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1.
Nat Commun ; 15(1): 3733, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740737

RESUMO

Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.


Assuntos
Quitina , Microvilosidades , Microvilosidades/ultraestrutura , Animais , Quitina/metabolismo , Quitina/química , Poliquetos/ultraestrutura , Actinas/metabolismo , Morfogênese
2.
Toxins (Basel) ; 16(5)2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38787067

RESUMO

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.


Assuntos
Proteínas de Bactérias , Polissacarídeos Bacterianos , Spodoptera , Animais , Proteínas de Bactérias/toxicidade , Proteínas de Bactérias/metabolismo , Polissacarídeos Bacterianos/farmacologia , Polissacarídeos Bacterianos/química , Spodoptera/efeitos dos fármacos , Larva/efeitos dos fármacos , Inseticidas/toxicidade , Inseticidas/farmacologia , Bacillus thuringiensis/metabolismo , Microvilosidades/metabolismo , Microvilosidades/efeitos dos fármacos
3.
J Virol ; 98(6): e0023524, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38775478

RESUMO

Baculoviruses enter insect midgut epithelial cells via a set of occlusion-derived virion (ODV) envelope proteins called per os infectivity factors (PIFs). P74 of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), which was the first identified PIF, is cleaved by an endogenous proteinase embedded within the occlusion body during per os infection, but the target site(s) and function of the cleavage have not yet been ascertained. Here, based on bioinformatics analyses, we report that cleavage was predicted at an arginine and lysine-rich region in the middle of P74. A series of recombinant viruses with site-directed mutants in this region of P74 were generated. R325 or R334 was identified as primary cleavage site. In addition, we showed that P74 is also cleaved by brush border membrane vesicles (BBMV) of the host insect at R325 or R334, instead of R195, R196, and R199, as previously reported. Simultaneous mutations in R195, R196, and R199 lead to instability of P74 during ODV release. Bioassays showed that mutations at both R325 and R334 significantly affected oral infectivity. Taken together, our data show that both R325 and R334 of AcMNPV P74 are the primary cleavage site for both occlusion body endogenous proteinase and BBMV proteinase during ODV release and are critical for oral infection. IMPORTANCE: Cleavage of viral envelope proteins is usually an important trigger for viral entry into host cells. Baculoviruses are insect-specific viruses that infect host insects via the oral route. P74, a per os infectivity factor of baculoviruses, is cleaved during viral entry. However, the function and precise cleavage sites of P74 remain unknown. In this study, we found that R325 or R334 between the N- and C-conserved domains of P74 was the primary cleavage site by proteinase either from the occlusion body or host midgut. The biological significance of cleavage seems to be the release of the potential fusion peptide at the N-terminus of the cleaved C-terminal P74. Our results shed light on the cleavage model of P74 and imply its role in membrane fusion in baculovirus per os infection.


Assuntos
Nucleopoliedrovírus , Animais , Nucleopoliedrovírus/metabolismo , Nucleopoliedrovírus/genética , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/genética , Internalização do Vírus , Células Sf9 , Spodoptera , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Microvilosidades/metabolismo , Microvilosidades/virologia , Vírion/metabolismo , Corpos de Oclusão Virais/metabolismo
4.
J Biol Chem ; 300(5): 107279, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38588808

RESUMO

Actin bundling proteins crosslink filaments into polarized structures that shape and support membrane protrusions including filopodia, microvilli, and stereocilia. In the case of epithelial microvilli, mitotic spindle positioning protein (MISP) is an actin bundler that localizes specifically to the basal rootlets, where the pointed ends of core bundle filaments converge. Previous studies established that MISP is prevented from binding more distal segments of the core bundle by competition with other actin-binding proteins. Yet whether MISP holds a preference for binding directly to rootlet actin remains an open question. By immunostaining native intestinal tissue sections, we found that microvillar rootlets are decorated with the severing protein, cofilin, suggesting high levels of ADP-actin in these structures. Using total internal reflection fluorescence microscopy assays, we also found that purified MISP exhibits a binding preference for ADP- versus ADP-Pi-actin-containing filaments. Consistent with this, assays with actively growing actin filaments revealed that MISP binds at or near their pointed ends. Moreover, although substrate attached MISP assembles filament bundles in parallel and antiparallel configurations, in solution MISP assembles parallel bundles consisting of multiple filaments exhibiting uniform polarity. These discoveries highlight nucleotide state sensing as a mechanism for sorting actin bundlers along filaments and driving their accumulation near filament ends. Such localized binding might drive parallel bundle formation and/or locally modulate bundle mechanical properties in microvilli and related protrusions.


Assuntos
Actinas , Animais , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microvilosidades/metabolismo , Ligação Proteica
5.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 41(3): 339-344, 2024 Mar 10.
Artigo em Chinês | MEDLINE | ID: mdl-38448026

RESUMO

OBJECTIVE: To explore the clinical and genetic characteristics of a neonate with Microvillus inclusion disease (MVID). METHODS: A neonate with MVID admitted to the First Affiliated Hospital of Zhengzhou University in May 2019 was selected as the study subject. Clinical data were collected. Whole exome sequencing (WES) was carried out, and candidate variants were verified by Sanger sequencing and multiple ligation-dependent probe amplification (MLPA). A literature was also carried out to summarize the clinical and genetic characteristics of MVID. RESULTS: The prematurely born neonate had presented with unexplained refractory diarrhea and metabolic acidosis. Active symptomatic treatment was ineffective, and the child had died at 2 months old. WES revealed that he had harbored compound heterozygous variants of the MYO5B gene, namely c.1591C>T (p.R531W) and deletion of exon 9. Sanger sequencing showed that the R531W variant was inherited form his father, and MLPA confirmed that the exon 9 deletion was inherited from his mother. Seven children with MVID were reported in China, of which one was lost during follow-up and six had deceased. One hundred eighty eight patients were reported worldwide and only one was cured. The clinical features of MVID had included refractory diarrhea, metabolic acidosis and poor prognosis. CONCLUSION: The child was diagnosed with MVID due to the compound heterozygous variants of the MYO5B gene, which has provided a basis for genetic counseling and prenatal diagnosis.


Assuntos
Acidose , Síndromes de Malabsorção , Microvilosidades , Mucolipidoses , Miosina Tipo V , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Gravidez , Diarreia/genética , Síndromes de Malabsorção/genética , Microvilosidades/patologia , Mucolipidoses/genética , Cadeias Pesadas de Miosina , Miosina Tipo V/genética
6.
Biochem Biophys Res Commun ; 706: 149762, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38484572

RESUMO

Revealing the mechanisms of glucose transport is crucial for studying pathological diseases caused by glucose toxicities. Numerous studies have revealed molecular functions involved in glucose transport in the nematode Caenorhabditis elegans, a commonly used model organism. However, the behavior of glucose in the intestinal lumen-to-cell remains elusive. To address that, we evaluated the diffusion coefficient of glucose in the intestinal apical brush border of C. elegans by using fluorescent glucose and fluorescence recovery after photobleaching. Fluorescent glucose taken in the intestine of worms accumulates in the apical brush border, and its diffusion coefficient of ∼10-8 cm2/s is two orders of magnitude slower than that in bulk. This result indicates that the intestinal brush border is a viscous layer. ERM-1 point mutations at the phosphorylation site, which shorten the microvilli length, did not significantly affect the diffusion coefficient of fluorescent glucose in the brush border. Our findings imply that glucose enrichment is dominantly maintained by the viscous layer composed of the glycocalyx and molecular complexes on the apical surface.


Assuntos
Caenorhabditis elegans , Mucosa Intestinal , Animais , Microvilosidades , Caenorhabditis elegans/genética , Glucose , Intestinos
7.
Acta Physiol (Oxf) ; 240(4): e14125, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38533975

RESUMO

AIM: Trafficking, membrane retention, and signal-specific regulation of the Na+/H+ exchanger 3 (NHE3) are modulated by the Na+/H+ Exchanger Regulatory Factor (NHERF) family of PDZ-adapter proteins. This study explored the assembly of NHE3 and NHERF2 with the cGMP-dependent kinase II (cGKII) within detergent-resistant membrane microdomains (DRMs, "lipid rafts") during in vivo guanylate cycle C receptor (Gucy2c) activation in murine small intestine. METHODS: Small intestinal brush border membranes (siBBMs) were isolated from wild type, NHE3-deficient, cGMP-kinase II-deficient, and NHERF2-deficient mice, after oral application of the heat-stable Escherichia coli toxin (STa) analog linaclotide. Lipid raft and non-raft fractions were separated by Optiprep density gradient centrifugation of Triton X-solubilized siBBMs. Confocal microscopy was performed to study NHE3 redistribution after linaclotide application in vivo. RESULTS: In the WT siBBM, NHE3, NHERF2, and cGKII were strongly raft associated. The raft association of NHE3, but not of cGKII, was NHERF2 dependent. After linaclotide application to WT mice, lipid raft association of NHE3 decreased, that of cGKII increased, while that of NHERF2 did not change. NHE3 expression in the BBM shifted from a microvillar to a terminal web region. The linaclotide-induced decrease in NHE3 raft association and in microvillar abundance was abolished in cGKII-deficient mice, and strongly reduced in NHERF2-deficient mice. CONCLUSION: NHE3, cGKII, and NHERF2 form a lipid raft-associated signal complex in the siBBM, which mediates the inhibition of salt and water absorption by Gucy2c activation. NHERF2 enhances the raft association of NHE3, which is essential for its close interaction with the exclusively raft-associated activated cGKII.


Assuntos
Microdomínios da Membrana , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio , Animais , Camundongos , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Intestino Delgado/metabolismo , Microdomínios da Membrana/metabolismo , Microvilosidades/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo
8.
Food Res Int ; 178: 113932, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38309864

RESUMO

The Digestible Indispensable Amino Acid Score (DIAAS) is the new gold standard method for the assessment of protein nutritional quality. The DIAAS is evaluated with in vivo models, that are complex, constraining and costly. There is still no established method to assess it in vitro. In this study, we proposed to add a jejunal-ileal digestion phase to the standardized in vitro gastrointestinal digestion protocol developed by the International Network of Excellence on the Fate of Food in the Gastrointestinal Tract (INFOGEST protocol) to mimic brush border digestion and to enable DIAAS assessment in vitro in a more physiologically relevant manner. This jejunal-ileal digestion phase was performed with a porcine intestinal aminopeptidase as an alternative to brush border membrane extract, which is more difficult to obtain in a standardized way. This modified INFOGEST protocol was applied to various food matrices (faba bean, pea and soy flours, whey protein isolate and caseins) and the results were compared to published in vivo data to assess the model's physiological relevance. The addition of the jejunal-ileal digestion phase lead to a significant (p < 0.05) increase of 31 and 29 % in free and total amino acid digestibility, respectively, and of 83 % on average for the in vitro DIAAS values for all food matrices. Although the in vitro DIAAS remained underestimated compared to the in vivo ones, a strong correlation between them was observed (r = 0.879, p = 0.009), stating the relevance of this last digestion phase. This improved digestion protocol is proposed as a suitable alternative to evaluate the DIAAS in vitro when in vivo assays are not applicable.


Assuntos
Aminoácidos Essenciais , Aminoácidos , Suínos , Animais , Aminoácidos/metabolismo , Microvilosidades/metabolismo , Proteínas Alimentares/metabolismo , Digestão
10.
Cell Mol Gastroenterol Hepatol ; 17(6): 983-1005, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38307491

RESUMO

Microvillus inclusion disease (MVID) is a rare condition that is present from birth and affects the digestive system. People with MVID experience severe diarrhea that is difficult to control, cannot absorb dietary nutrients, and struggle to grow and thrive. In addition, diverse clinical manifestations, some of which are life-threatening, have been reported in cases of MVID. MVID can be caused by variants in the MYO5B, STX3, STXBP2, or UNC45A gene. These genes produce proteins that have been functionally linked to each other in intestinal epithelial cells. MVID associated with STXBP2 variants presents in a subset of patients diagnosed with familial hemophagocytic lymphohistiocytosis type 5. MVID associated with UNC45A variants presents in most patients diagnosed with osteo-oto-hepato-enteric syndrome. Furthermore, variants in MYO5B or STX3 can also cause other diseases that are characterized by phenotypes that can co-occur in subsets of patients diagnosed with MVID. Recent studies involving clinical data and experiments with cells and animals revealed connections between specific phenotypes occurring outside of the digestive system and the type of gene variants that cause MVID. Here, we have reviewed these patterns and correlations, which are expected to be valuable for healthcare professionals in managing the disease and providing personalized care for patients and their families.


Assuntos
Síndromes de Malabsorção , Microvilosidades , Mucolipidoses , Fenótipo , Humanos , Mucolipidoses/genética , Mucolipidoses/patologia , Microvilosidades/patologia , Microvilosidades/genética , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/patologia , Animais , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Mutação , Predisposição Genética para Doença
12.
Anim Sci J ; 95(1): e13919, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38287469

RESUMO

We investigated the role of dietary carbohydrates in the maintenance of the enterocyte microvillar structure in the chicken ileum. Male chickens were divided into the control and three experimental groups, and the experimental groups were fed diets containing 50%, 25%, and 0% carbohydrates of the control diet. The structural alterations in enterocytes were examined using transmission electron microscopy and immunofluorescent techniques for ß-actin and villin. Glucagon-like peptide (GLP)-2 and proglucagon mRNA were detected by immunohistochemistry and in situ hybridization, respectively. Fragmentation and wide gap spaces were frequently observed in the microvilli of the 25% and 0% groups. The length, width, and density of microvilli were also decreased in the experimental groups. The experimental groups had shorter terminal web extensions, and there were substantial changes in the mitochondrial density between the control and experimental groups. Intensities of ß-actin and villin immunofluorescence observed on the apical surface of enterocytes were lower in the 0% group. The frequency of GLP-2-immunoreactive and proglucagon mRNA-expressing cells decreased with declining dietary carbohydrate levels. This study revealed that dietary carbohydrates contribute to the structural maintenance of enterocyte microvilli in the chicken ileum. The data from immunohistochemistry and in situ hybridization assays suggest the participation of GLP-2 in this maintenance system.


Assuntos
Galinhas , Enterócitos , Masculino , Animais , Galinhas/genética , Proglucagon/genética , Actinas , Carboidratos da Dieta , Íleo , Peptídeo 2 Semelhante ao Glucagon , RNA Mensageiro/genética , Microvilosidades
13.
Mol Biol Cell ; 35(3): ar36, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38170579

RESUMO

Transporting epithelial cells of the gut and kidney interact with their luminal environment through a densely packed collection of apical microvilli known as a brush border (BB). Proper brush border assembly depends on the intermicrovillar adhesion complex (IMAC), a protocadherin-based adhesion complex found at the distal tips of microvilli that mediates adhesion between neighboring protrusions to promote their organized packing. Loss of the IMAC adhesion molecule Cadherin-related family member 5 (CDHR5) results in significant brush border defects, though the functional properties of this protocadherin have not been thoroughly explored. Here, we show that the cytoplasmic tail of CDHR5 contributes to its correct apical targeting and functional properties in an isoform-specific manner. Library screening identified the Ezrin-associated scaffolds EBP50 and E3KARP as cytoplasmic binding partners for CDHR5. Consistent with this, loss of EBP50 disrupted proper brush border assembly with cells exhibiting markedly reduced apical IMAC levels. Together, our results shed light on the apical targeting determinants of CDHR5 and further define the interactome of the IMAC involved in brush border assembly.


Assuntos
Células Epiteliais , Protocaderinas , Microvilosidades/metabolismo , Células Epiteliais/metabolismo
14.
Indian J Pediatr ; 91(6): 598-605, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38105403

RESUMO

Congenital diarrhea and enteropathies (CODEs) constitute a group of rare genetic disorders characterized by severe diarrhea and malabsorption in the neonatal period or early infancy. Timely diagnosis and treatment is essential to prevent life-threatening complications, including dehydration, electrolyte imbalance, and malnutrition. This review offers a simplified approach to the diagnosis of CODEs, with a specific focus on microvillus inclusion disease (MVID), congenital tufting enteropathy (CTE), congenital chloride diarrhea (CLD), and congenital sodium diarrhea (CSD). Patients with CODEs typically present with severe watery or occasionally bloody diarrhea, steatorrhea, dehydration, poor growth, and developmental delay. Therefore, it is crucial to thoroughly evaluate infants with diarrhea to rule out infectious, allergic, or anatomical causes before considering CODEs as the underlying etiology. Diagnostic investigations for CODEs encompass various modalities, including stool tests, blood tests, immunological studies, endoscopy and biopsies for histology and electron microscopy, and next-generation sequencing (NGS). NGS plays a pivotal role in identifying the genetic mutations responsible for CODEs. Treatment options for CODEs are limited, often relying on total parenteral nutrition for hydration and nutritional support. In severe cases, intestinal transplantation may be considered. The long-term prognosis varies among specific CODEs, with some patients experiencing ongoing intestinal failure and associated complications. In conclusion, the early recognition and accurate diagnosis of CODEs are of paramount importance for implementing appropriate management strategies. Further research and advancements in genetic testing hold promise for enhancing diagnostic accuracy and exploring potential targeted therapies for these rare genetic disorders.


Assuntos
Diarreia , Síndromes de Malabsorção , Humanos , Diarreia/terapia , Diarreia/etiologia , Diarreia/congênito , Síndromes de Malabsorção/terapia , Síndromes de Malabsorção/diagnóstico , Síndromes de Malabsorção/genética , Recém-Nascido , Lactente , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/terapia , Erros Inatos do Metabolismo/genética , Mucolipidoses/diagnóstico , Mucolipidoses/terapia , Mucolipidoses/genética , Microvilosidades/patologia , Enteropatias/diagnóstico , Enteropatias/terapia , Enteropatias/genética , Anormalidades Múltiplas , Diarreia Infantil
15.
Dev Cell ; 58(20): 2048-2062.e7, 2023 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-37832537

RESUMO

To maximize solute transport, epithelial cells build an apical "brush border," where thousands of microvilli are linked to their neighbors by protocadherin-containing intermicrovillar adhesion complexes (IMACs). Previous studies established that the IMAC is needed to build a mature brush border, but how this complex contributes to the accumulation of new microvilli during differentiation remains unclear. We found that early in differentiation, mouse, human, and porcine epithelial cells exhibit a marginal accumulation of microvilli, which span junctions and interact with protrusions on neighboring cells using IMAC protocadherins. These transjunctional IMACs are highly stable and reinforced by tension across junctions. Finally, long-term live imaging showed that the accumulation of microvilli at cell margins consistently leads to accumulation in medial regions. Thus, nascent microvilli are stabilized by a marginal capture mechanism that depends on the formation of transjunctional IMACs. These results may offer insights into how apical specializations are assembled in diverse epithelial systems.


Assuntos
Células Epiteliais , Humanos , Animais , Camundongos , Suínos , Microvilosidades/metabolismo , Células Epiteliais/metabolismo
16.
Int J Mol Sci ; 24(17)2023 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-37685917

RESUMO

Membrane nanotubes are cell protrusions that grow to tens of micrometres and functionally connect cells. Actin filaments are semi-flexible polymers, and their polymerisation provides force for the formation and growth of membrane nanotubes. The molecular bases for the provision of appropriate force through such long distances are not yet clear. Actin filament bundles are likely involved in these processes; however, even actin bundles weaken when growing over long distances, and there must be a mechanism for their regeneration along the nanotubes. We investigated the possibility of the formation of periodic molecular relay stations along membrane nanotubes by describing the interactions of actin with full-length IRSp53 protein and its N-terminal I-BAR domain. We concluded that I-BAR is involved in the early phase of the formation of cell projections, while IRSp53 is also important for the elongation of protrusions. Considering that IRSp53 binds to the membrane along the nanotubes and nucleates actin polymerisation, we propose that, in membrane nanotubes, IRSp53 establishes molecular relay stations for actin polymerisation and, as a result, supports the generation of force required for the growth of nanotubes.


Assuntos
Actinas , Nanotubos , Citoesqueleto de Actina , Estruturas da Membrana Celular , Microvilosidades , Animais , Camundongos , Chlorocebus aethiops/metabolismo
17.
J Clin Invest ; 133(20)2023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37643022

RESUMO

Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.


Assuntos
Síndromes de Malabsorção , Mucolipidoses , Miosina Tipo V , Humanos , Microvilosidades/genética , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Enterócitos/metabolismo , Síndromes de Malabsorção/genética , Síndromes de Malabsorção/terapia , Síndromes de Malabsorção/metabolismo , Mucolipidoses/genética , Mucolipidoses/terapia , Mucolipidoses/metabolismo
18.
Cells ; 12(14)2023 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-37508507

RESUMO

In vitro organotypic cell-based intestinal platforms, able to faithfully recapitulate the complex functions of the organ in vivo, would be a great support to search for more sustainable feed ingredients in aquaculture. We previously demonstrated that proliferation or differentiation of rainbow trout intestinal cell lines is dictated by the culture environment. The aim of the present work was to develop a culture platform that can efficiently promote cell differentiation into mature enterocytes. We compared four options, seeding the RTpiMI cell line derived from the proximal intestine on (1) polyethylene terephthalate (PET) culture inserts ThinCert™ (TC), (2) TC coated with the solubilized basement membrane matrix Matrigel® (MM), (3) TC with the rainbow trout fibroblast cell line RTskin01 embedded within the Matrigel® matrix (MMfb), or (4) the highly porous polystyrene scaffold Alvetex® populated with the abovementioned fibroblast cell line (AV). We evaluated the presence of columnar cells with a clear polarization of brush border enzymes, the formation of an efficient barrier with a significant increase in transepithelial electrical resistance (TEER), and its ability to prevent the paracellular flux of large molecules but allow the transit of small compounds (proline and glucose) from the apical to the basolateral compartment. All parameters improved moving from the simplest (TC) through the more complex platforms. The presence of fibroblasts was particularly effective in enhancing epithelial cell differentiation within the AV platform recreating more closely the complexity of the intestinal mucosa, including the presence of extracellular vesicles between fibroblasts and epithelial cells.


Assuntos
Oncorhynchus mykiss , Animais , Oncorhynchus mykiss/metabolismo , Intestinos , Mucosa Intestinal/metabolismo , Linhagem Celular , Microvilosidades
19.
Gastroenterology ; 165(4): 986-998.e11, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37429363

RESUMO

BACKGROUND & AIMS: Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. METHODS: A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine in vitro and in vivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. RESULTS: N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3',5'-cyclic monophosphate, guanosine 3',5'-cyclic monophosphate, and Ca2+ in cell lines and in in vitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine in vivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. CONCLUSIONS: These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.


Assuntos
Enterotoxinas , Trocadores de Sódio-Hidrogênio , Camundongos , Animais , Humanos , Trocador 3 de Sódio-Hidrogênio/metabolismo , Enterotoxinas/farmacologia , Enterotoxinas/metabolismo , Células CACO-2 , Trocadores de Sódio-Hidrogênio/metabolismo , Enterócitos/metabolismo , Sódio/metabolismo , Diarreia/tratamento farmacológico , Diarreia/prevenção & controle , Diarreia/induzido quimicamente , Peptídeos/efeitos adversos , Microvilosidades/metabolismo
20.
J Anim Physiol Anim Nutr (Berl) ; 107(6): 1456-1464, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37448186

RESUMO

Digestible carbohydrates differ in glycaemic response, therewith having the potential to influence metabolic conditions such as insulin resistance and diabetes mellitus. Isomaltulose has been proven to lower the glycaemic response in humans, which to date has not been studied in dogs. Therefore, the aim of the present study was to characterise the digestibility, as well as the physiological effects of isomaltulose in dogs, in comparison to other saccharides. To this end, three studies were performed. Study 1 was an in vitro study, evaluating the small intestinal hydrolysis of isomaltulose compared to other relevant carbohydrate sources. Three of these saccharides, having close and low-moderate degrees of hydrolysis by brush border enzymes, were also evaluated in vivo for their glycaemic effects by measuring plasma levels of glucose, insulin and glucagon-like peptide 1 (GLP-1) 0-180 min after administration of a single dosage after an overnight fast (i.e., isomaltulose, sucrose and maltodextrin in a 3 × 3 Latin-square design, in 9 dogs, Study 2). To understand if digestive enzymes, underlying glycaemic responses for isomaltulose and sucrose can be upregulated, we exposed dogs to these saccharides for 2 weeks and repeated the measurements after an overnight fast in 18 dogs (Study 3). Isomaltulose was hydrolysed by intestinal enzyme preparation from all three dogs, but the degrading activity was low (e.g., 3.95 ± 1.03 times lower vs. sucrose), indicating a slower rate of hydrolysis. Isomaltulose had a low glycaemic response, in line with in vitro data. In vitro hydrolysis of sucrose was comparable or even higher than maltodextrin in contrast to the more pronounced glycaemic response to maltodextrin observed in vivo. The numerically higher blood glucose response to sucrose after continuous consumption, might indicate an adaptive response. In conclusion, the current work provides valuable insights into the digestion physiology of various saccharides in dogs. Further investigations on related benefits are thus warranted.


Assuntos
Glicemia , Sacarose , Humanos , Cães , Animais , Hidrólise , Microvilosidades/metabolismo
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