV and GF mice, followed by large intestine (LI), duodenum, and ileum. Gene set association analysis identified section-specific alterations in pathways associated with the absence of commensal microbiota. For example, in GF mice, cytochrome P450 (Cyp)-mediated xenobiotic metabolism was preferably down-regulated in duodenum and ileum, whereas intermediary metabolism pathways such as protein digestion and amino acid metabolism were preferably up-regulated in duodenum, jejunum, and LI. In GF mice, carboxypeptidase A1 (Cpa1), which is important for protein digestion, was the top most up-regulated gene within the entire transcriptome in duodenum (53-fold) and LI (142-fold). Conversely, fatty acid binding protein 6 (Fabp6/Ibabp), which is important for bile acid intestinal reabsorption, was the top most down-regulated gene in jejunum (358-fold), and the drug-metabolizing enzyme Cyp1a1 was the top most down-regulated gene in ileum (40-fold). Section-specific host transcriptomic response to the absence of intestinal microbiota was also observed for other important physiological pathways such as cell junction, the absorption of small molecules, bile acid homeostasis, and immune response. In conclusion, the present study has revealed section-specific host gene transcriptional alterations in GF mice, highlighting the importance of intestinal microbiota in facilitating the physiological and drug responses of the host intestine.

Evidence-Based Guidelines for Drug Interaction Studies: Model-Informed Time Course of Intestinal and Hepatic CYP3A4 Inhibition by Clarithromycin.

Drug-drug interaction (DDI) studies are mandated in drug development; however, protocols for evaluating the impact of cytochrome P450 (CYP) inhibition on new molecular entities are currently inconsistent. This study utilised validated physiologically based pharmacokinetic (PBPK) software to define the optimal dose, frequency, and duration of clarithromycin to achieve optimal characterisation of CYP3A4 inhibition in a study population. The Simcyp® Simulator (Version 19.0) was used to simulate clarithromycin-mediated CYP3A4 inhibition in healthy virtual cohorts. Between trial variability in magnitude and time course of CYP3A4 activity was assessed following clarithromycin dosing strategies obtained from the University of Washington Drug Interaction Database. Heterogeneity in CYP3A4 inhibition was evaluated across sex, race, and age. Literature review identified 500 mg twice daily for 5 days as the most common clarithromycin dosing protocol for CYP3A4 inhibition studies. On simulation, clarithromycin 500 mg twice daily resulted in the largest steady-state inhibition of hepatic (percent mean inhibition [95%CI] = 80 [77-83]) and small intestine (94 [94-95]) CYP3A4 activity (as compared to 500 mg once daily, 400 mg once/twice daily, or 250 mg once/twice daily). Additionally, 500 mg twice daily was associated with the shortest time for 90% of individuals to reach 90% of their minimum hepatic (4 days) and small intestine (1 days) CYP3A4 activity. The study presented herein supports that clarithromycin dosing protocol of 500 mg twice daily for 5 days is sufficient to achieve maximal hepatic and small intestine CYP3A4 inhibition. These findings were consistent between sex, race, and age differences.

Deficiency of AMPKα1 Exacerbates Intestinal Injury and Remote Acute Lung Injury in Mesenteric Ischemia and Reperfusion in Mice.

Mesenteric ischemia and reperfusion (I/R) injury can ensue from a variety of vascular diseases and represents a major cause of morbidity and mortality in intensive care units. It causes an inflammatory response associated with local gut dysfunction and remote organ injury. Adenosine monophosphate-activated protein kinase (AMPK) is a crucial regulator of metabolic homeostasis. The catalytic α1 subunit is highly expressed in the intestine and vascular system. In loss-of-function studies, we investigated the biological role of AMPKα1 in affecting the gastrointestinal barrier function. Male knock-out (KO) mice with a systemic deficiency of AMPKα1 and wild-type (WT) mice were subjected to a 30 min occlusion of the superior mesenteric artery. Four hours after reperfusion, AMPKα1 KO mice exhibited exaggerated histological gut injury and impairment of intestinal permeability associated with marked tissue lipid peroxidation and a lower apical expression of the junction proteins occludin and E-cadherin when compared to WT mice. Lung injury with neutrophil sequestration was higher in AMPKα1 KO mice than WT mice and paralleled with higher plasma levels of syndecan-1, a biomarker of endothelial injury. Thus, the data demonstrate that AMPKα1 is an important requisite for epithelial and endothelial integrity and has a protective role in remote organ injury after acute ischemic events.

Phospholipase Cγ1 represses colorectal cancer growth by inhibiting the Wnt/ß-catenin signaling axis.

As essential phospholipid signaling regulators, phospholipase C (PLC)s are activated by various extracellular ligands and mediate intracellular signal transduction. PLCγ1 is involved in regulating various cancer cell functions. However, the precise in vivo link between PLCγ1 and cancer behavior remains undefined. To investigate the role of PLCγ1 in colorectal carcinogenesis, we generated an intestinal tissue-specific Plcg1 knock out (KO) in adenomatous polyposis coli (Apc) Min/+ mice. Plcg1 deficiency in ApcMin/+ mice showed earlier death, with a higher colorectal tumor incidence in both number and size than in wild-type mice. Mechanistically, inhibition of PLCγ1 increased the levels of its substrate phosphoinositol 4,5-bisphosphate (PIP2) at the plasma membrane and promoted the activation of Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3ß (GSK3ß) to enhance ß-catenin signaling. Enhanced cell proliferation and Wnt/ß-catenin signaling were observed in colon tumors from Plcg1 KO mice. Furthermore, low PLCγ1 expression was associated with a poor prognosis of colon cancer patients. Collectively, we demonstrated the role of PLCγ1 in vivo as a tumor suppressor relationship between the regulation of the PIP2 level and Wnt/ß-catenin-dependent intestinal tumor formation.

Assessment of ameliorative effect of Trigonella foenum-graecum against CuO-NPs induced toxicity in Oreochromis mossambicus.

The current study assessed the ameliorative effect of Trigonella foenum graceum extract against copper oxide nanoparticles (CuO-NPs) induced toxicity in Oreochromis mossambicus. For this purpose 100 healthy fish weighing 20±2.34g were randomly divided into five different groups in duplicates and designated as control (C) no treatment, positive control (G*) treated with 0.12mg/L of CuO-NPs, experimental co-treated groups G1, G2 and G3 were treated with Trigonella foenum-graecum extract @ 18, 26 and 52mg/L along with 0.12 mg/L of CuO-NPs, respectively. In this study significant (P<0.05) changes were observed in the antioxidant activity of enzymes and histological alterations in the liver and intestine of fish in G*, G1 and G2 groups while a good ameliorative response of Trigonella foenum-graecum was observed in G3. Dose dependent alterations in glutathione, lipid peroxides, catalase, and malondialdehyde as well as histological architecture of liver and intestine were observed in treated groups, where more alterations were observed in positive control and low dose treated groups of Trigonella foenum-graecum. Moreover, more ameliorative effect was observed in high dose of Trigonella foenum-graecum treated group (G3). This study is novel as no previous data is available on the amelioration of Trigonella foenum-graecum extract against CuO-NPs induced toxicity in fish.

Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells.

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and ß1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and ß1 subunits, mediated via the Ca-activated PKC pathway.

Stocking density alters growth performance, serum biochemistry, digestive enzymes, immune response, and muscle quality of largemouth bass (Micropterus salmoides) in in-pond raceway system.

The effects of stocking density on growth performance, serum biochemistry, digestive enzymes, immune response, and muscle quality of largemouth bass (Micropterus salmoides) reared in nine in-pond raceway systems (IPRS, 22.0 m × 5.0 m × 2.0 m) were studied. M. salmoides with initial an body weight of 8.25 ± 0.51 g and body length of 6.99 ± 0.44 cm were reared at an initial stocking density of 90.91 ind./m3 (low stocking density, LSD), 113.63 ind./m3 (middle stocking density, MSD), and 136.36 ind./m3 (high stocking density, HSD) with triplication. After 300 days of culture, MSD recorded the highest final body weight, weight gain, specific growth rate, and yield, but the food conversion ratio in MSD was the lowest. The viscerosomatic index in LSD was significantly higher than other groups. The fish serum reared at HSD showed significantly lower total protein, higher total cholesterol, triglyceride, total bilirubin, glucose content, alanine transaminase, and aspartate transaminase activity. Significantly lower intestinal amylase, lipase, trypsin activities, hepatic superoxide dismutase (SOD) and catalase (CAT) activities, and higher malondialdehyde content were detected in HSD compared to others. The content of crude lipid, saturated fatty acid decreased, and total essential amino acid, delicious amino acid, and polyunsaturated fatty acid increased in muscle with stocking density increase. No significant difference was observed in muscle texture. Profitability analysis indicated the benefit-to-cost ratio varied between 1.10 and 1.68, of which MSD was significantly higher than others. The optimal stocking density for M. salmoides should be 113.63 ind./m3 in an IPRS farm.