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.

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.

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.

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.

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.

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.

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.

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.

Coping strategies in response to different levels of elevated water hardness in channel catfish (Ictalurus punctatus): Insight into ion-regulatory and histopathological modulations.

Water hardness above the optimal level can incite toxic effects in fish, which are often species specific. Hence, we aimed at obtaining insights on the potential effects of elevated water hardness as well as coping strategies in channel catfish (Ictalurus punctatus). First, a toxicity assay was performed where the 96 h-LC50 was calculated as 4939 mg/L CaCO3. Thereafter, to gain knowledge on the underlying adaptive strategies to high water hardness, fish were exposed to seven hardness levels (150, 600, 1000, 1500, 2000, 3000 and 4000 mg/L CaCO3 at pH 8.15) for 15 days. Results showed that branchial activities of Ca2+-ATPase and Na+/K+-ATPase, which facilitate Ca2+ uptake, reduced starting respectively from 1000 mg/L and 1500 mg/L CaCO3. Nevertheless, Ca2+ burden in plasma and tissue (gills, liver and intestine) remained elevated. Hardness exposure also disturbed cations (Na+, K+, Mg2+) and minerals (iron and phosphorus) homeostasis in a tissue-specific and dose-dependent manner. Both hemoglobin content and hematocrit dropped significantly at 3000-4000 mg/L CaCO3, with a parallel decline in iron content in plasma and gills. Muscle water content rose dramatically at 4000 mg/L CaCO3, indicating an osmo-regulation disruption. Higher hardness of 3000-4000 mg/L CaCO3 also incited a series of histopathological modifications in gills, liver and intestine; most likely due to excess Ca2+ accumulation. Overall, these data suggest that channel catfish can adapt to a wide range of elevated hardness by modulating Ca2+ regulatory pathways and histomorphological alterations, however, 1500 mg/L CaCO3 and above can impair the performance of this species.

Berberine and/or zinc protect against methotrexate-induced intestinal damage: Role of GSK-3ß/NRF2 and JAK1/STAT-3 signaling pathways.

AIM: The present study was undertaken to elucidate the potential protective mechanism of berberine (BBR) and/or zinc (Zn) against methotrexate (MTX)-induced intestinal injury. METHODS: Five groups of rats were assigned; normal group (received vehicle), MTX group (20 mg/kg; i.p. single dose), and the other three groups received a single daily oral dose of BBR (50 mg/kg), Zn (5 mg/kg), and BBR plus Zn respectively, for 5 days before MTX and 5 days after. RESULTS: Our results emphasized the toxic effect of MTX on rat's intestine as shown by disturbance of oxidant/antioxidant status, down-regulation of NRF2, SIRT1, FOXO-3, Akt, and mTOR expressions, along with up-regulation of GSK-3ß, JAK1, and STAT-3 expressions. Besides, severe intestinal histopathological changes were also observed. On the contrary, BBR and/or Zn produced marked protection against MTX-induced intestinal toxicity via amelioration of oxidative stress, improving NRF2, SIRT1, FOXO-3, GSK-3ß, Akt, mTOR, JAK1, and STAT-3 alterations. Moreover, our treatments significantly restored histopathological abnormalities. Interestingly, combination therapy of BBR plus Zn exhibited higher effectiveness than mono-therapy. SIGNIFICANCE: BBR plus Zn could be used as a novel therapy for the treatment of MTX-induced intestinal damage through modulation of GSK-3ß/NRF2, Akt/mTOR, JAK1/STAT-3, and SIRT1/FOXO-3 signaling pathways.

Contribution of Extrahepatic Aldehyde Oxidase Activity to Human Clearance.

Aldehyde oxidase (AOX) is a soluble, cytosolic enzyme that metabolizes various N-heterocyclic compounds and organic aldehydes. It has wide tissue distribution with highest levels found in liver, kidney, and lung. Human clearance projections of AOX substrates by in vitro assessments in isolated liver fractions (cytosol, S9) and even hepatocytes have been largely underpredictive of clinical outcomes. Various hypotheses have been suggested as to why this is the case. One explanation is that extrahepatic AOX expression contributes measurably to AOX clearance and is at least partially responsible for the often observed underpredictions. Although AOX expression has been confirmed in several extrahepatic tissues, activities therein and potential contribution to overall human clearance have not been thoroughly studied. In this work, the AOX enzyme activity using the S9 fractions of select extrahepatic human tissues (kidney, lung, vasculature, and intestine) were measured using carbazeran as a probe substrate. Measured activities were scaled to a whole-body clearance using best-available parameters and compared with liver S9 fractions. Here, the combined scaled AOX clearance obtained from the kidney, lung, vasculature, and intestine is very low and amounted to <1% of liver. This work suggests that AOX metabolism from extrahepatic sources plays little role in the underprediction of activity in human. One of the notable outcomes of this work has been the first direct demonstration of AOX activity in human vasculature. SIGNIFICANCE STATEMENT: This work demonstrates aldehyde oxidase (AOX) activity is measurable in a variety of extrahepatic human tissues, including vasculature, yet activities and potential contributions to human clearance are relatively low and insignificant when compared with the liver. Additionally, the modeling of the tissue-specific in vitro kinetic data suggests that AOX may be influenced by the tissue it resides in and thus show different affinity, activity, and modified activity over time.

Inhibition of α-glucosidases by tea polyphenols in rat intestinal extract and Caco-2 cells grown on Transwell.

Inhibition of maltase, sucrase, isomaltase and glucoamylase activity by acarbose, epigallocatechin gallate, epicatechin gallate and four polyphenol-rich tea extract from white, green, oolong, black tea, were investigated by using rat intestinal enzymes and human Caco-2 cells. Regarding rat intestinal enzyme mixture, all four tea extracts were very effective in inhibiting maltase and glucoamylase activity, but only white tea extract inhibited sucrase and isomaltase activity and the inhibition was limited. Mixed-type inhibition on rat maltase activity was observed. Tea extracts in combination with acarbose, produced a synergistic inhibitory effect on rat maltase activity. Caco-2 cells experiments were conducted in Transwells. Green tea extract and epigallocatechin gallate show dose-dependent inhibition on human sucrase activity, but no inhibition on rat sucrase activity. The opposite was observed on maltase activity. The results highlighted the different response in the two investigated model systems and show that tea polyphenols are good inhibitors for α-glucosidase activity.

Intestinal proteases profiling from Anticarsia gemmatalis and their binding to inhibitors.

Although the importance of intestinal hydrolases is recognized, there is little information on the intestinal proteome of lepidopterans such as Anticarsia gemmatalis. Thus, we carried out the proteomic analysis of the A. gemmatalis intestine to characterize the proteases by LC/MS. We examined the interactions of proteins identified with protease inhibitors (PI) using molecular docking. We found 54 expressed antigens for intestinal protease, suggesting multiple important isoforms. The hydrolytic arsenal featured allows for a more comprehensive understanding of insect feeding. The docking analysis showed that the soybean PI (SKTI) could bind efficiently with the trypsin sequences and, therefore, insect resistance does not seem to involve changing the sequences of the PI binding site. In addition, a SERPIN was identified and the interaction analysis showed the inhibitor binding site is in contact with the catalytic site of trypsin, possibly acting as a regulator. In addition, this SERPIN and the identified PI sequences can be targets for the control of proteolytic activity in the caterpillar intestine and serve as a support for the rational design of a molecule with greater stability, less prone to cleavage by proteases and viable for the control of insect pests such as A. gemmatalis.

Expression Profiles of Digestive Genes in the Gut and Salivary Glands of Tarnished Plant Bug (Hemiptera: Miridae).

Host plant preference of agricultural pests may shift throughout the growing season, allowing the pests to persist on wild hosts when crops are not available. Lygus Hahn (Hemiptera: Miridae) bugs are severe pests of cotton during flowering and fruiting stages, but can persist on alternative crops, or on weed species. Diversity of digestive enzymes produced by salivary glands and gut tissues play a pivotal role in an organism's ability to utilize various food sources. Polyphagous insects produce an array of enzymes that can process carbohydrates, lipids, and proteins. In this study, the digestive enzyme repertoire of the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois), was identified by high-throughput sequencing followed by cDNA cloning and sequencing. This study identified 87 digestive genes, including 30 polygalacturonases (PG), one ß-galactosidase, three α-glucosidases, six ß-glucosidases, 28 trypsin-like proteases, three serine proteases, one apyrase-like protease, one cysteine protease, 12 lipases, and two transcripts with low similarity to a xylanase A-like genes. RNA-Seq expression profiles of these digestive genes in adult tarnished plant bugs revealed that 57 and 12 genes were differentially expressed in the salivary gland and gut (≥5-fold, P ≤ 0.01), respectively. All polygalacturonase genes, most proteases, and two xylanase-like genes were differentially expressed in salivary glands, while most of the carbohydrate and lipid processing enzymes were differentially expressed in the gut. Seven of the proteases (KF208689, KF208697, KF208698, KF208699, KF208700, KF208701, and KF208702) were not detected in either the gut or salivary glands.