Brush Border Research Articles

Lrig1+ small intestinal progenitor cells require MYO5B for proper crypt proliferation and enterocyte maturation

Microvillus inclusion disease (MVID) is a congenital disorder characterized by severe diarrhea. MVID is caused by inactivating mutations in myosin Vb (MYO5B), a motor protein. Mice lacking MYO5B in their intestinal epithelium have demonstrated the importance of MYO5B in enterocyte brush border formation and epithelial cell differentiation. Additionally, the proliferative zone of the intestinal crypt of MYO5B-deficient mice is elongated compared to controls. We hypothesize that the progenitor cell-specific Cre driver under Lrig1 demonstrates crypt-specific effects caused by MYO5B loss. Lrig1-CreERT2;R26R-YFP and Myo5bfl/fl mice were crossbred to generate Lrig1-CreERT2;R26R-YFP;MYO5B fl/fl (Lrig1 ΔMYO5B ) mice. Adult Lrig1 ΔMYO5B mice and control littermates received tamoxifen injection at day 0. GI tissues were collected 2.5, 3, 4, and 5 days after tamoxifen administration for histological characterization. Lrig1 ΔMYO5B mice began experiencing significant weight loss 4 days after tamoxifen injection and lost 20% of their starting body weight on day 5. At day 5, the Lrig1 ΔMYO5B mouse colon was devoid of solid feces and their small intestine contained clear luminal contents, together suggesting a watery diarrheal phenotype. Enterocytes of Lrig1DMYO5B mice on day 5 contained abnormal cytoplasmic accumulation of PAS staining. Fewer goblet cells were apparent in intestinal sections from day 3 and 5 Lrig1 ΔMYO5B mice compared to controls. Crypts were elongated starting at day 3. Similarly, the PCNA+ epithelial region was expanded in Lrig1 ΔMYO5B mice, even reaching into the villi at day 5. The time course of the PCNA+ region expansion corresponded to the wave of MYO5B-loss beginning at the base of crypts. MYO5B expression was diminished in the crypts at day 3 and nearly absent from the epithelium by day 5. Interestingly, expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), the rate-limited enzyme of ketogenesis that is important for stem cell function, was expanded in the crypt at day 3 and 5 after tamoxifen. Sodium/glucose cotransporter 1 (SGLT1), which is present along the apical surface of mature enterocytes, was progressively diminished and mis-localized away from the apical surface after tamoxifen administration. Apical sodium-dependent bile acid transporter (ASBT), found in the distal small intestine, demonstrated a similar mis-localization away from the apical membrane in enterocytes.We established a novel mouse model that demonstrates progressive MYO5B loss in the intestinal epithelium, starting at the base of the crypt and reaching villi tips over the course of 5 days following Cre induction. This Lrig1 ΔMYO5B strain will allow for lineage tracing studies to better understand the effect of MYO5B loss on intestinal epithelial differentiation and progenitor function. NSF GFRP, NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Alteration of secretory cell lineages and immune cell migration induced by acute tuft cell ablation in mouse intestine

Background: Intestinal tuft cells have only been recently investigated thoroughly due to their roles in type 2 immunity and chemosensory function. This rare population of intestinal epithelium is implicated in extensively interacting with other cell types and involved in nutrient absorption. Doublecortin-like kinase 1 (DCLK1) is widely used for tuft cell identification in the mouse intestine. However, the effects of tuft cell deletion on mouse intestines have not been fully understood. Here, we investigated the effects of tuft cell deletion on the epithelial cell lineages and overall impact on mouse intestine. Methods: Tuft cell deletion was induced in Dclk1-IRES-GFP-CreERT2/+; Rosa-DTA (Dclk1-DTA) mice by a single dose of tamoxifen injection. Cre-negative DTA/+ mice and DTA-negative Dclk1-CreERT2 mice of littermates received tamoxifen injection at same time as controls. Mouse bodyweight was monitored daily, and intestinal tissues were sampled 2, 4, or 7 days after the tamoxifen injection. Immunostaining of intestinal tissue sections was performed for lysozyme (LYZ), trefoil factor 3 (TFF3), cluster of differentiation 3 (CD3), DCLK1, mast cell protease 1 (MCPT1), Ly6G, and F4/80. Image analysis was conducted using Qupath. Results: Dclk1-DTA mice showed a significant decrease in bodyweight to 11% of their original weight on day 4 after tamoxifen injection. They showed significantly shortened small intestinal length compared to control mice, however, colonic length showed no significant difference. Immunofluorescent staining of induced Dclk1-DTA mouse tissues revealed that DCLK1+ tuft cells were decreased on day 2, almost completely deleted on day 4, and recovered on day 7. Overall brush border structures defined by villin and SGLT1 staining were intact. Correlated with the tuft cell reduction, mislocalized Paneth-like cells, identified with LYZ+ granules, were observed in the middle or upper part of villi with increasing frequency on day 4 and decreasing on day 7. To confirm goblet/Paneth intermediate cells, multiplexed immunofluorescent (MxIF) staining was employed with LYZ and TFF3. Double-positive (LYZ+/TFF3+) cells were significantly increased in the small intestine of tuft cell-depleted mice on day 4. Intraepithelial lymphocytes, mucosal neutrophils, and F4/80+ macrophages/dendritic cells did not show significant changes after the tuft cell deletion. This indicates that there is no inflammatory reactions following tuft cell deletion. Conclusion: Acute ablation of intestinal tuft cell may cause nutrient malabsorption and changes in mucosal defense response in vivo due to the alteration of cell differentiation lineage and the decrease in absorptive area. Our findings support the importance of tuft cell function in nutrient sensing and mucosal defense mechanism. NIH R01DK128190 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Application of enzymatically treated Artemisia annua L. on adult grass carp (Ctenopharyngodon idella): Improved growth performance, intestinal antioxidant capacity and apical junctional complex

The present study evaluated the effects of enzymatically treated Artemisia annua L. (EA) on the growth performance, intestinal antioxidant capacity, and apical junctional complex (AJC) of adult grass carp. In this experiment, 375 healthy grass carp (initial body weight 673.96 ± 1.24 g) were selected and randomly divided into 5 treatments, and fish in each group were fed diets with different levels of EA (0, 400, 800, 1200, and 1600 mg/kg) 9 weeks. The results showed that compared with the control group (0 mg/kg EA diet), the EA supplementation in the diet increased the percentage of weight gain (PWG), specific growth rate (SGR), and feed intake (FI) (P < 0.05), indicating that dietary EA supplementation improved the growth performance of adult grass carp. The dietary EA supplementation also enhanced the digestion and absorption ability of adult grass carp, which might be reflected by improving the activities of hepatopancreas digestive enzymes (trypsin, chymotrypsin, lipase, and amylase) and intestinal brush border enzymes (alkaline phosphatase, Na+/K+-ATPase, and γ-glutamyl transpeptidase), but EA did not affect the creatine kinase enzyme activity. Meanwhile, we explored the effect of dietary EA addition on the structural integrity of the grass carp intestine. We observed that dietary EA supplementation improved the antioxidant capacity of the intestine which be reflected by increasing the activities levels of superoxide dismutase (SOD) and catalase (CAT), which might be related to the binding protein Keap1a (not Keap1b)/ NF-E2-related factor-2 (Nrf2) signaling pathway. In addition, dietary EA supplementation enhanced the integrity of AJC by increasing the mRNA levels of intestinal tight junction proteins (TJs) (except claudin 11, claudin 12, claudin 15a) and adhere junction proteins (AJs) (E-cadherin, α-catenin, β-catenin, nectin, and afadin), which might be related to the Rho family GTPases (RhoA)/ Rho-associated protein kinase (ROCK)/ myosin light chain kinase (MLCK) signaling pathway. Collectively, dietary EA supplementation improved growth performance, digestion and absorption ability, and intestinal structural integrity in grass carp, which were associated with the activation of related signaling pathways and regulatory factors. Finally, by the growth indicator percent weight gain (PWG) and intestinal oxidative damage index protein carbonyl (PC), the optimum amounts of EA supplementation of adult grass carp (673–1726 g) were assessed to be 808.09 and 730.38 mg/kg diet, respectively.

Developing a multiscale mathematical model of protein uptake along the mouse proximal tubule

The proximal tubule (PT) efficiently reclaims albumin and other proteins that escape the glomerular filtration barrier to maintain a protein free urine. The recovery of filtered proteins occurs via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. PT health is impacted by excess albumin uptake that occurs when the glomerular barrier is breached and by nephrotoxic drugs that enter cells in a megalin/cubilin dependent manner. Understanding where and how endocytic uptake occurs along the PT axis is key to predicting disease outcomes and to devising strategies to protect PT cells from nephrotoxic insults. Transcriptomic and proteomic studies in male rodents have demonstrated a difference in the expression of megalin and cubilin across the S1, S2, and S3 sub-segments that comprise the PT. Based on these differences in expression, we previously developed an axial model of protein uptake in mice which predicts that most uptake under normal conditions occurs in the S1 segment, with later PT segments providing excess uptake capacity under nephrotic conditions. Separately, we created a kinetic model that predicts rates of megalin trafficking rates in S1 segment cells. This model demonstrated that both the actual level and the fraction of total receptor present at the apical surface are critical determinants of endocytic capacity. To integrate our cell-based kinetic model of megalin traffic into the axial model, we used imaging-based approaches to quantify the expression and distribution of megalin and cubilin in SGLT2-positive S1 and SGLT2-negative S2 segments in fixed cortical kidney sections from wild-type C57BL/6 age-matched male and female mice. The fraction of megalin and cubilin at the apical surface was determined by colocalization with the brush border marker, villin, in regions where AP2, a marker for endocytic vesicles, was excluded. These data will be incorporated into our current axial model to develop a multiscale model of protein uptake along the length of the PT. The expanded model will improve our understanding of how endocytic capacity along the PT in vivo is regulated under normal and disease conditions and allow us to determine whether there are sex-based differences in the recovery of filtered proteins. National Institutes of Health T32 DK007052, F31 DK121394, R01 DK125049, R01 DK118726 and ASN Foundation for Kidney Research Pre-Doctoral Fellowship Award This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Сучасні уявлення про значущість забезпечення цинком при інфекційній патології в дітей

Zinc (Zn) is an essential trace element that is critical for growth, development and maintenance of immune function. Zn is an indispensable trace element for humans, which is part of more than 20 metalloenzymes, including DNA and RNA polymerase, phosphatase, carbonic anhydrase and some others. The cycle of cell development requires this trace element. The permeability of cell membranes is also related to Zn. It has antioxidant properties and delays the apoptosis of peripheral cells. The formation of immunity depends on the body’s supply of Zn, and its deficiency causes atrophy of the thymic-lymphatic system. Zn is part of insulin, accelerates the regeneration of the mucous layer of the intestines, increases the activity of enzymes of the brush border of enterocytes, increases the level of secretory antibodies and the intensity of cellular immunity. The provision of this element cannot but affect the course of various infectious pathologies. Purpose - by analyzing modern literature data, to find out the importance of Zn provision in infectious pathologies in children. It is analyzed a review of the literature on the pathogenetic role of Zn in the diagnosis, pathogenesis and treatment of infectious diseases, based on a search for articles in the Science, Medline and PubMed databases published from January 2017 to June 2021. An analysis of current literature has shown that Zn is an important micronutrient involved in the regulation of innate and adaptive immune responses. Zn is necessary to ensure the barrier function of membranes. It is involved in the modulation of the pro-inflammatory response. Zn homeostasis is essential for many aspects of the immune system, including hematopoiesis, cell maturation and differentiation, cell cycle progression, and proper immune cell function. Consequently, Zn deficiency leads to cell-mediated immune dysfunctions. Such dysfunctions lead to deterioration of the body's response to bacterial infection. Zn is known to modulate antiviral immunity. Due to zinc, the production of interferon-α is activated and its antiviral activity is increased. Studies show that during the infectious process there is a decrease in the concentration of zinc in the blood plasma. Such changes, on the one hand, provide the needs of immune protection factors, and on the other hand, reduce the availability of the trace element for pathogens. Conclusions. Zn is one of the important trace elements that plays a leading role in maintaining homeostasis and is an integral component of the pathogenesis of various pathological conditions of infectious origin. The most studied is Zn deficiency in infectious diarrhea in children, but studies have been conducted in developing countries with limited resources. No works studying the prevalence of Zn deficiency in Ukraine were found. No conflict of interests was declared by the authors.

Determining ACE-2 Level and Some Relevant Biochemical Parameters and studying the effect of Gender in Iraqi Diabetic Patients with Glomeruli and Renal Tubules Fibrosis as Early Prediction Marker

Diabetic kidney disease is an illness of the glomerulus that interferes with the glomerular filtration barrier (GFB), which is worked to enable kidney to selective purification of water and solutes in addition to limiting the movement of large macromolecules such as albumin. In the glomerular endothelium, mesangial cells, foot cells, and the brush border of the proximal tubules, ACE-2 is expressed and that the kidneys represent the highest-expressing region of this enzyme. Thus, the current study aimed to evaluate ACE-2 level in this case compared to healthy condition. The study Conducted with 120 male and female ranging in age (30-65) years old. Ninety patients with type 2 diabetes subdivided into three groups on the basis of ACR criteria including normoalbuminuria, microalbuminuria, macroalbuminuria (30 patients for each group) and 30 healthy people served as the control group, all visited Baghdad Teaching Hospital / Medical City and Al-Yarmouk Teaching Hospital, at the period between December 2021 and May 2022. ACE-2 levels were determined using the ELISA technique. Urea results showed significant differences between diabetic nephropathy in patient and control group in female cases but no significant differences in male patients with diabetic nephropathy and control group. Similar results were obtained in K ion. Also the results revealed significant differences in Na ion, ACR, eGFR, Urea, FBS, creatinine between diabetic nephropathy groups and healthy group.ACE-2 represents a good marker for early prediction in diabetic nephropathy case. ROC data analysis support the importance of ACE-2 in diagnosis of the studied disease case.

Effects of Intra-Amniotic Administration of the Hydrolyzed Protein of Chia (Salvia hispanica L.) and Lacticaseibacillus paracasei on Intestinal Functionality, Morphology, and Bacterial Populations, In Vivo (Gallus gallus).

As a protein source, chia contains high concentrations of bioactive peptides. Probiotics support a healthy digestive tract and immune system. Our study evaluated the effects of the intra-amniotic administration of the hydrolyzed chia protein and the probiotic Lacticaseibacillus paracasei on intestinal bacterial populations, the intestinal barrier, the inflammatory response, and brush border membrane functionality in ovo (Gallus gallus). Fertile broiler (Gallus gallus) eggs (n = 9/group) were divided into 5 groups: (NI) non-injected; (H2O) 18 MΩ H2O; (CP) 10 mg/mL hydrolyzed chia protein; (CPP) 10 mg/mL hydrolyzed chia protein + 106 colony-forming unit (CFU) L. paracasei; (P) 106 CFU L. paracasei. The intra-amniotic administration was performed on day 17 of incubation. At hatching (day 21), the animals were euthanized, and the duodenum and cecum content were collected. The probiotic downregulated the gene expression of NF-κβ, increased Lactobacillus and E. coli, and reduced Clostridium populations. The hydrolyzed chia protein downregulated the gene expression of TNF-α, increased OCLN, MUC2, and aminopeptidase, reduced Bifidobacterium, and increased Lactobacillus. The three experimental groups improved in terms of intestinal morphology. The current results suggest that the intra-amniotic administration of the hydrolyzed chia protein or a probiotic promoted positive changes in terms of the intestinal inflammation, barrier, and morphology, improving intestinal health.

Histochemical research of enzymes involved in cellular digestion in the digestive tract of tub gurnard, Chelidonichthys lucerna.

The tub gurnard Chelidonichthys lucerna (Linnaeus, 1758), Triglidae, is an opportunistic, demersal carnivorous fish. Data on the digestive enzymes of tub gurnard have not been reported in the literature. Therefore, the aim of this research was to investigate the distribution and intensity of alkaline phosphatase, acid phosphatase, non-specific esterase, and aminopeptidase in the digestive tract of tub gurnard. To investigate data about those enzymes tissue samples of the esophagus, anterior and posterior part of the stomach, pyloric caeca, anterior, middle and posterior part of the intestine proper, and rectum were taken. Azo-coupling methods were used to detect the enzymatic reactions. The intensities of the reactions were measured using ImageJ software. Alkaline phosphatase, acid phosphatase, and non-specific esterase activities were found in all parts of the digestive tract. The brush border of the pyloric caeca and intestine proper were the main sites of alkaline phosphatase reaction, with intensity decreasing toward the posterior parts of the digestive tract. The high intensities of acid phosphatase were found in the epithelium of the anterior part of the stomach, pyloric caeca, anterior part of the intestine proper, and in the rectum. The intensity of non-specific esterase was mainly increased from the anterior to the posterior parts of the digestive tract. Aminopeptidase activity was found in the esophagus, pyloric caeca, and intestine proper. Our results suggest that the entire digestive tract of the tub gurnard is involved in the digestion and absorption of dietary components.

Abstract 2636: Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer

Abstract Background: Amatoxin-based antibody drug conjugates (so called ATAC®s) constitute a new class of antibody-drug conjugates (ADCs) which utilize amanitin as toxic payload. Amanitin binds to the RNA pol II and thereby efficiently inhibits cellular transcription. In the current study, in vitro and in vivo data of an ATAC® targeting GCC (guanylyl cyclase C) are presented. GCC is a cell surface receptor expressed in &amp;gt;95% of colorectal cancer, and in approximately 65% of esophageal, gastric, and pancreatic tumors. In healthy conditions GCC expression is restricted to the gastrointestinal tract, and more specific to the apical brush border of the intestinal epithelium (luminal site). Thus, GCC in healthy tissue is not exposed to the circulation but upon tumor progression in GI malignancies it becomes accessible for i.v. injected targeted therapeutics. This tumor-specific accessibility for drugs in circulation makes GCC a highly attractive target for ATAC®s. Material and Methods: ATAC®s: Cysteine-reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of a proprietary anti-GCC antibody yielding ATAC®s with a DAR of 2.0. Animal models: Subcutaneous (s.c.) mouse xenograft models with the GCC overexpressing cell line HEK293-GUCY2C mono2 or colon cancer PDX models (Charles River) were performed in single-dose and multiple-dosing experiments. Tolerability was assessed in NMRI nude mice and non-human primates (NHP). Results: All anti-GCC ATAC®s with amanitin-linker derivatives, optimized for use in solid tumors, showed favorable in vitro cytotoxicity with picomolar activity on GCC+ cell lines and no cytotoxic activity on target-negative cells. In mouse xenograft models, the optimized anti-GCC ATAC®s caused dose-dependent tumor regression in HEK293-GUCY2C mono2 s.c. xenografts. Even in colorectal cancer PDX models, anti- GCC ATAC®s led to a substantial anti-tumor effect. Multiple dosing improved this anti-tumor efficacy even further without negative impact on toxicity. The MTD of the ATAC®s tested in mice differed between 7.5 and 50 mg/kg depending on linker chemistry. Safety profiling in cynomolgus monkeys revealed good tolerability for all selected ATAC®s. Hematology and clinical chemistry parameters were unaffected apart from a moderate and transient increase in liver enzymes. Conclusions: Targeted cytotoxic drug delivery to GCC+ cell lines was achieved by using anti-GCC ATAC®s optimized for the use in solid tumors. The mode of action of the payload amanitin led to a pronounced anti-tumor effect in vitro and in vivo in PDX models with good tolerability in non-human primates. The use of anti-GCC ATAC®s with their novel mode of action in the therapy of GCC positive cancers, such as colorectal cancer represents a promising approach in cancer therapy. Citation Format: Alexandra Braun, Andreas Pelz, Franziska Ebeling, Kristin Decker, Anikó Pálfi, Michael Kulke, Andreas Pahl, Torsten Hechler. Amanitin-based ADCs targeting Guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2636.

Maturation of Nephrons by Implanting hPSC-derived Kidney Progenitors Under Kidney Capsules of Unilaterally Nephrectomized Mice.

Human pluripotent stem cell (hPSC)-derived kidney organoids may contribute to disease modeling and the generation of kidney replacement tissues. However, the realization of such applications requires the induction of hPSCs into functional mature organoids. One of the key questions for this process is whether a specific vascular system exists for nephrogenesis. Our previous study showed that short-term (2 weeks) implantation of hPSC-derived organoids below the kidney capsules of unilaterally nephrectomized and immunodeficient mice resulted in the enlargement of organoids and production of vascular cells, although signs of maturation were lacking. Organoids were induced for 15 days in vitro and then grafted below kidney capsules of the same unilaterally nephrectomized immunodeficient mouse model to examine whether medium-term (4 weeks) implantation could improve organoid maturation and vascularization, as evaluated by immunofluorescence and transmission electron microscopy. We demonstrated that after 2-4 weeks of implantation, renal organoids formed host-derived vascularization and matured without any exogenous vascular endothelial growth factor. Glomerular filtration barrier maturation was evidenced by glomerular basement membrane deposition, perforated glomerular endothelial cell development, and apical, basal podocyte polarization. A polarized monolayer epithelium and extensive brush border were also observed for tubular epithelial cells. Our results indicate that the in vivo microenvironment is important for the maturation of human kidney organoids. Stromal expansion and a reduction of nephron structures were observed following longer-term (12 weeks) implantation, suggesting effects on off-target cells during the induction process. Accordingly, induction efficiency and transplantation models should be improved in the future.

Biological effects of exposure to copper nanoparticles: markers of exposure

Introduction. Aerosol nanoparticles at operating metallurgical plants are potent of the significant risk factor that needs to be studied.&#x0D; The purpose of the work was to study biological effects of exposure to copper nanoparticles compared to microparticles and identify potential markers of the exposure.&#x0D; Materials and methods. Two experimental groups of rats, twelve animals each, were subchronically exposed to copper-containing nanoparticles and microparticles injected intraperitoneally. Changes in their biochemical parameters, tissues, and organs were then compared to those in the control rats administered with deionized water. During periodic medical examinations of workers occupationally exposed to copper-containing aerosols with different particle size distribution, additional laboratory and instrumental methods of testing were used to identify possible exposure effects to health manifested by changes in biochemical parameters.&#x0D; Results. The experimental study showed that in liver, the exposure to copper nanoparticles, compared to microparticles, induced a more significant inhibition of replication of hepatocytes in regeneration, as demonstrated by the number of binuclear cells, and an increase in the number of Kupffer cells (tissue macrophages), while in kidney, it caused a more pronounced convoluted tubule brush border loss. The exposure to both micro- and nano-sized copper particles was found to induce pathological changes in neurons of the ganglionic layer of the cortex and basal nuclei. Test results showed a significant increase in the blood concentration of ceruloplasmin in the core personnel of the metallurgical plant following the exposure to copper aerosols rich in nanoparticles.&#x0D; Limitations. The assessment of the results of the impact of copper nanoparticles in the composition of the aerosol on the health of 224 employees of the enterprise and the results of the experiment obtained on 36 animals corresponded to the current social and ethical standards and norms.&#x0D; Conclusion. Specifics of the exposure to nanoparticles require a revision of approaches to regulating aerosols with account for particle size distribution.

A Novel Model of Pathogenesis of Metarhizium anisopliae Propagules through the Midguts of Aedes aegypti Larvae.

We assessed the effect of the entomopathogenic fungus Metarhizium anisopliae against Aedes aegypti. Conidia of M. anisopliae strains CG 489, CG 153, and IBCB 481 were grown in Adamek medium under different conditions to improve blastospore production. Mosquito larvae were exposed to blastospores or conidia of the three fungal strains at 1 × 107 propagules mL-1. M. anisopliae IBCB 481 and CG 153 reduced larval survival by 100%, whereas CG 489 decreased survival by about 50%. Blastospores of M. anisopliae IBCB 481 had better results in lowering larval survival. M. anisopliae CG 489 and CG 153 reduced larval survival similarly. For histopathology (HP) and scanning electron microscopy (SEM), larvae were exposed to M. anisopliae CG 153 for 24 h or 48 h. SEM confirmed the presence of fungi in the digestive tract, while HP confirmed that propagules reached the hemocoel via the midgut, damaged the peritrophic matrix, caused rupture and atrophy of the intestinal mucosa, caused cytoplasmic disorganization of the enterocytes, and degraded the brush border. Furthermore, we report for the first time the potential of M. anisopliae IBCB 481 to kill Ae. aegypti larvae and methods to improve the production of blastospores.

The Influence of Alcohol Consumption on Intestinal Nutrient Absorption: A Comprehensive Review.

Chronic alcohol use has been attributed to the development of malnutrition. This is in part due to the inhibitory effect of ethanol on the absorption of vital nutrients, including glucose, amino acids, lipids, water, vitamins, and minerals within the small intestine. Recent advances in research, along with new cutting-edge technologies, have advanced our understanding of the mechanism of ethanol's effect on intestinal nutrient absorption at the brush border membrane (BBM) of the small intestine. However, further studies are needed to delineate how ethanol consumption could have an impact on altered nutrient absorption under various disease conditions. Current research has elucidated the relationship of alcohol consumption on glucose, glutamine, vitamins B1 (thiamine), B2 (riboflavin), B9 (folate), C (ascorbic acid), selenium, iron, and zinc absorption within the small intestine. We conducted systematic computerized searches in PubMed using the following keywords: (1) "Alcohol effects on nutrient transport"; (2) "Alcohol mediated malabsorption of nutrients"; (3) "Alcohol effects on small intestinal nutrient transport"; and (4) "Alcohol mediated malabsorption of nutrients in small intestine". We included the relevant studies in this review. The main objective of this review is to marshal and analyze previously published research articles and discuss, in-depth, the understanding of ethanol's effect in modulating absorption of vital macro and micronutrients in health and disease conditions. This could ultimately provide great insights in the development of new therapeutic strategies to combat malnutrition associated with alcohol consumption.

A human Caco-2-based co-culture model of the inflamed intestinal mucosa for particle toxicity studies

The intestinal barrier is a complex interface of the human body, possessing the largest contact surface to nutrients and antigens and containing a major part of the immune system. It has to deal with continuous exposure to a broad mixture of essential, harmful, or useless substances and particles. In the context of plastic pollution and the ubiquitous occurrence of micro- and nanoplastics, oral exposure to such particles is of particular interest. Standard intestinal in vitro models, however, are unable to mimic the role of the immune system in the particle-exposed intestine. To allow for a closer look on the effect of particles on the intestinal immune system, we here developed a co-culture model to enable investigation of the epithelial brush border monolayer in a healthy and inflamed state. The model is based on well-established Caco-2 intestinal epithelial cells cultured in a Transwell™ system. Intraepithelial immune cells were mimicked by THP-1-derived M0-macrophages and MUTZ-3-derived dendritic cells. To fulfill the requirements needed for the investigation of particles, the co-culture system was developed without an additional matrix layer. Cell–cell contacts were established between interstitial and immune cells, and the Caco-2 standard cell culture medium was used, which is well-characterized for its role in defining the identity of particle dispersions. The model was characterized using confocal microscopy, membrane integrity measurements, and cytokine release assays from inflamed and healthy cells. Finally, the new co-culture model was used for investigation on polylactic acid, melamine formaldehyde resin, and polymethylmethacrylate plastic micro- and nanoparticles.

Nitrogen transporters along the intestinal spiral valve of cloudy catshark (Scyliorhinus torazame): Rhp2, Rhbg, UT

As part of their osmoregulatory strategy, marine elasmobranchs retain large quantities of urea to balance the osmotic pressure of the marine environment. The main source of nitrogen used to synthesize urea comes from the digestion and absorption of food across the gastrointestinal tract. In this study we investigated possible mechanisms of nitrogen movement across the spiral valve of the cloudy catshark (Scyliorhinus torazame) through the molecular identification of two Rhesus glycoprotein ammonia transporters (Rhp2 and Rhbg) and a urea transporter (UT). We used immunohistochemistry to determine the cellular localizations of Rhp2 and UT. Within the spiral valve, Rhp2 was expressed along the apical brush border membrane, and UT was expressed along the basolateral membrane and the blood vessels. The mRNA abundance of Rhp2 was significantly higher in all regions of the spiral valve of fasted catsharks compared to fed catsharks. The mRNA abundance of UT was significantly higher in the anterior spiral valve of fasted catsharks compared to fed. The mRNA transcript of four ornithine urea cycle (OUC) enzymes were detected along the length of the spiral valve and in the renal tissue, indicating the synthesis of urea via the OUC occurs in these tissues. The presence of Rhp2, Rhbg, and UT along the length of the spiral valve highlights the importance of ammonia and urea movement across the intestinal tissues, and increases our understanding of the mechanisms involved in maintaining whole-body nitrogen homeostasis in the cloudy catshark.

Effects of supplemental fulvic acid on survival, growth performance, digestive ability and immunity of large yellow croaker (Larimichthys crocea) larvae.

A 30-day feeding trial was designed to evaluate the effect of supplemental fulvic acid (FA) on survival, growth performance, digestive ability and immunity of large yellow croaker (Larimichthys crocea) larvae (initial body weight 11.33 ± 0.57mg). Four isonitrogenous and isolipids diets containing 0.00%, 0.01%, 0.02% and 0.04% FA were formulated, respectively. Results showed that the supplementation of 0.04% FA significantly improved survival rate of large yellow croaker larvae. Meanwhile, supplemental FA significantly increased final body weight and specific growth rate. Based on the specific growth rate, the optimal supplementation was 0.0135% FA. Larvae fed the diet with 0.01% FA had significantly higher villus height than the control. The supplementation of 0.01%-0.02% FA significantly increased the muscular thickness of intestine. Moreover, supplementation of FA significantly increased mRNA expression of intestinal epithelial proliferation and barrier genes (pcna, zo-1 and zo-2). Diets supplemented with 0.02%-0.04% FA significantly increased the activity of trypsin in the intestinal segment, while 0.01%-0.02% FA significantly increased the activity of trypsin in the pancreatic segment. Compared with the control, supplementation of FA remarkably increased activities of alkaline phosphatase and leucine aminopeptidase in the brush border membrane of intestine. Larvae fed the diet with 0.01% FA significantly increased activities of lysozyme and total nitric oxide synthase. Furthermore, the supplementation of 0.01% to 0.02% FA significantly decreased the mRNA expression of pro-inflammatory cytokines (tnf-α and il-6). Concurrently, supplemental FA significantly increased anti-inflammatory cytokine (il-10) mRNA expression level. In conclusion, this study indicated that the supplementation of FA could improve the survival rate and growth performance of larvae by promoting intestinal development, digestive enzymes activities and innate immunity.

Singed and vinculin play redundant roles in cell migration by regulating F-actin.

Drosophila Singed (mammalian Fascin) is an actin-binding protein that is known mainly for bundling parallel actin filaments. Among many functions of Singed, it is required for cell motility for both Drosophila and mammalian systems. Increased Fascin-1 levels positively correlate with greater metastasis and poor prognosis in human cancer. Border cell cluster, forms and migrates during Drosophila egg chamber development, shows higher expression of Singed compared with other follicle cells. Interestingly, loss of singed in border cells does not lead to any effect other than delay. In this work, we have screened many actin-binding proteins in search of functional redundancy with Singed for border cell migration. We have found that Vinculin works with Singed to regulate border cell migration, albeit mildly. Although Vinculin is known for anchoring F-actin to the membrane, knockdown of both singed and vinculin leads to a reduced level of F-actin and changes in protrusion characteristics in border cells. We have also observed that they may act together to control microvilli length of brush border membrane vesicles and the shape of egg chambers in Drosophila. We may conclude that singed and vinculin work together to control F-actin and these interactions are consistent across multiple platforms.

Transplantation of fecal filtrate to neonatal pigs reduces post-weaning diarrhea: A pilot study.

Post-weaning diarrhea (PWD) remains a major source of mortality and morbidity in swine production. Transplantation of bacteria-free filtrate of feces (fecal filtrate transplant, FFT) has shown gut protective effects in neonatal pigs, and early postnatal establishment of the gut microbiome is suggested to determine later stability and robustness of the gut. We, therefore, hypothesized that early postnatal transplantation of bacteria-free feces would have a protective effect against PWD. Using fecal filtrates derived from healthy lactating sows, we compared oral administration of fecal filtrate transplantation (FFT, n = 20) and saline (CON, n = 18) in newborn piglets. We assessed growth, diarrhea prevalence, blood parameters, organ measurements, morphology, and gut brush border enzymes and analyzed luminal bacterial composition using 16S rRNA gene amplicon sequencing. The two groups showed similar average daily gain (ADG) during the suckling period, whereas in the post-weaning period, a negative ADG was observed in both groups. While diarrhea was largely absent in both groups before weaning, there was a lower diarrhea prevalence on days 27 (p = 2.07*10-9), 28 (p = 0.04), and 35 (p = 0.04) in the FFT group relative to CON. At weaning on day 27, the FFT group had higher numbers of red blood cells, monocytes, and lymphocytes, while on day 35, i.e., 1 week after weaning, the two groups were similar regarding hematology. The biochemical profile was largely similar between FFT and CON on days 27 and 35, except for a higher level of alanine aminotransferase and a lower level of Mg in the FFT group. Likewise, organ weights relative to body weight were largely similar on day 35, albeit with a lower stomach weight and more colon content in FFT relative to CON. Gut mucosal percentage and mucosal enzyme activity were similar between the two groups on days 27 and 35. Gut bacterial composition was slightly different on day 35 but not on day 27. In conclusion, early postnatal administration of FFT, showed positive clinical effects in post-weaning pigs, albeit with subtle effects on the gut mucosa and microbiome. Prophylactic treatment with FFT may offer a means to reduce morbidity, yet larger studies are required to document effect size.

Fluorescent visualization and evaluation of NPC1L1-mediated vesicular endocytosis during intestinal cholesterol absorption in mice

Abstract Excessive cholesterol absorption from intestinal lumen contributes to the pathogenesis of hypercholesterolemia, which is an independent risk factor for atherosclerotic cardiovascular disease. Niemann-Pick C1-like 1 (NPC1L1) is a major membrane protein responsible for cholesterol absorption, in which the physiological role of vesicular endocytosis is still controversial, and it lacks a feasible tool to visualize and evaluate the endocytosis of NPC1L1 vesicles in vivo. Here, we genetically labeled endogenous NPC1L1 protein with EGFP in a knock-in mouse model, and demonstrated fluorescent visualization and evaluation of the endocytic vesicles of NPC1L1-cago during intestinal cholesterol absorption. The homozygous NPC1L1-EGFP mice have normal NPC1L1 expression pattern as well as cholesterol homeostasis on chow or high-cholesterol diets. The fluorescence of NPC1L1-EGFP fusion protein localizes at the brush border membrane of small intestine, and EGFP-positive vesicles is visualized beneath the membrane as early as 5 min post oral gavage of cholesterol. Of note, the vesicles colocalize with the early endosomal marker early endosome antigen 1 (EEA1) and the filipin-stained free cholesterol. Pretreatment with NPC1L1 inhibitor ezetimibe inhibits the formation of these cholesterol-induced endocytic vesicles. Our data support the notion that NPC1L1-mediated cholesterol absorption is a vesicular endocytic process. NPC1L1-EGFP mice are a useful model for visualizing cellular NPC1L1-cargo vesicle itineraries and for evaluating NPC1L1 activity in vivo in response to diverse pharmacological agents and nutrients.

Interaction between the α-glucosidases, sucrase-isomaltase and maltase-glucoamylase, in human intestinal brush border membranes and its potential impact on disaccharide digestion.

The two major intestinal α-glycosidases, sucrase-isomaltase (SI) and maltase-glucoamylase (MGAM), are active towards α-1,4 glycosidic linkages that prevail in starch. These enzymes share striking structural similarities and follow similar biosynthetic pathways. It has been hypothesized that starch digestion can be modulated via "toggling" of activities of these mucosal α-glycosidases, suggesting a possible interaction between these two enzyme complexes in the intestinal brush border membrane (BBM). Here, the potential interaction between SI and MGAM was investigated in solubilized BBMs utilizing reciprocal pull down assays, i.e., immunoprecipitation with anti-SI antibody followed by Western blotting with anti-MGAM antibody and vice versa. Our results demonstrate that SI interacts avidly with MGAM concomitant with a hetero-complex assembly in the BBMs. This interaction is resistant to detergents, such as Triton X-100 or Triton X-100 in combination with sodium deoxycholate. By contrast, inclusion of sodium deoxycholate into the solubilization buffer reduces the enzymatic activities towards sucrose and maltose substantially, most likely due to alterations in the quaternary structure of either enzyme. In view of their interaction, SI and MGAM regulate the final steps in starch digestion in the intestine, whereby SI assumes the major role by virtue of its predominant expression in the intestinal BBMs, while MGAM acts in auxiliary supportive fashion. These findings will help understand the pathophysiology of carbohydrate malabsorption in functional gastrointestinal disorders, particularly in irritable bowel syndrome, in which gene variants of SI are implicated.