Brush Border Research Articles

Fine structure of the posterior midgut in the mite Anystis baccarum (L.)

Homology of the posterior midgut regions (PMG) in different phylogenetic lineages of acariform mites (superorder Acariformes) remains unresolved. In the order Trombidiformes, the ultrastructure of the PMG is known primarily in derived groups; thus this study focuses on species belonging to a relatively basal trombidiform family.PMG of Anystis baccarum consists of the colon and postcolon separated by a small intercolon. The fine structure of the colon and postcolon is close to that of the corresponding organs of sarcoptiform mites with the epithelium showing absorptive and endocytotic activity. The epithelial cells produce a variety of excretory vacuoles and a peritrophic matrix around the feces. Morover, the epithelium of the postcolon is characterized by the highest apical brush border and especially numerous mitochondria suggesting involvement in water and ion absorption. The intercolon functions as a sphincter lined with an epithelium capable of producing excretory granules. A pair of short blind extensions arises assimmetrically from the intercolon into the body cavity. Ultrastructurally, these extensions are similar to the arachnid Malpighian tubules and may be their reduced version. Rare endocrine-like cells have been observed in the colon and postcolon.

Bile components affect the functions and transcriptome of the rainbow trout intestinal epithelial cell line RTgutGC

The concomitant increase in cultivation of fish and decrease in supply of marine ingredients, have greatly increased the demand for new nutrient sources. This also regards so-called functional ingredients which may benefit health and welfare of the fish. In vitro cell line-based intestinal epithelial barrier models may serve as tools for narrowing down the broad range of ingredient options, to identify the most promising candidates before in vivo feeding trials are run. In vivo, differentiation of the various epithelial cells in the fish intestine, from the multipotent stem cells, takes place in the presence of a variety of substances from dietary and endogenous origin. Among these, bile salts have recently received attention as regulators of epithelial function in health and disease but have not, until now, been included in the medium when culturing fish gut epithelial cells in vitro. As bile salts are present at high levels in the chyme of the fish intestine, in particular in salmon and rainbow trout, mostly as taurocholate (>90%), their role for effects of diet ingredients on the in vitro gut cell model should be understood.With this study, we wanted to investigate whether inclusion of bile from rainbow trout or pure taurocholate in the culture media would modulate functions of the RTgutGC epithelial cells. Here, we demonstrated that the rainbow trout intestinal epithelial cell line RTgutGC responded significantly to the presence of bile components. Treatment with rainbow trout bile taken from the gall bladder (RTbile) or pure taurocholate (TC) at taurocholate concentrations of ≤0.5 mg/mL retained normal cell morphology, cell viability as in cell oxidation-reduction metabolic activity and membrane integrity, and barrier features, while high concentrations of bile salts (≥1 mg/mL) were cytotoxic to the cells. After long-term (4 days) bile treatment, transcriptome responses showed how bile salts play important roles in intestinal epithelial cell metabolism. qPCR data demonstrated that barrier function genes, brush border enzyme genes and immune genes were significantly affected. Although similar trends were seen, treatment with bile salt as a component of rainbow trout bile or pure taurocholate, induced somewhat different effects. In conclusion, this study clearly indicates that bile salts should be included in the cell medium when running in vitro studies of gut cell functions, not at least immune functions, preferably at the level of ∼0.5 mg/mL supplemented as pure taurocholate to ensure reproducibility.

Fucoidan Improves Growth, Digestive Tract Maturation, and Gut Microbiota in Large Yellow Croaker (Larimichthys crocea) Larvae.

The early life period is considered an essential period for gut microbial colonization. Manipulating gut microbiota interventions during early life periods has been proven to be a promising method to boost healthy growth. Therefore, the aim of the present study was to investigate the effects of dietary fucoidan (Fuc) on the growth, digestive tract maturation, and gut microbiota of large yellow croaker (Larimichthys crocea) larvae. Four diets were formulated with different levels of Fuc (0.00%, 0.50%, 1.00%, and 2.00%). Results showed that dietary Fuc significantly improved the growth performance of larvae. Meanwhile, dietary Fuc promoted digestive tract maturation. Dietary 1.00% Fuc significantly improved intestinal morphology. Dietary Fuc upregulated the expression of intestinal cell proliferation and differentiation related-genes and intestinal barrier related-genes. Dietary 2.00% Fuc significantly increased the activities of brush border membranes enzymes and lipase while inhibiting α-amylase. Furthermore, dietary Fuc maintained healthy intestinal micro-ecology. In detail, dietary 1.00% and 2.00% Fuc altered the overall structure of the gut microbiota and increased the relative abundance of Bacteroidetes while decreasing the relative abundance of opportunistic pathogens and facultative anaerobe. In conclusion, appropriate dietary Fuc (1.00-2.00%) could improve the growth of large yellow croaker larvae by promoting digestive tract maturation and maintaining an ideal intestinal micro-ecology.

The morphological and functional diversity of apical microvilli.

Sensory neurons use specialized apical processes to perceive external stimuli and monitor internal body conditions. The apical apparatus can include cilia, microvilli, or both, and is adapted for the functions of the particular cell type. Photoreceptors detect light through a large, modified cilium (outer segment), that is supported by a surrounding ring of microvilli-like calyceal processes (CPs). Although first reported 150 years ago, CPs remain poorly understood. As a basis for future study, we therefore conducted a review of existing literature about sensory cell microvilli, which can act either as the primary sensory detector or as support for a cilia-based detector. While all microvilli are finger-like cellular protrusions with an actin core, the processes vary across cell types in size, number, arrangement, dynamics, and function. We summarize the current state of knowledge about CPs and the characteristics of the microvilli found on inner ear hair cells (stereocilia) and cerebral spinal fluid-contacting neurons, with comparisons to the brush border of the intestinal and renal epithelia. The structure, stability, and dynamics of the actin core are regulated by a complement of actin-binding proteins, which includes both common components and unique features when compared across cell types. Further, microvilli are often supported by lateral links, a glycocalyx, and a defined extracellular matrix, each adapted to the function and environment of the cell. Our comparison of microvillar features will inform further research into how CPs support photoreceptor function, and also provide a general basis for investigations into the structure and functions of apical microvilli found on sensory neurons.

Helical ultrastructure of the metalloprotease meprin α in complex with a small molecule inhibitor

The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin β, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin β reveal unique features of the active site of meprin α, and helical assembly more broadly.

Proteomics profiling of kidney brush border membrane from rats using LC-MS/MS analysis.

Chronic kidney disease (CKD) is defined by a reduced renal function, that is, glomerular filtration rate, and the extent of kidney damage is assessed by determining serum creatinine levels and proteins in urine, diagnosed as proteinuria/albuminuria. Albuminuria increases with age and can result from glomerular and/or proximal tubule (PT) alterations. Brush border membranes (BBMs) on PT cells are important in maintaining the stability of PT functions. An LC-MS/MS bottom-up proteomics analysis of BBMs from four groups of rat models was applied to investigate protein abundance alterations associated with CKD progression. Moreover, systems biology analyses were used to identify key proteins that can provide insight into the different regulated molecular pathways and processes associated with CKD. Our results indicated that 303 proteins showed significantly altered expressions from the severe CKD BBM group when compared to the control. Focusing on renal diseases, several proteins including Ctnnb1, Fah, and Icam1 were annotated to kidney damage and urination disorder. The up-regulation of Ctnnb1 (β-catenin) could contribute to CKD through the regulation of the WNT signaling pathway. Overall, the study of protein abundance changes in BBMs from rat models helps to reveal protein corrections with important pathways and regulator effects involved in CKD. Although this study is focused on rat models, the results provided more information for a deeper insight into possible CKD mechanisms in humans.

The emerging double-edged sword role of Sirtuins in the gastric inflammation-carcinoma sequence revealed by bulk and single-cell transcriptomes.

Histone modification and the inflammation-carcinoma sequence (ICS) have been acknowledgedly implicated in gastric carcinogenesis. However, the extremum expression of some histone modification genes (HMGs) in intestinal metaplasia (IM) rather than GC obscures the roles of HMGs in ICS. In this study, we assumed an explanation that the roles of HMGs in ICS were stage specific. Bulk RNA-seq on endoscopy biopsy samples from a total of 50 patients was accompanied by reanalysis of a set of published single-cell transcriptomes, which cross-sectionally profiled the transcriptomic features of chronic superficial gastritis (SG), atrophy gastritis (AG), IM, and early gastric cancer (GC). Differential analysis observed significantly peaked expression of SIRT6 and SIRT7 at IM. Weighted correlation network analysis on bulk transcriptome recognized significant correlations between SIRT1/6 and IM. The single-cell atlas identified one subgroup of B cells expressing high level of TFF1 (TFF1 hi naive B cell) that theoretically played important roles in defending microbial infection, while SIRT6 displayed a positive correlation with TFF1 low naive B cells. Moreover, gene set enrichment analysis at different lesions (SG-AG, AG-IM, and IM-GC) highlighted that gene sets contributing to IM, e.g., Brush Border, were largely enriched from co-expressing genes of Sirtuins (SIRTs) in AG-IM. Surveys of the genes negatively correlated with SIRT6 in public databases considered SIRT6 as tumor suppressors, which was confirmed by the cell proliferation and migration assays after transient transfection of SIRT6 overexpression vector into AGS cells. All the above observations were then confirmed by serial section-based immunohistochemistry against Ki-67, MUC2, MUC5AC, p53, and SIRT6 on the endoscopic submucosal dissection tissue. By contrast, the expression of the other HMGs varied even opposite within same family. Taken together, this study preliminarily demonstrated the two-edged sword role of SIRTs in ICS and, by extension, showed that the roles of HMGs in ICS were probably stage specific. Our study may provide new insights into and attract attention on gastric prevention and therapy targeting HMGs.

The beneficial effects of exogenous protease K originated from Parengyodontium album on growth performance of grass carp (Ctenopharyngodon idella) in relation to the enhanced intestinal digestion and absorption capacities

The study was primarily designed to analyze the effect of exogenous protease K on growth performance, amino acid metabolism, digestion and absorption capacities, expression of amino acid transporters related genes and proteins in intestine of sub-adult grass carp (Ctenopharyngodon idella). Six experimental diets with protease K (0, 3000, 6000, 9000, 12,000 and 15,000 U/kg) were fed to 450 grass carp (619.51 ± 0.54 g) for 60 days. The results indicated that the highest value of final body weight, percent weight gain (PWG), specific growth rate, protein efficiency ratio, activity of hepatopancreas glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase, and the lowest concentration of plasma ammonia were observed in groups with protease K of 6000 and 9000 U/kg. Meanwhile, the groups with 6000 and 9000 U/kg protease K exhibited the highest activities of digestive and brush border membrane enzymes in the three intestinal segments, as well as the highest value of intestinal somatic index and intestinal length index. The highest concentration of free amino acid in the serum and the lowest in intestine were observed in groups with supplementation of protease K 6000 and 9000 U/kg, which might be related to the up-regulated mRNA level and protein expression of intestinal amino acid transporters (SLC1A5, SLC7A6, SLC1A3 et al) in these groups. Furthermore, optimal levels of protease K up-regulated the mRNA levels of target of rapamycin (TOR), S6 kinase 1 (S6K1), and protein expression of TOR and phosphorylation TORSer2448, but reduced the mRNA level of 4E binding protein 1 (4EBP1). In summary, optimal levels of exogenous protease K was found to be conducive to facilitate the improvement of growth performance probably via enhancing digestion and absorption capacity, and utilization of amino acid in sub-adult grass carp. In addition, using quadratic regression analysis of PWG and alkaline phosphatase in the midgut, the optimum addition of protease K for the sub-adult grass carp were noted to be 7380.82 and 7406.44 U/kg, respectively.

Ojeoksan Ameliorates Cisplatin-Induced Acute Kidney Injury in Mice by Downregulating MAPK and NF-κB Pathways.

Acute kidney injury (AKI) is a major side effect of cisplatin, a crucial anticancer agent. Therefore, it is necessary to develop drugs to protect against cisplatin-induced nephrotoxicity. Ojeoksan (OJS), a traditional blended herbal prescription, is mostly used in Korea; however, there are no reports on the efficacy of OJS against cisplatin-induced AKI. To investigate the reno-protective effect of OJS on AKI, we orally administered 50, 100, and 200 mg/kg of OJS to mice 1 h before intraperitoneal injection with 20 mg/kg of cisplatin. OJS inhibited the increase of blood urea nitrogen (BUN) and serum creatinine (SCr) levels and reduced histological changes in the kidney, like loss of brush borders, renal tubular necrosis, and cast formation. Administration of OSJ reduced the levels of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α. In addition, OJS inhibited the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways in cisplatin-induced AKI. These results suggest that OJS attenuates cisplatin-induced AKI by downregulating the MAPK and NF-κB pathways.

The intermicrovillar adhesion complex in gut barrier function and inflammation.

The surface of intestinal epithelial cells is covered by the brush border, which consists of densely packed cellular extrusions called microvilli. Until recently, microvilli have not been known to be interconnected. In 2014, a protein complex, called the intermicrovillar adhesion complex (IMAC) which is located at the tips of the microvilli and responsible for the regular spatial organization of the brush border, was identified. Deletion of IMAC components such as cadherin-related family member-2 (CDHR2) in mice resulted in microvillus disorganization and fanning, a structural aberration that is also found in the brush border of patients with inflammatory bowel disease. The etiology of inflammatory bowel disease has been primarily associated with dysfunctional mucosal immunity, but the discovery of the IMAC may encourage theories of an epithelial origin. Here, possible effects of the brush border on the gut barrier function and intestinal inflammation are discussed proposing that the IMAC protects against inflammation through its microvillus cross-linking function.

Myosin 5b is required for proper localization of the intermicrovillar adhesion complex in the intestinal brush border.

Intestinal enterocytes have an elaborate apical membrane of actin-rich protrusions known as microvilli. The organization of microvilli is orchestrated by the intermicrovillar adhesion complex (IMAC), which connects the distal tips of adjacent microvilli. The IMAC is composed of CDHR2 and CDHR5 as well as the scaffolding proteins USH1C, ANKS4B, and Myosin 7b (MYO7B). To create an IMAC, cells must transport the proteins to the apical membrane. Myosin 5b (MYO5B) is a molecular motor that traffics ion transporters to the apical membrane of enterocytes, and we hypothesized that MYO5B may also be responsible for the localization of IMAC proteins. To address this question, we used two different mouse models: 1) neonatal germline MYO5B knockout (MYO5B KO) mice and 2) adult intestinal-specific tamoxifen-inducible VillinCreERT2;MYO5Bflox/flox mice. In control mice, immunostaining revealed that CDHR2, CDHR5, USH1C, and MYO7B were highly enriched at the tips of the microvilli. In contrast, neonatal germline and adult MYO5B-deficient mice showed loss of apical CDHR2, CDHR5, and MYO7B in the brush border and accumulation in a subapical compartment. Colocalization analysis revealed decreased Mander's coefficients in adult inducible MYO5B-deficient mice compared with control mice for CDHR2, CDHR5, USH1C, and MYO7B. Scanning electron microscopy images further demonstrated aberrant microvilli packing in adult inducible MYO5B-deficient mouse small intestine. These data indicate that MYO5B is responsible for the delivery of IMAC components to the apical membrane.NEW & NOTEWORTHY The intestinal epithelium absorbs nutrients and water through an elaborate apical membrane of highly organized microvilli. Microvilli organization is regulated by the intermicrovillar adhesion complexes, which create links between neighboring microvilli and control microvilli packing and density. In this study, we report a new trafficking partner of the IMAC, Myosin 5b. Loss of Myosin 5b results in a disorganized brush border and failure of IMAC proteins to reach the distal tips of microvilli.

Enteropathy endoscopic findings in small intestine in patients with severe COVID-19

Background and aim: Small intestine study is a challenge in the field of clinical research. However, capsule endoscopy is a safe and minimally invasive alternative. During the COVID-19 pandemic, this endoscopic procedure acquires added value. Although SARS-CoV-2 has mainly respiratory manifestations, gastrointestinal symptoms are observed in 30% of cases. ACE-2 used by SARS-CoV-2 to infect cells is highly expressed in the brush border of enterocytes. This enzyme transports essential amino acids in the small intestine and regulates intestinal inflammation through antimicrobial peptides production. Here we describe the endoscopic changes in the mucosa of the small intestine secondary to severe SARS-CoV-2 infection in patients with and without gastrointestinal symptoms. Methods: We performed a prospective and observational study in hospitalized patients with severe COVID-19 disease. Capsule endoscopy and detection of SARS-CoV-2 RNA in feces were performed in each participant. Each capsule was reviewed separately by two trained endoscopists. Results: Twenty individuals were enrolled. Diarrhea was observed in 78% of participants. In 45% of capsule endoscopy studies, changes characterized by shortening of villi, atrophy or denuded areas accompanied by red spots or patches, hyperemia or edema were found. Individuals showing these changes in the intestinal mucosa were positive for SARS-CoV-2 in stool. Conclusions: Here we provide evidence of macroscopic changes along the small intestine in COVID-19 patients. We proposed the term of COVID-19 enteropathy to denote involvement of the mucosa and in the villi of the small intestine in SARS-CoV-2 infected individuals. Longitudinal follow-up will be valuable for a better understanding of COVID-19 enteropathy.

An adherent-invasive Escherichia coli-colonized mouse model to evaluate microbiota-targeting strategies in Crohn's disease.

Adherent-invasive Escherichia coli (AIEC) were investigated for their involvement in the induction/chronicity of intestinal inflammation in Crohn's disease (CD). AIEC gut establishment is favoured by overexpression of the glycoprotein CEACAM6 in the ileal epithelium. We generated a transgenic mouse model, named 'Vill-hCC6', in which the human CEACAM6 gene was under the control of the villin promoter, conditioning expression in the small intestine. We demonstrated that CEACAM6 is strongly expressed in the small intestine mucosa and is correlated with numerous glycosylations displayed at the brush border of enterocytes. Ex vivo, the AIEC-enterocyte interaction was enhanced by CEACAM6 expression and necessitated the presence of the bacterial adhesive factor FimH. Finally, AIEC bacteria preferentially persisted in a FimH-dependent manner in the ileal mucosa of Vill-hCC6 mice compared to wild-type mice. This preclinical model opens new perspectives in the mechanistic study of the AIEC pathobiont and represents a valuable tool to evaluate the efficacy of new strategies to eliminate AIEC implanted in the ileal mucosa, such as phages, inhibitory and/or anti-virulence molecules, or CRISPR-based strategies targeting virulence or fitness factors of AIEC bacteria.

Effect of Pomegranate Peels Aqueous Extract on the Histological Structure of Small Intestine of Local Male Rabbits (Oryctolagus cuniculus).

Most feed materials are predominantly complex and in insoluble forms as animals use them. The aim of digestion processes, therefore, is to sequentially modify the feed substances into simple and soluble forms that are appropriate for absorption and ingestion. This study aimed to investigate the possible effect of pomegranate peel aqueous extract on the histological structure of the small intestine of local male rabbits (Oryctolagus cuniculus). Twelve healthy adult male rabbits acquired from a local market in Iraq- Baghdad was used in this study. The animals were divided into two main groups, six animals in the control group, while the other six were considered as treated groups with pomegranate peel extract. The pomegranate extract was prepared by the maceration method. The experiment lasted for two months, from 1/10/2020 to 1/12/2020. The results showed that the tunica mucosa of the duodenum of the experimental group has huge overcrowded intestinal villi associated with hypertrophy of mucosal villi, in addition to a marked increase in the population of goblet cells and their secretory activities. The tunica submucosa was occupied by a thick layer of Brunner's glands, which were compound tubular mucous alveolar type and had clear cytoplasm. The jejunum of the experimental group showed that the mucosal layer was characterized by a marked increase of intestinal plica circularis, which is associated with the projection of pyramidal-shaped intestinal mucosa. The epithelial mucosa was (pseudostratified columnar epithelium) covered by a thick brush border of microvilli. The histological arrangement of various layers of the ileum was nearly analogous to that of the jejunum except for some distinctive differences. An evident aggregated lymphocytes forming Payers patches occupied the tunica submucosa in both the control and treated groups. The most historical parameters in all segments of the small intestine revealed a significant increase in experimental animals compared to control animals. In conclusion, the aqueous extract of pomegranate peels have apparent positive effects on the histological structure of the small intestine of rabbits improving, positively the digestive efficiency of animals and enhancing the efficiency of the immune system in the animal through an apparent increase in the numbers of goblet cell that plays importance for immunity of the body.

Measuring key human carbohydrate digestive enzyme activities using high-performance anion-exchange chromatography with pulsed amperometric detection.

Carbohydrate digestion in the mammalian gastrointestinal tract is catalyzed by α-amylases and α-glucosidases to produce monosaccharides for absorption. Inhibition of these enzymes is the major activity of the drugs acarbose and miglitol, which are used to manage diabetes. Furthermore, delaying carbohydrate digestion via inhibition of α-amylases and α-glucosidases is an effective strategy to blunt blood glucose spikes, a major risk factor for developing metabolic diseases. Here, we present an in vitro protocol developed to accurately and specifically assess the activity of α-amylases and α-glucosidases, including sucrase, maltase and isomaltase. The assay is especially suitable for measuring inhibition by compounds, drugs and extracts, with minimal interference from impurities or endogenous components, because the substrates and digestive products in the enzyme activity assays are quantified directly by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). Multiple enzyme sources can be used, but here we present the protocol using commercially available human α-amylase to assess starch hydrolysis with maltoheptaose as the substrate, and with brush border sucrase-isomaltase (with maltase, sucrase and isomaltase activities) derived from differentiated human intestinal Caco-2(/TC7) cells to assess hydrolysis of disaccharides. The wet-lab assay takes ~2-5 h depending on the number of samples, and the HPAE-PAD analysis takes 35 min per sample. A full dataset therefore takes 1-3 d and allows detection of subtle changes in enzyme activity with high sensitivity and reliability.

Effects of supplemental ferulic acid (FA) on survival, growth performance, digestive enzyme activities, antioxidant capacity and lipid metabolism of large yellow croaker (Larimichthys crocea) larvae.

A 30-day feeding trial was conducted to investigate the effects of supplemental ferulic acid (FA) on survival, growth performance, digestive enzyme activities, antioxidant capacity and lipid metabolism of the large yellow croaker larvae (initial weight: 2.58 ± 0.30mg). Four isonitrogenous and isolipidic micro-diets were formulated with graded levels of FA (0, 20, 40, and 80mg/kg) and fed to the experimental larvae seven times daily. Results showed that larvae fed the diet with 40mg/kg FA had significantly higher survival rate, while the specific growth rate was higher in larvae fed diets with 40 and 80mg/kg FA than the control group (P < 0.05). Activities of trypsin in pancreatic segments (PS) and intestinal segments, lipase in PS and alkaline phosphatase in brush border membrane were significantly increased by supplementation of FA compared to the control group (P < 0.05). Supplementation of FA significantly increased activities of total superoxide dismutase and catalase, and reduced the malondialdehyde content compared to the control group (P < 0.05). Meanwhile, activities of lysozyme, total nitric oxide synthase and nitric oxide content were significantly improved by supplemental FA in diets. Furthermore, supplementation of 40mg/kg FA reduced the triglyceride content in larval visceral mass probably through down-regulating expression of lipogenesis-related genes (scd1, fas and dgat2) and up-regulating expression of lipid catabolism-related genes (aco, cpt-1 and hl). In conclusion, appropriate supplementation of 40mg/kg FA could improve the survival and growth performance of large yellow croaker larvae through increasing digestive function, antioxidant capacity and promoting lipid metabolism.

Dual MVK cleavable linkers effectively reduce renal retention of 111In-fibronectin-binding peptides

BackgroundPreviously, we have exploited bacterial adhesins-derived fibronectin-binding peptides (FnBPs) for targeting mechanically altered fibronectin (Fn) fibrils within the cancer-associated extra-cellular matrix (ECM). However, despite the ability of FnBP probes to visualize pathological lesions, when labeled with metallic radionuclides and administered for targeted imaging, they exhibit high and persistent retention of radioactivity within the kidneys. Intending to overcome this issue towards a future translation of FnBPs to the clinic, the goal of the present study was to reduce the renal retention of 111In-labelled FnBPs employing dual renal brush border membrane (BBM) enzyme-sensitive Met-Val-Lys-based linkers, enabling a rapid washout of radioactivity from the kidneys. MethodsThree maleimide-activated NOTA-conjugated brush border-enzyme cleavable linkers equipped with either single or dual consecutive MVK-based cleavable moieties were designed and synthesized. Their respective NOTA-MVK-based FnBPA5.1 conjugates were obtained by means of maleimide-thiol mediated conjugation at the N-terminus of the Fn-binding sequence, radiolabeled with indium-111, and further evaluated in vitro and in vivo in comparison to the control [111In]In-FnBPA5.1. ResultsThe linker equipped with two MVK sites displayed a two-fold more effective cleavage rate than the single MVK featuring linker in vitro, as revealed by the quantification of the released Met-containing radiometabolites. SPECT/CT imaging and biodistribution studies of the series of FnBPA5.1 radioconjugates performed at 24 h post-injection (p.i.) confirmed the in vitro results, indicating that the renal retention of 111In-labelled FnBPs can be significantly lowered through the interposition of a single MVK-based sequence between the Fn-targeting moiety and the chelating unit (52.75 ± 9.79 vs 92.88 ± 4.85 % iA/g, P < 0.001), and even further reduced by the addition of a second one (down to 34.82 ± 6.04, P < 0.001), with minor influence on the biodistribution in other organs, such as tumors. ConclusionsIn summary, we report here promising 111In-labelled FnBP radiotracers equipped with dual MVK-based cleavable linkers leading to a more effective reduction of renal retention and improved tumor-to-kidney ratios compared to the single MVK-featuring derivative. Our dual MVK strategy is a crucial step towards the clinical translation of mechano-sensory FnBPs and might as well be adopted for other radiopharmaceuticals suffering from persistent renal retention of radioactivity.

De novo apical domain formation inside the Drosophila adult midgut epithelium.

In the adult Drosophila midgut, basal intestinal stem cells give rise to enteroblasts that integrate into the epithelium as they differentiate into enterocytes. Integrating enteroblasts must generate a new apical domain and break through the septate junctions between neighbouring enterocytes, while maintaining barrier function. We observe that enteroblasts form an apical membrane initiation site (AMIS) when they reach the septate junction between the enterocytes. Cadherin clears from the apical surface and an apical space appears between above the enteroblast. New septate junctions then form laterally with the enterocytes and the AMIS develops into an apical domain below the enterocyte septate junction. The enteroblast therefore forms a pre-assembled apical compartment before it has a free apical surface in contact with the gut lumen. Finally, the enterocyte septate junction disassembles and the enteroblast/pre-enterocyte reaches the gut lumen with a fully formed brush border. The process of enteroblast integration resembles lumen formation in mammalian epithelial cysts, highlighting the similarities between the fly midgut and mammalian epithelia.

Butyrate Glycerides Protect against Intestinal Inflammation and Barrier Dysfunction in Mice.

This study investigates the attenuating effects of butyrate glycerides (BG) on intestinal inflammatory responses and barrier dysfunction induced by LPS stimulation. An initial dose-response test was carried out to identify the optimal dose of BG for further testing. The mice were given intragastric administration of BG at different doses followed by lipopolysaccharide (LPS) intraperitoneal injection. The small intestinal morphology and cytokine mRNA expression were measured. With 1.5 g/kg BW BG administration, it was possible to alleviate the injury of duodenal morphology, attenuate ileum villus height reduction and promote IL-10 mRNA expression. Therefore, the optimal dosage of 1.5 g/kg BW BG was selected for the main experiment. The ultrastructure image of jejunum and ileum epithelial cells, mRNA expression, the level of cytokine and immunofluorescence in the ileum were analyzed. The results showed that BG maintain the ileac brush border, tight junction structures and protein expression. BG attenuated the increased inflammatory cytokines, TLR4 and JNK mRNA expression. Taken together, 1.5 g/kg BW BG administration maintained intestinal barrier function and reduced intestinal and body inflammation responses induced by LPS in mice. The mechanism by which BG alleviated intestinal inflammatory response and maintained intestinal barrier function may be related to the JNK signaling pathway.

Alterations in Intestinal Brush Border Membrane Functionality and Bacterial Populations Following Intra-Amniotic Administration (Gallus gallus) of Catechin and Its Derivatives.

Catechin is a flavonoid naturally present in numerous dietary products and fruits (e.g., apples, berries, grape seeds, kiwis, green tea, red wine, etc.) and has previously been shown to be an antioxidant and beneficial for the gut microbiome. To further enhance the health benefits, bioavailability, and stability of catechin, we synthesized and characterized catechin pentaacetate and catechin pentabutanoate as two new ester derivatives of catechin. Catechin and its derivatives were assessed in vivo via intra-amniotic administration (Gallus gallus), with the following treatment groups: (1) non-injected (control); (2) deionized H2O (control); (3) Tween (0.004 mg/mL dose); (4) inulin (50 mg/mL dose); (5) Catechin (6.2 mg/mL dose); (6) Catechin pentaacetate (10 mg/mL dose); and (7) Catechin pentabutanoate (12.8 mg/mL dose). The effects on physiological markers associated with brush border membrane morphology, intestinal bacterial populations, and duodenal gene expression of key proteins were investigated. Compared to the controls, our results demonstrated a significant (p < 0.05) decrease in Clostridium genera and E. coli species density with catechin and its synthetic derivative exposure. Furthermore, catechin and its derivatives decreased iron and zinc transporter (Ferroportin and ZnT1, respectively) gene expression in the duodenum compared to the controls. In conclusion, catechin and its synthetic derivatives have the potential to improve intestinal morphology and functionality and positively modulate the microbiome.