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Although fat is a crucial source of energy in diets, excessive intake leads to obesity. Fat absorption in the gut is prevailingly thought to occur organ-autonomously by diffusion 1-3 . Whether the process is controlled by the brain-to-gut axis, however, remains largely unknown. Here we demonstrate that the dorsal motor nucleus of vagus (DMV) plays a key part in this process. Inactivation of DMV neurons reduces intestinal fat absorption and consequently causes weight loss, whereas activation of the DMV increases fat absorption and weight gain. Notably, the inactivation of a subpopulation of DMV neurons that project to the jejunum shortens the length of microvilli, thereby reducing fat absorption. Moreover, we identify a natural compound, puerarin, that mimics the suppression of the DMV-vagus pathway, which in turn leads to reduced fat absorption. Photoaffinity chemical methods and cryogenic electron microscopy of the structure of a GABA A receptor-puerarin complex reveal that puerarin binds to an allosteric modulatory site. Notably, conditional Gabra1 knockout in the DMV largely abolishes puerarin-induced intestinal fat loss. In summary, we discover that suppression of the DMV-vagus-jejunum axis controls intestinal fat absorption by shortening the length of microvilli and illustrate the therapeutic potential of puerarin binding to GABRA1 in fat loss., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea , Spodoptera frugiperda , and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development.

Bacillus thuringiensis (Bt) secretes the nutritional insecticidal protein Vip3Aa11, which exhibits high toxicity against the fall armyworm ( Spodoptera frugiperda ). The Bt HD270 extracellular polysaccharide (EPS) enhances the toxicity of Vip3Aa11 protoxin against S. frugiperda by enhancing the attachment of brush border membrane vesicles (BBMVs). However, how EPS-HD270 interacts with Vip3Aa11 protoxin in vivo and the effect of EPS-HD270 on the toxicity of activated Vip3Aa11 toxin are not yet clear. Our results indicated that there is an interaction between mannose, a monosaccharide that composes EPS-HD270, and Vip3Aa11 protoxin, with a dissociation constant of Kd = 16.75 ± 0.95 mmol/L. When EPS-HD270 and Vip3Aa11 protoxin were simultaneously fed to third-instar larvae, laser confocal microscopy observations revealed the co-localization of the two compounds near the midgut wall, which aggravated the damage to BBMVs. EPS-HD270 did not have a synergistic insecticidal effect on the activated Vip3Aa11 protein against S. frugiperda . The activated Vip3Aa11 toxin demonstrated a significantly reduced binding capacity (548.73 ± 82.87 nmol/L) towards EPS-HD270 in comparison to the protoxin (34.96 ± 9.00 nmol/L). Furthermore, this activation diminished the affinity of EPS-HD270 for BBMVs. This study provides important evidence for further elucidating the synergistic insecticidal mechanism between extracellular polysaccharides and Vip3Aa11 protein both in vivo and in vitro.

Saffron ( Crocus sativus L.) is known as the most expensive spice. C. sativus dried red stigmas, called threads, are used for culinary, cosmetic, and medicinal purposes. The rest of the flower is often discarded, but is now being used in teas, as coloring agents, and fodder. Previous studies have attributed antioxidant, anti-inflammatory, hepatoprotective, neuroprotective, anti-depressant, and anticancer properties to C. sativus floral bio-residues. The aim of this study is to assess C. sativus flower water extract (CFWE) for its effects on hemoglobin, brush boarder membrane (BBM) functionality, morphology, intestinal gene expression, and cecal microbiome in vivo ( Gallus gallus ), a clinically validated model. For this, Gallus gallus eggs were divided into six treatment groups (non-injected, 18 Ω H 2 O, 1% CFWE, 2% CFWE, 5% CFWE, and 10% CFWE) with n ~10 for each group. On day 17 of incubation, 1 mL of the extracts/control were administered in the amnion of the eggs. The amniotic fluid along with the administered extracts are orally consumed by the developing embryo over the course of the next few days. On day 21, the hatchlings were euthanized, the blood, duodenum, and cecum were harvested for assessment. The results showed a significant dose-dependent decrease in hemoglobin concentration, villus surface area, goblet cell number, and diameter. Furthermore, we observed a significant increase in Paneth cell number and Mucin 2 (MUC2) gene expression proportional to the increase in CFWE concentration. Additionally, the cecum microbiome analysis revealed C. sativus flower water extract altered the bacterial populations. There was a significant dose-dependent reduction in Lactobacillus and Clostridium sp., suggesting an antibacterial effect of the extract on the gut in the given model. These results suggest that the dietary consumption of C. sativus flower may have negative effects on BBM functionality, morphology, mineral absorption, microbial populations, and iron status.

Postprandial hyperglycemia is an important causative factor of type 2 diabetes mellitus, and permanent localization of intestinal GLUT2 in the brush border membrane is an important reason of postprandial hyperglycemia. Berberine, a small molecule derived from Coptidis rhizome, has been found to be potent at lowering blood glucose, but how berberine lowers postprandial blood glucose is still elusive. Here, we investigated the effect of berberine on intestinal glucose transporter 2 (GLUT2) translocation and intestinal glucose absorption in type 2 diabetes mouse model. Type 2 diabetes was induced by feeding of a high-fat diet and injection of streptozotocin and diabetic mice were treated with berberine for 6 weeks. The effects of berberine on intestinal glucose transport and GLUT2 translocation were accessed in isolated intestines and intestinal epithelial cells (IEC-6), respectively. We found that berberine treatment improved glucose tolerance and systemic insulin sensitivity in diabetic mice. Furthermore, berberine decreased intestinal glucose transport and inhibited GLUT2 translocation from cytoplasm to brush border membrane in intestinal epithelial cells. Mechanistically, berberine inhibited intestinal insulin-like growth factor 1 (IGF-1R) phosphorylation and thus reduced localization of PLC-β2 in the membrane, leading to decreased GLUT2 translocation. These results suggest that berberine reduces intestinal glucose absorption through inhibiting IGF-1R-PLC-β2-GLUT2 signal pathway.

Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis). In this study, we have set up the conditions to analyze the specific binding of 125 I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with 125 I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using 125 I-DI-III. In addition, the truncated protein 125 I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement in vivo. IMPORTANCE This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two Spodoptera spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained in vivo (bioassays against larvae), domains IV and V are not necessary for ex vivo toxicity to Sf21 cells.

Mechanical stiffness is closely related to cell adhesion and rounding in some cells. In leukocytes, dephosphorylation of ezrin/radixin/moesin (ERM) proteins is linked to cell adhesion events. To elucidate the relationship between surface stiffness, cell adhesion, and ERM dephosphorylation in leukocytes, we examined the relationship in the myelogenous leukemia line, KG-1, by treatment with modulation drugs. KG-1 cells have ring-shaped cortical actin with microvilli as the only F-actin cytoskeleton, and the actin structure constructs the mechanical stiffness of the cells. Phorbol 12-myristate 13-acetate and staurosporine, which induced cell adhesion to fibronectin surface and ERM dephosphorylation, caused a decrease in surface stiffness in KG-1 cells. Calyculin A, which inhibited ERM dephosphorylation and had no effect on cell adhesion, did not affect surface stiffness. To clarify whether decreasing cell surface stiffness and inducing cell adhesion are equivalent, we examined KG-1 cell adhesion by treatment with actin-attenuated cell softening reagents. Cytochalasin D clearly diminished cell adhesion, and high concentrations of Y27632 slightly induced cell adhesion. Only Y27632 slightly decreased ERM phosphorylation in KG-1 cells. Thus, decreasing cell surface stiffness and inducing cell adhesion are not equivalent, but these phenomena are coordinately regulated by ERM dephosphorylation in KG-1 cells., (© 2021. Japan Human Cell Society.)

Bacillus thuringiensis (Bt) are entomopathogenic bacteria that produce different kinds of insecticidal proteins. However, studies on Bt exopolysaccharides are lacking. Here, we aimed to explore the characteristics and insecticidal synergism of EPS, an exopolysaccharide from Bt strain 4D19. The molecular weight of EPS-2 was 58.0 kDa, which consisted of mannose (44.2%), GlcN (35.5%), D-GalN (8.0%), glucose (5.5%), arabinose (5.1%), galactose (0.9%), Man-UA (0.3%) and Glc-UA (0.2%). The toxicity of insecticidal proteins against Plutella xylostella was increased by adding EPS. EPS-2 bound to Cry1Ac protoxin and promoted the binding of Cry1Ac protoxin to the gut membrane of P. xylostella, but did not bind to activated toxins. These results suggested that EPS-2 may bind to the protoxin C-terminal region to enhance insecticidal activity. Our findings indicated that Bt strains produce exopolysaccharide to enhance the toxicity of insecticidal crystal proteins, which could be applied in biopesticide research and product development., (Copyright © 2021. Published by Elsevier B.V.)

Oxidative stress (OS) is a key factor in the development of gastrointestinal disorders, in which the intestinal barrier is altered. However, the Multidrug resistance-associated protein 2 (Mrp2) status, an essential component of the intestinal transcellular barrier exhibiting pharmaco-toxicological relevance by limiting the orally ingested toxicants and drugs absorption, has not been investigated. We here evaluated the short-term effect of OS on Mrp2 by treatment of isolated rat intestinal sacs with tert-butyl hydroperoxide (TBH) for 30 min. OS induction by TBH (250 and 500 μM) was confirmed by increased lipid peroxidation end products, decreased reduced glutathione (GSH) content and altered antioxidant enzyme activities. Under this condition, assessment of Mrp2 distribution between brush border (BBM) and intracellular (IM) membrane fractions, showed that Mrp2 protein decreased in BBM and increased in IM, consistent with an internalization process. This was associated with decreased efflux activity and, consequently, impaired barrier function. Subsequent incubation with N-Acetyl-L-Cysteine (NAC, 1 mM) reestablished GSH content and reverted concomitantly the alteration in Mrp2 localization and function induced by TBH. Cotreatment with a specific inhibitor of classic calcium-dependent Protein Kinase C (cPKC) implicated this kinase in TBH-effects. In conclusion, we demonstrated a negative posttranslational regulation of rat intestinal Mrp2 after short-term exposition to OS, a process likely mediated by cPKC and dependent on intracellular GSH content. The concomitant impairment of the Mrp2 barrier function may have implications in xenobiotic absorption and toxicity in a variety of human diseases linked to OS, with notable consequences on the toxicity/safety of therapeutic agents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The intra-amniotic administration approach has been used to evaluate the effects of plant origin prebiotics on intestinal health and on brush border membrane functionality and morphology. Prebiotics are fermentable dietary fibers, which can positively affect the host by selectively stimulating the growth and activity of colon bacteria, thus improving intestinal health. The consumption of prebiotics increases digestive tract motility, which leads to hyperplasia and/or hypertrophy of intestinal cells, increasing nutrient digestive and absorptive surface area. This review collates information about the effects and relationship between prebiotic consumption on small intestinal brush border membrane functionality and morphology by utilizing the intra-amniotic administration approach. To date, research has shown that the intra-amniotic administration of prebiotics affects the expression of key brush border membrane functional proteins, intestinal surface area (villi height/width), and goblet cell number/size. These effects may improve brush border membrane functionality and digestive/absorptive capabilities.

Intestinal health relies on the association between the mucosal immune system, intestinal barrier and gut microbiota. Bioactive components that affect the gut microbiota composition, epithelial physical barrier and intestinal morphology were previously studied. The current systematic review evaluated evidence of anthocyanin effects and the ability to improve gut microbiota composition, their metabolites and parameters of the physical barrier; this was conducted in order to answer the question: "Does food source or extract of anthocyanin promote changes on intestinal parameters?". The data analysis was conducted following the PRISMA guidelines with the search performed at PubMed, Cochrane and Scopus databases for experimental studies, and the risk of bias was assessed by the SYRCLE tool. Twenty-seven studies performed in animal models were included, and evaluated for limitations in heterogeneity, methodologies, absence of information regarding allocation process and investigators' blinding. The data were analyzed, and the anthocyanin supplementation demonstrated positive effects on intestinal health. The main results identified were an increase of Bacteroidetes and a decrease of Firmicutes , an increase of short chain fatty acids production, a decrease of intestinal pH and intestinal permeability, an increase of the number of goblet cells and tight junction proteins and villi improvement in length or height. Thus, the anthocyanin supplementation has a potential effect to improve the intestinal health. PROSPERO (CRD42020204835).

In Inflammatory Bowel Disease (IBD), malabsorption of electrolytes (NaCl) results in diarrhea. Inhibition of coupled NaCl absorption, mediated by the dual operation of Na:H and Cl:HCO 3 exchangers on the brush border membrane (BBM) of the intestinal villus cells has been reported in IBD. In the SAMP1/YitFcs (SAMP1) mice model of spontaneous ileitis, representing Crohn's disease, DRA (Downregulated in Adenoma) mediated Cl:HCO 3 exchange was shown to be inhibited secondary to diminished affinity of the exchanger for Cl. However, NHE3 mediated Na:H exchange remained unaffected. Mast cells and their secreted mediators are known to be increased in the IBD mucosa and can affect intestinal electrolyte absorption. However, how mast cell mediators may regulate Cl:HCO 3 exchange in SAMP1 mice is unknown. Therefore, the aim of this study was to determine the effect of mast cell mediators on the downregulation of DRA in SAMP1 mice. Mast cell numbers and their degranulation marker enzyme (β-hexosaminidase) levels were significantly increased in SAMP1 mice compared to control AKR mice. However, treatment of SAMP1 mice with a mast cell stabilizer, ketotifen, restored the β-hexosaminidase enzyme levels to normal in the intestine, demonstrating stabilization of mast cells by ketotifen. Moreover, downregulation of Cl:HCO 3 exchange activity was restored in ketotifen treated SAMP1 mice. Kinetic studies showed that ketotifen restored the altered affinity of Cl:HCO 3 exchange in SAMP1 mice villus cells thus reinstating its activity to normal. Further, RT-qPCR, Western blot and immunofluorescence studies showed that the expression levels of DRA mRNA and BBM protein, respectively remained unaltered in all experimental conditions, supporting the kinetic data. Thus, inhibition of Cl:HCO 3 exchange resulting in chloride malabsorption leading to diarrhea in IBD is likely mediated by mast cell mediators.

Exposure to total body irradiation (TBI) causes dose- and tissue-specific lethality. However, there are few effective and nontoxic radiation countermeasures for the radiation injury. In the current study, mice were pretreated with a traditional antimicrobial agent, FZD, before TBI; the protective effects of FZD on radiation injury were evaluated by using parameters such as the spleen index and thymus index, immunohistochemical staining of intestinal tissue, and frequency of micronuclei in polychromatophilic erythrocytes of bone marrow. The intestinal epithelial cell line IEC-6 was used to investigate the underlying mechanisms. Our results indicated that FZD administration significantly improved the survival of lethal dose-irradiated mice, decreased the number of micronuclei, upregulated the number of leukocytes and immune organ indices, and restored intestinal integrity in mice after TBI. TUNEL and western blot showed that FZD protected intestinal tissue by downregulating radiation-induced apoptosis and autophagy. Meanwhile, FZD protected IEC-6 cells from radiation-induced cell death by inhibiting apoptosis and autophagy. To sum up, FZD protected against radiation-induced cell death both in vitro and in vivo through antiapoptosis and antiautophagy mechanisms., Competing Interests: The authors declare no competing interests., (Copyright © 2021 Shumei Ma et al.)

Food grade titanium dioxide (TiO 2 ) containing nanofractions, is commonly applied to whiten and brighten food products, which put consumers under health risks of ingesting TiO 2 nanoparticles (NPs). Although the oral toxicity of TiO 2 -NPs has been evaluated in several studies, gaps in knowledge exist regarding interactions between NPs and food components. Therefore, this study aimed to estimate the influence of TiO 2 -NPs on nutrient absorption and metabolism through an in situ intestinal loop experiment which conducted on adult Sprague Dawley (SD) rats after 30-d gastrointestinal exposure to TiO 2 -NPs of two different sizes (N-TiO 2 and M-TiO 2 ). Results showed that exposure to TiO 2 -NPs caused flat apical membranes with sparse and short microvilli and inflammatory infiltration in small intestine. Both particles were absorbed into small intestinal cells, but N-TiO 2 with smaller size could more easily be transported through gut and raise the blood titanium (Ti) levels. Changes in serum levels of amino acid were also different after exposure to these two particles. After injecting mixed solution of nutrients into in situ intestinal loop, the N-TiO 2 exposure groups displayed significant absorption inhibition of the added histidine (His) and metabolism disorder of some non-added amino acid. However, no influence was observed on metal elements or glucose levels. This study identified TiO 2 -NPs with small sizes could affect nutrient absorption and metabolism by inducing intestinal epithelium injury, and amino acids were more susceptible than metal elements and glucose. These findings suggested that foods supplemented with TiO 2 -NPs should be carefully consumed by people with high protein requirements, such as children, the elderly, and patients with high metabolic disease or intestinal inflammation.

The farnesoid X receptor (FXR), as a bile acid (BA) sensor, plays an important role in the regulation of lipid metabolism. However, the effects and underlying molecular mechanisms of FXR on intestinal glucose homeostasis remain elusive. Herein, we demonstrated that FXR and glucose transporter 2 (GLUT2) are essential for BA-mediated glucose homeostasis in the intestine. BA-activated FXR enhanced glucose uptake in intestinal epithelial cells by increasing the expression of GLUT2, which depended on ERK1/2 phosphorylation via S1PR2. However, it also reduced the cell energy generation via inhibition of oxidative phosphorylation, which is crucial for intestinal glucose transport. Moreover, BA-activated FXR signalling potently inhibited specific glucose flux through the intestinal epithelium to the circulation, which reduced the increase in blood glucose levels in mice following oral glucose administration. This trend was supported by the changed ratio of GLUT2 to SGLT1 in the brush border membrane (BBM), including especially decreased GLUT2 abundance in the BBM. Furthermore, impaired intestinal FXR signalling was observed in the patients with intestinal bile acid deficiency (IBAD). These findings uncover a novel function by which FXR sustains the intestinal glucose homeostasis and provide a rationale for FXR agonists in the treatment of IBAD-related hyperglycaemia., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.

In age-related macular degeneration (AMD), both systemic and local zinc levels decline. Elevation of zinc in clinical studies delayed the progression to end-stage AMD. However, the molecular pathways underpinning this beneficial effect are not yet identified. In this study, we used differentiated primary human fetal retinal pigment epithelium (RPE) cultures and long-term zinc supplementation to carry out a combined transcriptome, proteome and secretome analysis from three genetically different human donors. After combining significant differences, we identified the complex molecular networks using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA). The cell cultures from the three donors showed extensive pigmentation, development of microvilli and basal infoldings and responded to zinc supplementation with an increase in transepithelial electrical resistance (TEER) (apical supplementation: 443.2 ± 79.3%, basal supplementation: 424.9 ± 116.8%, compared to control: 317.5 ± 98.2%). Significant changes were observed in the expression of 1044 genes, 151 cellular proteins and 124 secreted proteins. Gene set enrichment analysis revealed changes in specific molecular pathways related to cell adhesion/polarity, extracellular matrix organization, protein processing/transport, and oxidative stress response by zinc and identified a key upstream regulator effect similar to that of TGFB1.

The consumption of teff ( Eragrostis tef ), a gluten-free cereal grain, has increased due to its dense nutrient composition including complex carbohydrates, unsaturated fatty acids, trace minerals (especially Fe), and phytochemicals. This study utilized the clinically-validated Gallus gallus intra amniotic feeding model to assess the effects of intra-amniotic administration of teff extracts versus controls using seven groups: (1) non-injected; (2) 18Ω H 2 O injected; (3) 5% inulin; (4) teff extract 1%; (5) teff extract 2.5%; (6) teff extract 5%; and (7) teff extract 7.5%. The treatment groups were compared to each other and to controls. Our data demonstrated a significant improvement in hepatic iron (Fe) and zinc (Zn) concentration and LA:DGLA ratio without concomitant serum concentration changes, up-regulation of various Fe and Zn brush border membrane proteins, and beneficial morphological changes to duodenal villi and goblet cells. No significant taxonomic alterations were observed using 16S rRNA sequencing of the cecal microbiota. Several important bacterial metabolic pathways were differentially enriched in the teff group, likely due to teff's high relative fiber concentration, demonstrating an important bacterial-host interaction that contributed to improvements in the physiological status of Fe and Zn. Therefore, teff appeared to represent a promising staple food crop and should be further evaluated.

Background: Insufflation with CO 2 can employ continuous flow, recirculated gas and/or additional warming and humidification. The ability to compare these modes of delivery depends upon the assays employed and opportunities to minimize subject variation. The use of pigs to train colorectal surgeons provided an opportunity to compare three modes of CO 2 delivery under controlled circumstances., Methods: Sixteen pigs were subjected to rectal resection, insufflated with dry-cold CO 2 (DC-CO 2 ) (n = 5), recirculated CO 2 by an AirSeal device (n = 5) and humidification and warming (HW-CO2) by a HumiGard device (n = 6). Peritoneal biopsies were harvested from the same region of the peritoneum for fixation for immunohistochemistry for hypoxia-inducible factor 1 alpha (HIF-1α) and scanning electron microscopy (SEM) to evaluate hypoxia induction or tissue/cellular damage, respectively., Results: DC-CO 2 insufflation by both modes leads to significant damage to mesothelial cells as measured by cellular bulging and retraction as well as microvillus shortening compared with HW-CO 2 at 1 to 1.5 h. DC-CO 2 also leads to a rapid and significant induction of HIF-1α compared with HW-CO 2 ., Conclusions: DC-CO 2 insufflation induces substantive cellular damage and hypoxia responses within the first hour of application. The use of HW-CO 2 insufflation ameliorates these processes for the first one to one and half hours in a large mammal used to replicate surgery in humans.

Background: An integral intestinal barrier is essential for intestinal homeostasis. Yet, as a side effect of cancer treatment, chemotherapeutic drugs have been reported to cause mucositis. In a recent study, we found that alginate oligosaccharides (AOS) prevent busulfan induced intestinal mucositis. However, it is not known if AOS improves small intestine epithelial cell integrity and migration, which are two essential processes for maintaining the mechanical barrier function of the small intestine. In the current investigation, we aimed to explore the effects of AOS on the integrity and migration of small intestine cells using swine intestinal epithelial IPEC-J2 cells., Methods: Cell integrity was determined using the TEER assay. Cell migration capability was detected using a wound healing experiment. Small interfering RNA (siRNA) was used to inhibit mannose receptor (MR) expression. Western blotting and immunofluorescence staining were used to determine protein expression., Results: Increasing levels of AOS improved cell integrity as measure by TEER. At the same time, AOS improved IPEC-J2 cell migration capacity as shown in the wound closure assay. It is interesting to note that AOS increased the expression of intestinal microvillus proteins and junction proteins to benefit cell integrity. MR siRNA blocked the action of AOS on cell integrity and cell migration and inhibited the expression of microvillus and cell junction proteins., Conclusion: We identified the underlying mechanisms by which AOS improved small intestinal mucositis. As a novel, natural food additive, AOS may be administered to prevent intestinal mucositis induced by chemotherapy or other issues., Competing Interests: Declaration of competing interest All authors disclosed no financial relationships relevant to this publication., (Copyright © 2020. Published by Elsevier Inc.)

Toosendanin (TSN), which is extracted from the root bark of Melia toosendan Siebold and Zuccarini, has multiple modes of action against insects. Especially, this compound has a potent stomach poisoning activity against several lepidoptera pests. In this paper, the signs of toxicity, digestive enzymes activity, the histopathological changes and immuno-electron microscopic localization of TSN in the midgut epithelium of Mythimna separate Walker larvae were investigated for better understanding its action mechanism against insects. The bioassay results indicated that TSN has strong stomach poisoning against the fifth-instar larvae of M. separata (LC 50  = 252.23 μg/mL). The typical poisoned symptom were regurgitation and paralysis. Activities of digestive enzymes had no obvious changes after treatment with LC 80 dose of TSN. The midgut epithelial cells of insect were damaged by TSN, showing the degeneration of microvilli, hyperplasia of smooth endoplasmic reticulum and condensation of chromatin. Immunohistochemical analysis revealed that the gold particles existed on the microvilli of columnar cells and goblet cells, and gradually accumulated with the exacerbation of poisoning symptoms, showing that TSN targets on the microvilli of the midgutcells. Therefore, TSN acts on digestive system and locates in the microvilli of midgutcells of M. separata., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest about this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Microvilli are finger-like membrane protrusions, supported by the actin cytoskeleton, and found on almost all cell types. A growing body of evidence suggests that the dynamic lymphocyte microvilli, with their highly curved membranes, play an important role in signal transduction leading to immune responses. Nevertheless, challenges in modulating local membrane curvature and monitoring the high dynamicity of microvilli hampered the investigation of the curvature-generation mechanism and its functional consequences in signaling. These technical barriers have been partially overcome by recent advancements in adapted super-resolution microscopy. Here, we review the up-to-date progress in understanding the mechanisms and functional consequences of microvillus formation in T cell signaling. We discuss how the deformation of local membranes could potentially affect the organization of signaling proteins and their biochemical activities. We propose that curved membranes, together with the underlying cytoskeleton, shape microvilli into a unique compartment that sense and process signals leading to lymphocyte activation., (Copyright © 2020 Orbach and Su.)

The inclusion of pulses in traditional wheat-based food products is increasing as the food industry and consumers are recognizing the nutritional benefits due to the high protein, antioxidant activity, and good source of dietary fiber of pulses. Iron deficiency is a significant global health challenge, affecting approximately 30% of the world's population. Dietary iron deficiency is the foremost cause of anemia, a condition that harms cognitive development and increases maternal and infant mortality. This study intended to demonstrate the potential efficacy of low-phytate biofortified pea varieties on dietary iron (Fe) bioavailability, as well as on intestinal microbiome, energetic status, and brush border membrane (BBM) functionality in vivo ( Gallus gallus ). We hypothesized that the low-phytate biofortified peas would significantly improve Fe bioavailability, BBM functionality, and the prevalence of beneficial bacterial populations. A six-week efficacy feeding ( n = 12) was conducted to compare four low-phytate biofortified pea diets with control pea diet (CDC Bronco), as well as a no-pea diet. During the feeding trial, hemoglobin (Hb), body-Hb Fe, feed intake, and body weight were monitored. Upon the completion of the study, hepatic Fe and ferritin, pectoral glycogen, duodenal gene expression, and cecum bacterial population analyses were conducted. The results indicated that certain low-phytate pea varieties provided greater Fe bioavailability and moderately improved Fe status, while they also had significant effects on gut microbiota and duodenal brush border membrane functionality. Our findings provide further evidence that the low-phytate pea varieties appear to improve Fe physiological status and gut microbiota in vivo, and they highlight the likelihood that this strategy can further improve the efficacy and safety of the crop biofortification and mineral bioavailability approach.

Epithelial ovarian cancer (EOC) metastasis occurs by exfoliation of cells and multicellular aggregates (MCAs) from the tumor into the peritoneal cavity, adhesion to and retraction of peritoneal mesothelial cells and subsequent anchoring. Elevated levels of lysophosphatidic acid (LPA) have been linked to aberrant cell proliferation, oncogenesis, and metastasis. LPA disrupts junctional integrity and epithelial cohesion in vitro however, the fate of free-floating cells/MCAs and the response of host peritoneal tissues to LPA remain unclear. EOC MCAs displayed significant LPA-induced changes in surface ultrastructure with the loss of cell surface protrusions and poor aggregation, resulting in increased dissemination of small clusters compared to untreated control MCAs. LPA also diminished the adhesive capacity of EOC single cells and MCAs to murine peritoneal explants and impaired MCA survival and mesothelial clearance competence. Peritoneal tissues from healthy mice injected with LPA exhibited enhanced mesothelial surface microvilli. Ultrastructural alterations were associated with restricted peritoneal susceptibility to metastatic colonization by single cells as well as epithelial-type MCAs. The functional consequence is an LPA-induced dissemination of small mesenchymal-type clusters, promoting a miliary mode of peritoneal seeding that complicates surgical removal and is associated with worse prognosis.

Adaptive regulation of epithelial transporters to nutrient intake is essential to decrease energy costs of their synthesis and maintenance, however such regulation is understudied. Previously we demonstrated that the transport function of the basolateral amino acid uniporter LAT4 (Slc43a2) is increased by dephosphorylation of serine 274 (S274) and nearly abolished by dephosphorylation of serine 297 (S297) when expressed in Xenopus oocytes. Phosphorylation changes in the jejunum of food-entrained mice suggested an increase in LAT4 transport function during food expectation. Thus, we investigated further how phosphorylation, expression and localization of mouse intestinal LAT4 respond to food-entrained diurnal rhythm and dietary protein content. In mice entrained with 18% protein diet, LAT4 mRNA was not submitted to diurnal regulation, unlike mRNAs of luminal symporters and antiporters. Only in duodenum, LAT4 protein expression increased during food intake. Concurrently, S274 phosphorylation was decreased in all three small intestinal segments, whereas S297 phosphorylation was increased only in jejunum. Interestingly, during food intake, S274 phosphorylation was nearly absent in ileum and accompanied by strong phosphorylation of mTORC1 target S6. Entraining mice with 8% protein diet provoked a shift in jejunal LAT4 localization from the cell surface to intracellular stores and increased S274 phosphorylation in both jejunum and ileum during food anticipation, suggesting decreased transport function. In contrast, 40% dietary protein content led to increased LAT4 expression in jejunum and its internalization in ileum. Ex vivo treatments of isolated intestinal villi fraction demonstrated that S274 phosphorylation was stimulated by protein kinase A. Rapamycin-sensitive insulin treatment and amino acids increased S297 phosphorylation, suggesting that the response to food intake might be regulated via the insulin-mTORC1 pathway. Ghrelin, an oscillating orexigenic hormone, did not affect phosphorylation of intestinal LAT4. Overall, we show that phosphorylation, expression and localization of intestinal mouse LAT4 responds to diurnal and dietary stimuli in location-specific manner., Competing Interests: The authors have declared that no competing interests exist.

Background: Chronic alcohol use often leads to malnutrition. However, how the intestinal absorption of nutrients such as glucose may be affected during moderate ethanol use has not been investigated. Glucose is absorbed via sodium (Na)-dependent glucose co-transport (SGLT1; SLC5A1) along the brush border membrane (BBM) of intestinal absorptive villus cells., Objective: The aim of this study was to investigate how moderate alcohol consumption affects the absorption of glucose via SGLT1., Methods: Intestinal epithelial cells (IEC-18; rat) were exposed to 8.64 mM ethanol over 1, 3, 6, and 12 h. Rats (16-wk-old, male, Sprague-Dawley) were administered 2 g/kg ethanol over 1, 3, and 6 h. Na-dependent 3H-O-methyl-d-glucose uptake was measured to assess SGLT1 activity. Na-K-ATPase activity was measured as a function of inorganic phosphate release. Protein expression was analyzed by Western blot analysis and immunohistochemical staining., Results: Ethanol significantly decreased Na-dependent glucose absorption in enterocytes in vitro (ethanol treatment: 48.4% of controls at 1 h; P < 0.01) and in vivo (ethanol treatment: 60.0% of controls at 1 h; P < 0.01). Na-K-ATPase activity was significantly inhibited in vitro (ethanol treatment: 36.9% of controls at 1 h; P < 0.01) and in vivo (ethanol treatment: 42.1% of controls at 1 h; P < 0.01). Kinetic studies showed that the mechanism of inhibition of Na-glucose co-transport was secondary to a decrease in the affinity (1/Km) of the co-transporter for glucose both in vitro and in vivo. Western blots and immunohistochemistry further demonstrated unaltered amounts of SGLT1 after ethanol treatment., Conclusions: Moderate ethanol significantly decreases glucose absorption in IEC-18 cells and in villus cells of Sprague-Dawley rats. The inhibition of SGLT1 is secondary to an altered Na gradient at the cellular level and secondary to diminished affinity of the co-transporter for glucose at the protein level in the BBM. These observations may, at least in part, explain 1 possible mechanism of the onset of malnutrition associated with alcohol consumption., (Copyright © The Author(s) 2019.)

The hepatopancreas of crustaceans species has been recognized as an essential target organ to assess trace elements' effects. Due to its dynamic and capability of detoxifying trace metal, this organ often indicates distinct pathological disturbances. In the present work, we intend to evaluate the bioaccumulation of trace metal in three Orchestia species (Orchestia montagui, Orchestia gammarellus, and Orchestia mediterranea) living in symmetry in the banks of Bizerte lagoon (37°13'8″N 09°55'1″E) after their exposure during 14 days to a mixture of copper and zinc, and to highlight the effect of these metals on their hepatopancreas ultrastructure using transmission electron microscopy. At the end of the experiment, results showed that the mortality and the body mass varied according to the used nominal concentrations. Significant alterations were noted in all the treatment groups. The degree of these alterations depends on the used concentration, and they are represented especially by the cells remoteness and the border lyses, the reduction of the nuclear volume, the increase in the cytoplasm density with the presence of trace metal in the nucleus as well as in the vacuole, the disorganization and the destruction of microvilli, the condensation of the majority of cellular organelles and mitochondria swelling. Through this study, Orchestia genus could be an attractive candidate for the biochemical study of trace metal toxicity in Tunisian wetlands., (© 2019 Wiley Periodicals, Inc.)

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of intestinal mucosa and submucosa, characterized by the disruption of the intestinal epithelial barrier, increased production of inflammatory mediators, and excessive tissue injury. Intestinal epithelial cells, as well as microvascular endothelial cells, play important roles in IBD. To study the potential effects of kaempferol in IBD progress, we established a novel epithelial-endothelial cells coculture model to investigate the intestinal inflammation and barrier function. Data demonstrated an obvious increased transepithelial electrical resistance (TEER) (1222 ± 60.40 Ω cm 2 vs 1371 ± 38.77 Ω cm 2 ), decreased flux of FITC (180.8 ± 20.06 μg/mL vs 136.7 ± 14.78 μg/mL), and up-regulated occludin and claudin-2 expression in Caco-2 that was specifically cocultured with endothelial cells. Meanwhile, 80 μM kaempferol alleviated the drop of TEER, the increase of FITC flux, and the overexpression of interleukin-8 (IL-8) induced by 1 μg/mL lipopolysaccharide (LPS). Additionally, kaempferol also ameliorated the LPS-induced decrease of protein expression of zonula occludens-1 (ZO-1), occludin, and claudin-2, together with the inhibited protein expressions of the phosphorylation level of NF-κB and I-κB induced by LPS. Our results suggest that kaempferol alleviates the IL-8 secretion and barrier dysfunction of the Caco-2 monolayer in the LPS-induced epithelial-endothelial coculture model via inhibiting the NF-κB signaling pathway activation.

In vitro studies showed that 1-(propan-2-ylamino)-4-propoxy-9H-thioxanthen-9-one (TX5) increases P-glycoprotein (P-gp) expression and activity in Caco-2 cells, preventing xenobiotic toxicity. The present study aimed at investigating TX5 effects on P-gp expression/activity using Wistar Han rats: a) in vivo, evaluating intestinal P-gp activity; b) ex vivo, evaluating P-gp expression in ileum brush border membranes (BBM) and P-gp activity in everted intestinal sacs; c) ex vivo, evaluating P-gp activity in everted intestinal sacs of the distal and proximal ileum. TX5 (30 mg/kg, b.w.), gavage, activated P-gp in vivo, given the significant decrease in the AUC of digoxin (0.25 mg/kg, b.w.). The efflux of rhodamine 123 (300 μM), a P-gp fluorescent substrate, significantly increased in TX5-treated everted sacs from the distal portion of the rat ileum, when P-gp activity was evaluated in the presence of TX5 (20 μM), an effect abolished by the P-gp inhibitor verapamil (100 μM). No increases on P-gp expression or activity were found in TX5-treated BBM of the distal ileum and everted distal sacs, respectively, 24 h after TX5 (10 mg/kg, b.w.) administration. In vivo, no differences were found on digoxin portal concentration between control (digoxin 0.025 mg/kg, b.w., intraduodenal) and TX5-treated (digoxin+TX5 20 μM, intraduodenal) rats. The observed discrepancies in digoxin results can be related to differences in TX5 dose administered and used methodologies. Thus, the results show that TX5 activates P-gp at the distal portion of the rat ileum, and, at the higher dose tested (30 mg/kg, b.w.), seems to modulate in vivo the AUC of P-gp substrates., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Metal oxide nanoparticles (NP) are increasingly used in the food and agriculture industries, making human consumption nearly unavoidable. The goal of this study was to use the Gallus gallus (broiler chicken) intra-amniotic administration of physiologically relevant concentrations of TiO 2 , SiO 2 , and ZnO to better understand the effects of NP exposure on gut health and function. Immediately after hatch, blood, cecum, and small intestine were collected for assessment of iron (Fe)-metabolism, zinc (Zn)-metabolism, brush border membrane (BBM) functional, and pro-inflammatory related proteins gene expression; blood Fe and Zn levels; cecum weight; and the relative abundance of intestinal microflora. NP type, dose, and the presence or absence of minerals was shown to result in altered mineral transporter, BBM functional, and pro-inflammatory gene expression. Metal oxide NP also altered the abundance of intestinal bacterial populations. Overall, the data suggest that the in vivo results align with in vitro studies, and that NP have the potential to negatively affect intestinal functionality and health., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Alterations of the microbial composition in the gut and the concomitant dysregulation of the mucosal immune response are associated with the pathogenesis of opportunistic infections, chronic inflammation, and inflammatory bowel disease. To create a platform for the investigation of the underlying mechanisms, we established a three-dimensional microphysiological model of the human intestine. This model resembles organotypic microanatomical structures and includes tissue resident innate immune cells exhibiting features of mucosal macrophages and dendritic cells. The model displays the physiological immune tolerance of the intestinal lumen to microbial-associated molecular patterns and can, therefore, be colonised with living microorganisms. Functional studies on microbial interaction between probiotic Lactobacillus rhamnosus and the opportunistic pathogen Candida albicans show that pre-colonization of the intestinal lumen of the model by L. rhamnosus reduces C. albicans-induced tissue damage, lowers its translocation, and limits fungal burden. We demonstrate that microbial interactions can be efficiently investigated using the in vitro model creating a more physiological and immunocompetent microenvironment. The intestinal model allows a detailed characterisation of the immune response, microbial pathogenicity mechanisms, and quantification of cellular dysfunction attributed to alterations in the microbial composition., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cholera toxin is commonly known to induce chloride secretion of the intestine. In recent years, effects on epithelial barrier function have been reported, indicating synergistic co-regulation of transporters and tight junction proteins. Our current study focused on the analysis of cholera toxin effects on transepithelial resistance and on tight junction proteins, the latter known as structural correlates of barrier function. Ligated segments of the rat jejunum were injected with buffered solution containing cholera toxin (1 μg/ml) and incubated for 4 h. Subsequently, selfsame tissue specimens were mounted in Ussing chambers, and cholera toxin (1 μg/ml) was added on the apical side. Transepithelial resistance and permeability of sodium fluorescein (376 Da) were analyzed. Subsequently, tissues were removed, expression and localization of claudins were analyzed, and morphological studies were performed employing transmission electron microscopy and confocal laser scanning microscopy. Cholera toxin induced a marked decrease in transepithelial resistance in the rat jejunal epithelium and an increase in paracellular permeability for sodium fluorescein. Immunoblotting of tight junction proteins revealed an increase in claudin-2 signals, which was verified by confocal laser scanning immunofluorescence microscopy, and a decrease in tricellulin, whereas other tight junction proteins remained unchanged. Transmission electron microscopy showed a reduction in the number of microvilli after incubation with cholera toxin. Moreover, cholera toxin led to a widening of the intercellular space between enterocytes. In accordance with the commonly known prosecretory effect of cholera toxin, our study revealed a complementary effect on small intestinal barrier function and integrity, which might constitute a pathomechanism with high relevance for prevention and therapeutic approaches.

Some nanoparticles (NPs) have been shown to disrupt intestinal microvilli morphology in vitro, an alteration that could potentially affect nutrient absorption and barrier properties. This study aimed at evaluating the potential effect of CeO 2 NPs (4-8 nm, citrate stabilized) on Caco-2 microvilli morphology. In addition to the standard Caco-2 cell clone, the C2BBe1 clone was used, as it is considered to develop a more homogeneous cellular morphology. Semiautomated microvilli density quantification and a new cell scoring approach were used to evaluate scanning electron microscopy (SEM) images. The quantification method made use of the whole micrograph surface, avoiding the need to choose subareas for analysis, and increasing the representativeness of the results when compared to previous studies. The main advantage of the scoring system is that it informs on the intercellular variability within a cell preparation. Benzalkonium was used as a positive control inducing toxicity and morphological alterations on microvilli. After three-week differentiation, Caco-2 cells were exposed to 100 μg/mL of CeO 2 NPs for 24 h. The integrity of the membrane was evaluated by transepithelial electrical resistance (TEER) and thereafter processed for its observation by SEM. Results showed that both the standard Caco-2 clone and the C2BBe1 clone present notable morphological heterogeneity. The two evaluation approaches were able to identify morphological effects caused by the positive control, but did not detect statistically significant morphological alterations after exposure to CeO 2 NPs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, extend laterally, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that microridges are formed of F-actin networks and require the function of the Arp2/3 complex for their maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.

Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) is mainly controlled with synthetic insecticides such as chlorantraniliprole. However, these compounds may affect non-target organs of insect metabolism. The objective of this study was to evaluate the toxic effect in the midgut goblet cells of A. gemmatalis caterpillars exposed to chlorantraniliprole. The midgut of these caterpillars, which ingested the insecticide in medium-lethal dose (LD 50 ), was dissected and evaluated by transmission electron microscopy. The goblet cells microvilli, after exposure to the insecticide, were disorganized and degenerated. This can compromise ionic homeostasis and nutrient absorption, impair physiological mechanisms of detoxification, and reduce the movement of food boluses throughout the insect midgut., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S -/- ) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake. In two clinically relevant mouse models of acute kidney injury caused by myoglobin as seen in rhabdomyolysis and the aminoglycoside gentamicin, Gb3S -/- mice showed a preserved renal function and morphology, compared to WT. Pharmacologic inhibition of glucosylceramide-based glycosphingolipids, including Gb3, in WT mice corroborated the results of genetically Gb3-deficient mice. In conclusion, our data significantly advance the current knowledge on the physiologic and pathophysiologic role of Gb3 in proximal tubules, showing an involvement in the reabsorption of filtered albumin, myoglobin and the aminoglycoside gentamicin., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

In the mammalian small intestine, sodium is primarily absorbed by Na + /H + exchange (NHE3) and Na-glucose cotransport (SGLT1) in the brush border membrane (BBM) of villus cells. However, how enhanced cellular constitutive nitric oxide (cNO) may affect NHE3 and SGLT1 remains unclear. Both in vivo in rabbit intestinal villus cells and in vitro IEC-18 cells, administration of NO donor, GSNAP, modestly increased cNO. GSNAP stimulated SGLT1 in villus and IEC-18 cells. The mechanism of stimulation was secondary to an increase in the affinity of SGLT1 for glucose. The change in SGLT1 was not secondary to altered Na-extruding capacity of the cell since Na + /K + -ATPase was decreased by GSNAP treatment. In contrast, GSNAP inhibited NHE3 activity in villus cell BBM. The mechanism of NHE3 inhibition was secondary to reduced BBM transporter numbers. These studies demonstrated that the physiological increase in cNO uniquely regulates mammalian small intestinal NHE3 and SGLT1 to maintain Na homeostasis., (© 2019 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Aims: To evaluate the protective efficacy of Pediococcus pentosaceus GS4 in cadmium (Cd)-induced toxicity in Swiss albino mice., Methods and Results: The previously reported probiotic P. pentosaceus GS4 interaction with Cd was evaluated experimentally. Swiss albino mice were segregated randomly into three different groups containing nine animals each. The binding ability of P. pentosaceus GS4 and Cd was demonstrated by atomic absorption spectrometry, Fourier transform infrared spectroscopy and scanning electron microscopy analysis respectively. Cd challenge was attenuated by the P. pentosaceus GS4 treatment and was evaluated by estimating Cd level in liver and faeces, corresponding biomarkers for toxicity in liver and intestines and histopathological analysis with brush border membrane (BBM) integrity. Results showed that P. pentosaceus GS4 bound Cd effectively. Pediococcus pentosaceus GS4 intervention significantly enhanced faecal evacuation of Cd with an effect of reduced tissue deposition. Histologically, Cd-induced gut was found significantly replenished with lactobacilli in addition to reduced hyperplasia, lowered lymphocytes infiltration and enhancement of BBM-based disaccharidases, proving its role in protecting membrane integrity., Conclusions: Probiotic P. pentosaceus GS4 efficiently alleviated toxicity from vital organs such as liver and intestine caused by Cd exposure in a murine model., Significance and Impact of the Study: Intervention of P. pentosaceus GS4 alleviated Cd-induced toxicity, and thus it may be employed therapeutically to protect Cd toxicity., (© 2019 The Society for Applied Microbiology.)

The uterine epithelium undergoes remodelling to become receptive to blastocyst implantation during pregnancy in a process known as the plasma membrane transformation. There are commonalities in ultrastructural changes to the epithelium, which, in eutherian, pregnancies are controlled by maternal hormones, progesterone and oestrogens. The aim of this study was to determine the effects that sex steroids have on the uterine epithelium in the fat-tailed dunnart Sminthopsis crassicaudata, the first such study in a marsupial. Females were exposed to exogenous hormones while they were reproductively quiescent, thus not producing physiological concentrations of ovarian hormones. We found that changes to the protein E-cadherin, which forms part of the adherens junction, are controlled by progesterone and that changes to the desmoglein-2 protein, which forms part of desmosomes, are controlled by 17β-oestradiol. Exposure to a combination of progesterone and 17β-oestradiol causes changes to the microvilli on the apical surface and to the ultrastructure of the uterine epithelium. There is a decrease in lateral adhesion when the uterus is exposed to progesterone and 17β-oestradiol that mimics the hormone environment of uterine receptivity. We conclude that uterine receptivity and the plasma membrane transformation in marsupial and eutherian pregnancies are under the same endocrine control and may be an ancestral feature of therian mammals.

Cholesterol homeostasis is maintained through a balance of de novo synthesis, intestinal absorption, and excretion from the gut. The small intestine contributes to cholesterol homeostasis by absorbing and excreting it, the latter of which is referred to as trans-intestinal cholesterol efflux (TICE). Because the excretion efficiency of endogenous cholesterol is inversely associated with the development of atherosclerosis, TICE provides an attractive therapeutic target. Thus, elucidation of the mechanism is warranted. We have shown that intestinal cholesterol absorption and TICE are inversely correlated in intestinal perfusion experiments in mice. In this review, we summarized 28 paired data sets for absorption efficiency and fecal neutral sterol excretion, a surrogate marker of TICE, obtained from 13 available publications in a figure, demonstrating the inverse correlation were nearly consistent with the assumption. We then offer a bidirectional flux model that accommodates absorption and TICE occurring in the same segment. In this model, the brush border membrane (BBM) of intestinal epithelial cells stands as the dividing ridge for cholesterol fluxes, making the opposite fluxes competitive and being coordinated by shared BBM-localized transporters, ATP-binding cassette G5/G8 and Niemann-Pick C1-like 1. Furthermore, the idea is applied to address how excess plant sterol/stanol (PS) intake reduces circulating cholesterol level, because the mechanism is still unclear. We propose that unabsorbable PS repeatedly shuttles between the BBM and lumen and promotes concomitant cholesterol efflux. Additionally, PSs, which are chemically analogous to cholesterol, may disturb the trafficking machineries that transport cholesterol to the cell interior., Competing Interests: The authors declare no competing interest.

Using X. laevis oocyte expression system, we investigated whether human Na + -coupled monocarboxylate transporter 1 (SLC5A8, hSMCT1) is involved in 2,4-dichlorophenoxyacetate (2,4-D) uptake by the renal tubular epithelial cells. 2,4-D is a herbicide that causes nephrotoxicity. Heterologous expression of hSMCT1 in X. laevis oocytes conferred the ability to take up 2,4-D; the induced uptake process was Na + -dependent and electrogenic. The Na + -dependent uptake of 2,4-D was inhibited not only by known hSMCT1 substrates, but also by many structural analogs of 2,4-D. The currents induced by 2,4-D, 4-chlorophenoxyacetate (4-CPA) and 2-methyl-4-chlorophenoxyacetate (MCPA) were saturable: the rank order of the maximal induced current and the affinity for hSMCT1was 2,4-D > 4-CPA > MCPA. The relationship between the structures of the derivatives and their transport activity implied specific structural features in a compound for recognition as a substrate by hSMCT1. Furthermore, we have demonstrated using purified rabbit renal brush-border membrane vesicles that 2,4-D potently inhibited the Na + -dependent uptake of pyroglutamate, a typical substrate for Smct1, and that 2,4-D uptake process was Na + -dependent, saturable and inhibitable by a potent blocker, ibuprofen. We conclude that hSMCT1 is involved partially in the renal reabsorption of 2,4-D and its derivatives and their nephrotoxicity., (Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.)

Sodium chlorate (NaClO 3 ) is widely used in paper and pulp industries and as a non-selective herbicide. Humans can be exposed to NaClO 3 through contaminated drinking water due to its improper and unchecked usage in industries and as herbicide. NaClO 3 is also present as a major stable by-product in drinking water that has been disinfected with chlorine dioxide. In this study, we have investigated the effect of a single acute oral dose of NaClO 3 on rat kidney. Adult male Wistar rats were divided into one control and four NaClO 3 treated groups that were orally given different doses of NaClO 3 and euthanized 24 hr after the treatment. Oral administration of NaClO 3 resulted in increased hydrogen peroxide levels, lipid, and protein oxidation while thiol and glutathione content and activities of brush border membrane enzymes were decreased in kidney in a NaClO 3 dose-dependent manner. Significant alterations in the activities of enzymes involved in carbohydrate metabolism and antioxidant defense were also observed. Administration of NaClO 3 induced DNA fragmentation and increased DNA-protein cross-linking. Histological studies showed marked damage in kidney from NaClO 3 treated animals. These results strongly suggest that NaClO 3 induces nephrotoxicity via redox imbalance that results in DNA and membrane damage, metabolic alterations and brush border membrane enzyme dysfunction., (© 2018 Wiley Periodicals, Inc.)

This study investigated the effect of enteral administration of obestatin on the development of small intestine, as well as oxidative stress markers and trancriptomic profile of gastrointestinal genes. Suckling rats were assigned to 3 groups treated with: C-saline solution; OL-obestatin (125 nmol/kg BW); OH-obestatin (250 nmol/kg BW) administered twice daily, from the 14th to the 21st day of life. Enteral administration of obestatin in both studied doses had no effect neither on the body weight of animals nor the BMI calculated in the day of euthanasia. Compared to the control group (C), treatment with obestatin resulted in significant changes in the histometry of the small intestinal wall as well as intestinal epithelial cell remodeling. The observed changes and their possible implications for intestinal development were dependent on the dosage of peptide. The enteral administration of high dose (OH) of obestatin significantly decreased its expression in the stomach and increased markers of oxidative stress. The gene profile revealed MAPK3 (mitogen-activated protein kinase-3) as the key regulator gene for obestatin action in the gastrointestinal track. In conclusion, we have showed that enteral administration of obestatin influences the gut mucosa remodeling. It is also suggested that the administration of high dose (OH) has inhibitory effect on the intestinal maturation of suckling rats., Competing Interests: The authors have declared that no competing interests exist.

Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight <3rd percentile, mean birth weight 1804±110 g, gestational age 35.7±0.61 weeks, n=25) and women delivering an appropriately-for gestational age infant (control group, birth weight 25th-75th centile, mean birth weight 2493±216 g, gestational age 33.9±0.95 weeks, n=19) were recruited and placentas were collected at delivery. MVM was isolated and folate transporter protein expression was measured using Western blot and transporter activity was determined using radiolabelled methyltetrahydrofolic acid and rapid filtration. Whereas the expression of FR-α was unaffected, MVM RFC protein expression was significantly decreased in the IUGR group (-34%, P<.05). IUGR MVM had a significantly lower folate uptake compared to the control group (-38%, P<.05). In conclusion, placental folate transport capacity is decreased in IUGR, which may contribute to the restricted fetal growth and intrauterine programming of childhood and adult disease. These findings suggest that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The obstructive renal radioactivity after injection of antibody fragments/constructs labeled with metallic radionuclides would be improved by liberating a radiometal chelate of urinary excretion from the antibody molecules by enzymes on the renal brush border membrane (BBM). A tripeptide GFK sequence was newly evaluated as an enzyme-cleavable linkage and conjugated to a 99m Tc chelate of an isonicotinic acid derivative of 2-picolylglycine ( 99m Tc-IPG). 99m Tc-IPG-glycine was liberated from 99m Tc-IPG-GFK by the enzymes, while 99m Tc-IPG-GK (where the tripeptide GFK was substituted with a dipeptide GK) did not. When injected into mice, 99m Tc-IPG-GFK-conjugated Fab exhibited lower renal radioactivity levels than directly radioiodinated Fab shortly after injection without reducing the tumor radioactivity levels, due to a release and excretion of 99m Tc-IPG-glycine by enzymes present on the renal BBM. These findings would provide insights to develop antibody fragments/constructs labeled with metallic radionuclides of the clinical relevance for improved renal radioactivity levels.

Although small intestinal epithelial stem cells form crypts when using intestinal culture conditions, colon stem cells usually form colonospheres. Colon mesenchymal cell feeder layers can stimulate colon crypts to form organoids and produce crypts. We have investigated whether conditioned medium from colon mesenchymal cells can also stimulate colonosphere and organoid cryptogenesis. We prepared conditioned medium (CM) from WEHI-YH2 cells (mouse colon myofibroblasts); the CM stimulated both colonosphere formation and organoid cryptogenesis in vitro. The colon organoid-stimulating factors in WEHI-YH2 CM are inactivated by heating and trypsin digestion and proteins can be concentrated by ultrafiltration. Both the colonosphere- and organoid cryptogenesis- stimulatory effects of the CM are independent of canonical Wnt and Notch signaling. In contrast, bone morphogenetic protein 4 (BMP4) abolishes colonosphere formation and organoid cryptogenesis. The Transforming Growth Factor beta (TGFβ) Type I receptor kinase inhibitor (A83-01) stimulates colonosphere formation, whereas the Epidermal Growth Factor receptor (EGFR) kinase inhibitor (AG1478) reduces the formation of colonospheres, but in the presence of EGF, a "just-right" concentration of AG1478 increases colon organoid cryptogenesis., Competing Interests: The authors have declared that no competing interests exist.

Background: Neonates on long-term parenteral nutrition (PN) may develop parenteral nutrition-associated liver disease (PNALD). Aluminum (Al) is a known contaminant of infant PN, and we hypothesize that it substantially contributes to PNALD. In this study, we aim to assess the impact of Al on hepatocytes in a piglet model. Methods: We conducted a randomized control trial using a Yucatan piglet PN model. Piglets, aged 3⁻6 days, were placed into two groups. The high Al group ( n = 8) received PN with 63 µg/kg/day of Al, while the low Al group ( n = 7) received PN with 24 µg/kg/day of Al. Serum samples for total bile acids (TBA) were collected over two weeks, and liver tissue was obtained at the end of the experiment. Bile canaliculus morphometry were studied by transmission electron microscopy (TEM) and ImageJ software analysis. Results: The canalicular space was smaller and the microvilli were shorter in the high Al group than in the low Al group. There was no difference in the TBA between the groups. Conclusions: Al causes structural changes in the hepatocytes despite unaltered serum bile acids. High Al in PN is associated with short microvilli, which could decrease the functional excretion area of the hepatocytes and impair bile flow.

Nanosized titanium dioxide (TiO 2 ) is a common additive in food and cosmetic products. The goal of this study was to investigate if TiO 2 nanoparticles affect intestinal epithelial tissues, normal intestinal function, or metabolic homeostasis using in vitro and in vivo methods. An in vitro model of intestinal epithelial tissue was created by seeding co-cultures of Caco-2 and HT29-MTX cells on a Transwell permeable support. These experiments were repeated with monolayers that had been cultured with the beneficial commensal bacteria Lactobacillus rhamnosus GG (L. rhamnosus). Glucose uptake and transport in the presence of TiO 2 nanoparticles was assessed using fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). When the cell monolayers were exposed to physiologically relevant doses of TiO 2 , a statistically significant reduction in glucose transport was observed. These differences in glucose absorption were eliminated in the presence of beneficial bacteria. The decrease in glucose absorption was caused by damage to intestinal microvilli, which decreased the surface area available for absorption. Damage to microvilli was ameliorated in the presence of L. rhamnosus. Complimentary studies in Drosophila melanogaster showed that TiO 2 ingestion resulted in decreased body size and glucose content. The results suggest that TiO 2 nanoparticles alter glucose transport across the intestinal epithelium, and that TiO 2 nanoparticle ingestion may have physiological consequences.

Inorganic arsenic (As), the most toxic form of As found in water and food, is considered a human carcinogen. Numerous studies show its systemic toxicity, describing pathologies associated with chronic exposure. The main pathway of exposure to inorganic As is oral, but many of the events that occur during its passage through the gastrointestinal tract are unknown. This study evaluates the effect of subchronic exposure to inorganic As [As(III): 0.025-0.1 mg/L; As(V): 0.25-1 mg/L, up to 21 days] on the intestinal epithelium, using Caco-2 cells as in vitro model. Inorganic As produces a pro-inflammatory response throughout the exposure time, with an increase in IL-8 release (up to 488%). It also causes changes in the program of cell proliferation and differentiation, which leads to impairment of the cell repair process. In addition, subchronic exposure affects the epithelial structure, causing loss of microvilli, fundamental structures in the processes of intestinal absorption and digestion. Moreover, the exposure affects the epithelial barrier function, evidenced by an increase of Lucifer Yellow transport (103-199%). Therefore, it can be concluded that subchronic exposure to inorganic As can alter intestinal homeostasis, affecting the mucosal layer, which performs the most important functions of the intestinal wall., (Copyright © 2018 Elsevier B.V. All rights reserved.)