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Phagocytosis is the necessary first step to sense foreign microbes or particles and enables activation of innate immune pathways such as inflammasomes. However, the molecular mechanisms underlying how phagosomes modulate inflammasome activity are not fully understood. We show that in murine dendritic cells (DCs), the lysosomal histidine/peptide solute carrier transporter SLC15A4, associated with human inflammatory disorders, is recruited to phagosomes and is required for optimal inflammasome activity after infectious or sterile stimuli. Dextran sodium sulfate-treated SLC15A4-deficient mice exhibit decreased colon inflammation, reduced IL-1ß production by intestinal DCs, and increased autophagy. Similarly, SLC15A4-deficient DCs infected with Salmonella typhimurium show reduced caspase-1 cleavage and IL-1ß production. This correlates with peripheral NLRC4 inflammasome assembly and increased autophagy. Overexpression of constitutively active mTORC1 rescues decreased IL-1ß levels and caspase1 cleavage, and restores perinuclear inflammasome positioning. Our findings support that SLC15A4 couples phagocytosis with inflammasome perinuclear assembly and inhibition of autophagy through phagosomal content sensing. Our data also reveal the previously unappreciated importance of mTORC1 signaling pathways to promote and sustain inflammasome activity.
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Células Dendríticas , Inflamasomas , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de Transporte de Membrana , Animales , Autofagia , Caspasa 1/metabolismo , Células Dendríticas/metabolismo , Dextranos/metabolismo , Histidina , Humanos , Interleucina-1beta/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Proteínas del Tejido Nervioso/metabolismo , Fagosomas/metabolismoRESUMEN
OBJECTIVE: Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with the majority of cases initiated by inactivation of the APC tumour suppressor. This results in the constitutive activation of canonical WNT pathway transcriptional effector ß-catenin, along with induction of WNT feedback inhibitors, including the extracellular palmitoleoyl-protein carboxylesterase NOTUM which antagonises WNT-FZD receptor-ligand interactions. Here, we sought to evaluate the effects of NOTUM activity on CRC as a function of driver mutation landscape. DESIGN: Mouse and human colon organoids engineered with combinations of CRC driver mutations were used for Notum genetic gain-of-function and loss-of-function studies. In vitro assays, in vivo endoscope-guided orthotopic organoid implantation assays and transcriptomic profiling were employed to characterise the effects of Notum activity. Small molecule inhibitors of Notum activity were used in preclinical therapeutic proof-of-principle studies targeting oncogenic Notum activity. RESULTS: NOTUM retains tumour suppressive activity in APC-null adenomas despite constitutive ß-catenin activity. Strikingly, on progression to adenocarcinoma with P53 loss, NOTUM becomes an obligate oncogene. These phenotypes are Wnt-independent, resulting from differential activity of NOTUM on glypican 1 and 4 in early-stage versus late-stage disease, respectively. Ultimately, preclinical mouse models and human organoid cultures demonstrate that pharmacological inhibition of NOTUM is highly effective in arresting primary adenocarcinoma growth and inhibiting metastatic colonisation of distal organs. CONCLUSIONS: Our findings that a single agent targeting the extracellular enzyme NOTUM is effective in treating highly aggressive, metastatic adenocarcinomas in preclinical mouse models and human organoids make NOTUM and its glypican targets therapeutic vulnerabilities in advanced CRC.
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Adenocarcinoma , Neoplasias Colorrectales , Humanos , Ratones , Animales , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Mutación , Vía de Señalización Wnt/genética , Cateninas/genética , Cateninas/metabolismo , Cateninas/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genéticaRESUMEN
Calorie restriction can enhance the regenerative capacity of the injured intestinal epithelium. Among other metabolic changes, calorie restriction can activate the autophagy pathway. Although independent studies have attributed the regenerative benefit of calorie restriction to downregulation of mTORC1, it is not known whether autophagy itself is required for the regenerative benefit of calorie restriction. We used mouse and organoid models with autophagy gene deletion to evaluate the contribution of autophagy to intestinal epithelial regeneration following calorie restriction. In the absence of injury, mice with intestinal epithelial-specific deletion of autophagy gene Atg7 (Atg7ΔIEC) exhibit weight loss and histological changes similar to wild-type mice following calorie restriction. Conversely, calorie-restricted Atg7ΔIEC mice displayed a significant reduction in regenerative crypt foci after irradiation compared with calorie-restricted wild-type mice. Targeted analyses of tissue metabolites in calorie-restricted mice revealed an association between calorie restriction and reduced glycocholic acid (GCA) in wild-type mice but not in Atg7ΔIEC mice. To evaluate whether GCA can directly modulate epithelial stem cell self-renewal, we performed enteroid formation assays with or without GCA. Wild-type enteroids exhibited reduced enteroid formation efficiency in response to GCA treatment, suggesting that reduced availability of GCA during calorie restriction may be one mechanism by which calorie restriction favors epithelial regeneration in a manner dependent upon epithelial autophagy. Taken together, our data support the premise that intestinal epithelial Atg7 is required for the regenerative benefit of calorie restriction, due in part to its role in modulating luminal GCA with direct effects on epithelial stem cell self-renewal.NEW & NOTEWORTHY Calorie restriction is associated with enhanced intestinal regeneration after irradiation, but the requirement of autophagy for this process is not known. Our data support the premise that intestinal epithelial autophagy is required for the regenerative benefit of calorie restriction. We also report that luminal levels of primary bile acid glycocholic acid are modulated by epithelial cell autophagy during calorie restriction with direct effects on epithelial stem cell function.
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Restricción Calórica , Intestinos , Ratones , Animales , Intestinos/fisiología , Mucosa Intestinal/metabolismo , Células Epiteliales , Autofagia/genéticaRESUMEN
Directional migration is inherently important for epithelial tissue regeneration and repair, but how it is precisely controlled and coordinated with cell proliferation is unclear. Here, we report that Ovol2, a transcriptional repressor that inhibits epithelial-to-mesenchymal transition (EMT), plays a crucial role in adult skin epithelial regeneration and repair. Ovol2-deficient mice show compromised wound healing characterized by aberrant epidermal cell migration and proliferation, as well as delayed anagen progression characterized by defects in hair follicle matrix cell proliferation and subsequent differentiation. Epidermal keratinocytes and bulge hair follicle stem cells (Bu-HFSCs) lacking Ovol2 fail to expand in culture and display molecular alterations consistent with enhanced EMT and reduced proliferation. Live imaging of wound explants and Bu-HFSCs reveals increased migration speed but reduced directionality, and post-mitotic cell cycle arrest. Remarkably, simultaneous deletion of Zeb1 encoding an EMT-promoting factor restores directional migration to Ovol2-deficient Bu-HFSCs. Taken together, our findings highlight the important function of an Ovol2-Zeb1 EMT-regulatory circuit in controlling the directional migration of epithelial stem and progenitor cells to facilitate adult skin epithelial regeneration and repair.
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Movimiento Celular/genética , Proliferación Celular/genética , Factores de Transcripción/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Animales , Diferenciación Celular , Células Epidérmicas/metabolismo , Transición Epitelial-Mesenquimal/genética , Regulación del Desarrollo de la Expresión Génica , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Queratinocitos/metabolismo , Ratones , Piel/crecimiento & desarrollo , Piel/metabolismo , Células Madre/citología , Células Madre/metabolismo , Cicatrización de Heridas/genéticaRESUMEN
Hairlessness is usually a rare trait in pigs; however, in this study, we found hairless (HR) pigs at a relatively high frequency in 1 pig herd. We observed that, the lower hair shaft density of HR pigs could be mainly attributed to the lower hair follicle density, and during the embryonic period, d 39-45 were a critical stage for the formation of the hair follicle. In this regard, d 41 during gestation was a particularly important point. Hair follicle morphogenesis occurring at an early stage of embryo development is similar to humans and mice. Further analyses of association studies based on single-nucleotide polymorphism chip as well as sequence data, mRNA sequencing, immunohistochemistry, and comparative genomics demonstrated that microtubule-associated protein 2 (MAP2) is a key gene responsible for hair follicle density and 1 missense mutation of A-to-G at rs328005415 in MAP2, causing a valine-to-methionine substitution leads to the HR phenotype. Considering the high homology between pigs and humans, our research has some significance for the study of the mechanisms of skin development, hair morphogenesis, and hair loss in humans by showing that the pig may be a more appropriate model in which to study these processes.-Jiang, Y., Jiang, Y., Zhang, H., Mei, M., Song, H., Ma, X., Jiang, L., Yu, Z., Zhang, Q., Ding, X. A mutation in MAP2 is associated with prenatal hair follicle density.
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Folículo Piloso/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Mutación Missense/fisiología , Porcinos/embriología , Porcinos/genética , Animales , Animales Recién Nacidos , Desarrollo Embrionario , Desarrollo Fetal , Regulación del Desarrollo de la Expresión Génica , Proteínas Asociadas a Microtúbulos/genéticaRESUMEN
Objective To investigate the relationship between interleukin-1ß (IL-1ß) and miR-185-5p in the process of joint injury in acute gouty arthritis (AGA). Methods The serum miR-185-5p levels of 89 AGA patients and 91 healthy volunteers were detected by real-time quantitative PCR. The correlation between miR-185-5p expression level and VAS score or IL-1ß expression level was evaluated by Pearson correlation coefficient method. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of miR-185-5p in AGA. THP-1 cells were induced by sodium urate (MSU) to construct an in vitro acute gouty inflammatory cell model. After the expression level of miR-185-5p in THP-1 cells was upregulated or downregulated by transfection of miR-185-5p mimics or inhibitors in vitro, inflammatory cytokines of THP-1 cells, such as IL-1ß, IL-8 and tumor necrosis factor α (TNF-α), were detected by ELISA. The luciferase reporter gene assay was used to determine the interaction between miR-185-5p and the 3'-UTR of IL-1ß. Results Compared with the healthy control group, the expression level of serum miR-185-5p in AGA patients was significantly reduced. The level of serum miR-185-5p was negatively correlated with VAS score and IL-1ß expression level. The area under the curve (AUC) was 0.905, the sensitivity was 80.17% and the specificity was 83.52%. Down-regulation of miR-185-5p significantly promoted the expression of IL-1ß, IL-8 and tumor necrosis factor (TNF-α), while overexpression of miR-185-5p showed the opposite results. Luciferase reporter gene assay showed that IL-1ß was the target gene of miR-185-5p, and miR-185-5p negatively regulated the expression of IL-1ß. Conclusion miR-185-5p alleviates the inflammatory response in AGA by inhibiting IL-1ß.
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Artritis Gotosa , MicroARNs , Humanos , Regiones no Traducidas 3' , Artritis Gotosa/genética , Interleucina-1beta/genética , Interleucina-8 , Luciferasas , MicroARNs/genética , Factor de Necrosis Tumoral alfaRESUMEN
BACKGROUND & AIMS: The intestinal epithelium interfaces with a diverse milieu of luminal contents while maintaining robust digestive and barrier functions. Facultative intestinal stem cells are cells that survive tissue injury and divide to re-establish the epithelium. Prior studies have shown autophagic state as functional marker of facultative intestinal stem cells, but regulatory mechanisms are not known. The current study evaluated a post-transcriptional regulation of autophagy as an important factor for facultative stem cell state and tissue regeneration. METHODS: We evaluated stem cell composition, autophagic vesicle content, organoid formation, and in vivo regeneration in mice with intestinal epithelial deletion of the RNA binding protein IGF2 messenger RNA binding protein 1 (IMP1). The contribution of autophagy to resulting in vitro and in vivo phenotypes was evaluated via genetic inactivation of Atg7. Molecular analyses of IMP1 modulation of autophagy at the protein and transcript localization levels were performed using IMP1 mutant studies and single-molecule fluorescent in situ hybridization. RESULTS: Epithelial Imp1 deletion reduced leucine rich repeat containing G protein coupled receptor 5 cell frequency but enhanced both organoid formation efficiency and in vivo regeneration after irradiation. We confirmed prior studies showing increased autophagy with IMP1 deletion. Deletion of Atg7 reversed the enhanced regeneration observed with Imp1 deletion. IMP1 deletion or mutation of IMP1 phosphorylation sites enhanced expression of essential autophagy protein microtubule-associated protein 1 light chain 3ß. Furthermore, immunofluorescence imaging coupled with single-molecule fluorescent in situ hybridization showed IMP1 colocalization with MAP1LC3B transcripts at homeostasis. Stress induction led to decreased colocalization. CONCLUSIONS: Depletion of IMP1 enhances autophagy, which promotes intestinal regeneration via expansion of facultative intestinal stem cells.
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Mucosa Intestinal , Intestinos , Animales , Ratones , Hibridación Fluorescente in Situ , Mucosa Intestinal/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Células Madre/metabolismoRESUMEN
OBJECTIVE: Interstitial lung disease (ILD) is a severe manifestation of rheumatoid arthritis (RA), which is characterized by low survival time post-diagnosis. Thus, it is important to explore the role of gene regulation related with ILD. METHOD: Constructed a RA-ILD-related long chain noncoding RNA - messenger RNA (lncRNA-mRNA) network (ILD-LMN), based on ILD- and RA-related genes. We analyzed the topological properties of the resulting network. RESULT: The results for network modularization and functional analysis showed that ILD-LMN performed basic and specific functions in ILD pathology. Furthermore, differential expression and correlation analysis of hub nodes revealed highly correlated competitive endogenous RNA regulatory relationships with important roles in pathological regulation. Following this, statistical analysis of disease-related single nucleotide polymorphisms (SNPs) in hub lncRNAs revealed that some of transcription factor-related SNPs were significantly associated with the expression of lncRNA. In fact, these SNPs exhibited significant differential expression in disease and normal samples. CONCLUSION: These results suggest that ILD-LMN has important implications in the study of disease. Altogether, the study of RA- and ILD-related lncRNA and genes on the basis of biological network would assist in providing better treatment opportunities for ILD patients. Additionally, it would promote further research on treatment of the disease.
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Artritis Reumatoide , Enfermedades Pulmonares Intersticiales , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , Artritis Reumatoide/diagnóstico , Artritis Reumatoide/genética , Artritis Reumatoide/complicaciones , Enfermedades Pulmonares Intersticiales/etiología , Enfermedades Pulmonares Intersticiales/genéticaRESUMEN
Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.
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Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.
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Proteínas Proto-Oncogénicas p21(ras) , Células Madre , Animales , Ratones , Proliferación Celular/fisiología , Supervivencia Celular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Intestinos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN/metabolismo , Transducción de Señal/fisiología , Células Madre/metabolismoRESUMEN
BACKGROUND: Psoraleae Fructus has been widely used in China and its surroundings; however, Psoraleae Fructus and its compound preparation have been reported recently to cause liver injury in clinics. Thus, its safe use has attracted increasing attention. The possible mechanism is related to the metabolism of psoralen, but it still needs further clarification. PURPOSE: The present study was designed to evaluate the toxicity of psoralen and investigate the potentially related molecular mechanisms using chemical biology methods combined with animal experiments to provide evidence for the rational clinical use of psoralen. METHODS: An in vivo experiment was conducted with a time series of 20-80 mg/kg psoralen to verify its toxic performance. Target capture and click reactions were used to investigate direct targets of psoralen. Selectivity for different glutathione-S-transferase (GST) subtypes in the liver and inhibition of cytochrome P450 (CYP450) were also detected. RESULTS: Psoralen build-up in the liver is the primary cause of liver damage. Our study revealed the mechanism by which psoralen induces liver injury. Psoralen can bind directly to CYP2D6, CYP3A4, GST-α, and GST-µ and inhibit their activities, causing the depletion of glutathione (GSH) in vivo, which in turn induces hepatic damage. The special structure of α,ß-unsaturated lactones in psoralen facilitates its attachment to its target; therefore, complementing psoralen with GSH can efficiently protect the liver from damage. CONCLUSIONS: Psoralen causes a disorder in drug metabolism by inhibiting the activity of CYPs and GSTs, causing exhaustion of GSH, and subsequently leading to liver damage. The co-administration of GSH and psoralen is an effective way to avoid liver injury in clinical settings.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Ficusina , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Sistema Enzimático del Citocromo P-450/metabolismo , Ficusina/metabolismo , Ficusina/farmacología , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , HígadoRESUMEN
The permeability of different strength grades of submerged non-dispersible concrete with different granulated slag admixtures in a saline soil environment simulated by different erosion solutions was investigated. The variation patterns of the chloride ion diffusion coefficient and pore characteristics were tested using NEL and MIP. The microscopic morphology of the specimens in different erosion environments and with slag doping was observed using SEM. The results showed that the impermeability of concrete in sulfate and complex salt environments was significantly reduced. The resistance of concrete to chloride ion penetration increased with the increase in strength grade, and the Cl- diffusion coefficient of C35 was 5-30% lower than those of C30 and C25 underwater non-dispersible concrete at 360 d. Meanwhile, the admixture of granulated blast-furnace slag optimized the pore size distribution and improved the matrix compactness and permeability.
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Skin epidermis constitutes the exterior barrier that protects the body from dehydration and environmental assaults. Barrier defects underlie common inflammatory skin diseases, but the molecular mechanisms that maintain barrier integrity and regulate epidermal-immune cell cross-talk in inflamed skin are not fully understood. In this study, we show that skin epithelia-specific deletion of Ovol1, which encodes a skin diseaseâlinked transcriptional repressor, impairs the epidermal barrier and aggravates psoriasis-like skin inflammation in mice in part by enhancing neutrophil accumulation and abscess formation. Through molecular studies, we identify IL-33, a cytokine with known pro-inflammatory and anti-inflammatory activities, and Cxcl1, a neutrophil-attracting chemokine, as potential weak and strong direct targets of Ovol1, respectively. Furthermore, we provide functional evidence that elevated Il33 expression reduces disease severity in imiquimod-treated Ovol1-deficient mice, whereas persistent accumulation and epidermal migration of neutrophils exacerbate it. Collectively, our study uncovers the importance of an epidermally expressed transcription factor that regulates both the integrity of the epidermal barrier and the behavior of neutrophils in psoriasis-like inflammation.
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Dermatitis , Psoriasis , Animales , Proteínas de Unión al ADN , Dermatitis/metabolismo , Modelos Animales de Enfermedad , Epidermis/metabolismo , Inflamación/metabolismo , Factor Intrinseco/metabolismo , Queratinocitos/metabolismo , Ratones , Neutrófilos , Piel/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Previous study showed that circular RNA Absent-Small-Homeotic-2--Like protein (circASH2L) was higher in rheumatoid arthritis (RA) patients. However, the roles and mechanisms of circASH2L in RA progression remain unclear. METHODS: Levels analysis was conducted using western blot and qRT-PCR. The proliferation, apoptosis, cell cycle progression, migration, invasiveness, and inflammation of RA fibroblast-like synoviocytes (RA-FLSs) were determined via MTT, flow cytometry, western blot, transwell, and ELISA assays. RESULTS: CircASH2L knockdown in RA-FLSs suppressed cell proliferative, migratory, and invasive capacities, triggered cell cycle arrest, promoted apoptosis, and inhibited inflammation. Mechanistically, circASH2L targeted miR-129-5p, and repression of miR-129-5p abolished the functions of circASH2L silencing on the growth, motility, and inflammation of RA-FLSs. Besides, miR-129-5p was found to directly target HIPK2, and suppressed the tumor-like biologic behaviors and inflammation of RA-FLSs via regulating HIPK2. Importantly, we proved that circASH2L could modulate HIPK2 expression via miR-129-5p. CONCLUSION: CircASH2L promoted RA-FLS growth, motility, and inflammation through miR-129-5p/HIPK2 axis.
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Artritis Reumatoide/genética , Artritis Reumatoide/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Fibroblastos/patología , Regulación del Desarrollo de la Expresión Génica/genética , Expresión Génica/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Circular/genética , ARN Circular/fisiología , Sinoviocitos/patología , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Ciclo Celular/genética , Movimiento Celular/genética , Proliferación Celular/genética , Células Cultivadas , Progresión de la Enfermedad , Humanos , InflamaciónRESUMEN
BACKGROUND: Defining epithelial cell contributions to inflammatory bowel disease (IBD) is essential for the development of much needed therapies for barrier repair. Children with very early onset (VEO)-IBD have more extensive, severe, and refractory disease than older children and adults with IBD and, in some cases, have defective barrier function. We therefore evaluated functional and transcriptomic differences between pediatric IBD (VEO and older onset) and non-IBD epithelium using 3-dimensional, biopsy-derived organoids. METHODS: We measured growth efficiency relative to histopathological and clinical parameters in patient enteroid (ileum) and colonoid (colon) lines. We performed RNA-sequencing on patient colonoids and subsequent flow cytometry after multiple passages to evaluate changes that persisted in culture. RESULTS: Enteroids and colonoids from pediatric patients with IBD exhibited decreased growth associated with histological inflammation compared with non-IBD controls. We observed increased LYZ expression in colonoids from pediatric IBD patients, which has been reported previously in adult patients with IBD. We also observed upregulation of antigen presentation genes HLA-DRB1 and HLA-DRA, which persisted after prolonged passaging in patients with pediatric IBD. CONCLUSIONS: We present the first functional evaluation of enteroids and colonoids from patients with VEO-IBD and older onset pediatric IBD, a subset of which exhibits poor growth. Enhanced, persistent epithelial antigen presentation gene expression in patient colonoids supports the notion that epithelial cell-intrinsic differences may contribute to IBD pathogenesis.
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Presentación de Antígeno , Enfermedades Inflamatorias del Intestino , Organoides/crecimiento & desarrollo , Niño , Humanos , Inflamación , Enfermedades Inflamatorias del Intestino/genética , Organoides/fisiopatología , Regulación hacia ArribaRESUMEN
The evolution of enzyme genes at the pathway level has attracted increasing attention in recent years. Most investigations have focused on microorganisms, plants and invertebrates but rarely on vertebrates. The dopamine pathway, which participates in almost every aspect of brain function, is an excellent candidate for study at the pathway level. Herein, we report data on the divergence of six dopamine metabolic enzyme genes (three anabolic, three catabolic enzymes) and five dopamine receptor genes across five mammals, namely Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, and Rattus norvegicus. For enzyme genes, our data confirm previous conclusion that the upstream genes have evolved more slowly than downstream genes. Moreover, we found that catabolic genes in the dopamine metabolic pathway have evolved faster than anabolic genes, and maximum likelihood analysis suggested that this difference in evolutionary rate may be explained by anabolic genes being more constrained during selection. For dopamine receptor genes, however, the broadly expressed genes have tended to evolve more slowly than the narrowly expressed genes; maximum likelihood analysis showed that the relatively rapid evolutionary rate of the narrowly expressed receptor genes was a consequence of relaxed selective constraints. Finally, our data imply that selective constraints on synonymous sites in enzyme genes are relaxed compared with those of receptor genes because of differences in their patterns of functional regulation.
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Dopamina/biosíntesis , Dopamina/genética , Evolución Molecular , Procesamiento Proteico-Postraduccional/genética , Receptores Dopaminérgicos/genética , Animales , Composición de Base/genética , Codón/genética , Exones/genética , Variación Genética , Humanos , Receptores Dopaminérgicos/metabolismo , Factores de TiempoRESUMEN
Mammary and extramammary Paget's Diseases (PD) are a malignant skin cancer characterized by the appearance of Paget cells. Although easily diagnosed, its pathogenesis remains unknown. Here, single-cell RNA-sequencing identified distinct cellular states, novel biomarkers, and signaling pathways - including mTOR, associated with extramammary PD. Interestingly, we identified MSI1 ectopic overexpression in basal epithelial cells of human PD skin, and show that Msi1 overexpression in the epidermal basal layer of mice phenocopies human PD at histopathological, single-cell and molecular levels. Using this mouse model, we identified novel biomarkers of Paget-like cells that translated to human Paget cells. Furthermore, single-cell trajectory, RNA velocity and lineage-tracing analyses revealed a putative keratinocyte-to-Paget-like cell conversion, supporting the in situ transformation theory of disease pathogenesis. Mechanistically, the Msi1-mTOR pathway drives keratinocyte-Paget-like cell conversion, and suppression of mTOR signaling with Rapamycin significantly rescued the Paget-like phenotype in Msi1-overexpressing transgenic mice. Topical Rapamycin treatment improved extramammary PD-associated symptoms in humans, suggesting mTOR inhibition as a novel therapeutic treatment in PD.
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Antibióticos Antineoplásicos/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Paget Extramamaria/tratamiento farmacológico , Proteínas de Unión al ARN/metabolismo , Sirolimus/administración & dosificación , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adulto , Anciano , Animales , Biomarcadores/metabolismo , Femenino , Humanos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Wound healing is essential for skin repair after injury, and it consists of hemostasis, inflammation, re-epithelialization, and remodeling phases. Successful re-epithelialization, which relies on proliferation and migration of epidermal keratinocytes, requires a reduction in tissue inflammation. Therefore, understanding the molecular mechanism underlying the transition from inflammation to re-epithelialization will help to better understand the principles of wound healing. Currently, the in vivo functions of specific microRNAs in wound healing are not fully understood. We observed that miR-31 expression is strongly induced in wound edge keratinocytes, and is directly regulated by the activity of NF-κB and signal transducer and activator of transcription 3 signaling pathways during the inflammation phase. We used miR-31 loss-of-function mouse models to demonstrate that miR-31 promotes keratinocyte proliferation and migration. Mechanistically, miR-31 activates the Ras/mitogen-activated protein kinase signaling by directly targeting Rasa1, Spred1, Spred2, and Spry4, which are negative regulators of the Ras/mitogen-activated protein kinase pathway. Knockdown of these miR-31 targets at least partially rescues the delayed scratch wound re-epithelialization phenotype observed in vitro in miR-31 knockdown keratinocytes. Taken together, these findings identify miR-31 as an important cell-autonomous mediator during the transition from inflammation to re-epithelialization phases of wound healing, suggesting a therapeutic potential for miR-31 in skin injury repair.
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Queratinocitos/metabolismo , MicroARNs/genética , Repitelización/fisiología , Cicatrización de Heridas/genética , Heridas y Lesiones/patología , Animales , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Hibridación in Situ , Queratinocitos/patología , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Transducción de Señal , Heridas y Lesiones/genética , Heridas y Lesiones/metabolismoRESUMEN
Polyclonal catalytic antibodies (abzymes) play an important role in immunology research. In this study, we report polyclonal antibodies IgYs isolated from chicken egg yolk with hydrolysis activity for the first time. The IgYs were raised in hens using HNPBV [4-(hydroxy (naphthalen-2-yloxy) phosphoryl) butanoic acid] attached to BSA (Bovine serum albumin) as an immunogen. Anti-(HNPBV-BSA) IgYs were isolated from yolks of the eggs laid using a two-step salt precipitation and one-step gel filtration protocol. NA (naphthalen-2-yl acetate) was selected as the substrate and the hydrolysis reaction of the IgYs for it was examined. The result reveals that the rate of the hydrolysis reaction is higher (Kcat/K (uncat) approximately 2x10(4)). The purified IgYs were digested with pepsin and the smaller fragment (Fab') with specific antigen binding properties was produced. The research indicates that the enzymatic properties of Fab' are similar to IgYs. The catalytic activity of the IgYs was further determined by measuring the rate of hydrolysis of NA in the presence of inhibitor. These findings show that chicken egg is an excellent donor for polyclonal catalytic antibodies.
Asunto(s)
Anticuerpos Catalíticos/química , Anticuerpos Catalíticos/inmunología , Yema de Huevo/inmunología , Inmunoglobulinas/inmunología , Animales , Sitios de Unión de Anticuerpos , Hidrólisis , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulinas/aislamiento & purificación , Inmunoglobulinas/metabolismo , Cinética , Pepsina A/metabolismoRESUMEN
Long-term nitroglycerin (NTG) therapy causes tolerance to its effects attributing to increased oxidative stress and endothelial dysfunction. Shenmai injection (SMI), which is clinically used to treat cardiovascular diseases, consists of two herbal medicines, Ginseng Rubra and Ophiopogonjaponicas, and is reported to have antioxidant effects. The present study was designed to investigate the potential preventive effects of Shenmai injection on development of nitroglycerin-induced tolerance. The present study involves both in vivo and in vitro experiments to investigate nitroglycerin-induced tolerance. We examined the effect of Shenmai injection on the cardiovascular oxidative stress by measuring the serum levels of malondialdehyde (MDA) and superoxide dismutase (SOD). Endothelial dysfunction was determined by an endothelium-dependent vasorelaxation method in aortic rings and NOS activity. Inhibition of the cGMP/cGK-I signalling pathway was determined from released serum levels of cGMP and the protein expression levels of sGC, cGK-I, PDE1A and P-VASP by western blot. Here, we showed that SMI ameliorated the decrease in AV Peak Vel, the attenuation in the vasodilation response to nitroglycerin and endothelial dysfunction. SMI also reduced the cardiovascular oxidative stress by reducing the release of MDA and increasing the activity of SOD. Shenmai injection further ameliorated inhibition of the cGMP/cGK-I signalling pathway triggered by nitroglycerin-induced tolerance through up-regulating the protein expression of sGC, cGK-I, and P-VASP and down- regulating the proteins expression of PDE1A. In vitro studies showed that Shenmai injection could recover the attenuated vasodilation response to nitroglycerin following incubation (of aortic rings) with nitroglycerin via activating the enzymes of sGC and cGK-I. Therefore, we conclude that Shenmai injection could prevent NTG nitroglycerin-induced tolerance at least in part by decreasing the cardiovascular oxidative stress, meliorating the endothelial dysfunction and ameliorating the inhibition of the cGMP/cGK-I signalling pathway. These findings indicate the potential of Shenmai injection (SMI) as a promising medicine for preventing the development of nitroglycerin-induced tolerance.