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Hematoporphyrin monomethyl ether-photodynamic therapy (HMME-PDT) has achieved encouraging clinical outcomes in adult port-wine stain (PWS). Optimal treatment option for children with PWS was minimal. To compare whether the clinical effectiveness of HMME-PDT with the 5-min (fast) administration treatment regimen (FATR) was better than the 20-min (slow) administration treatment regimen (SATR) for PWS of children in vivo and in vitro. Thirty-four children with PWS were divided into two groups including FATR and SATR. The two groups received three times HMME-PDT, respectively. Treatment efficacy and safety were evaluated in vivo and in vitro. Erythema index (EI) was used to evaluate the clinical outcomes. Both FATR and SATR were effective and safe in children with PWS after HMME-PDT. There were significance differences between the two groups in reductions of EI after the second treatment (p < 0.001) and the third treatment (p < 0.001) with HMME-PDT. The serum HMME concentration reach the peak level at short time compare with SATR group. A significance increased superoxide levels were observed in FATR group compare to SATR groups in vitro (p < 0.05). Our study suggested that HMME-PDT was effective and safe for children with PWS, the therapy regimen with FATR was better in clinical efficacy than that of the SATR.
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Hemangioma Capilar , Fotoquimioterapia , Mancha Vino de Oporto , Niño , Humanos , Pueblos del Este de Asia , Hemangioma Capilar/tratamiento farmacológico , Fotoquimioterapia/efectos adversos , Fármacos Fotosensibilizantes/efectos adversos , Mancha Vino de Oporto/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Cytosine arabinoside (Ara-C) is one of the most widely used chemotherapeutic agents for hematological malignancies. The residues of Ara-C have been detected in wastewater and river water with increased usage and discharge. As the ability to cross the placenta and the teratogenicity at low ng/L levels, the toxic effects on pregnant women and infants have been concerned. The toxicity of Ara-C exposure on early embryonic neurodevelopment has not been fully elucidated. In this study, pregnant C57BL/6 mice were injected with different doses of Ara-C on Gestation day (GD) 7.5 and assessed on GD11.5 and GD13.5 to explore the neural developmental effects of Ara-C. HE staining, immunofluorescence, western blot, EdU assay, and flow cytometry were utilized to determine the toxic effects of Ara-C in vivo and in vitro. Our results showed that Ara-C (15-22.5 mg/kg body weight) induced the occurrence of neural tube defects (NTDs). The expression of PH3 was markedly reduced in embryos with Ara-C-induced NTDs, compared to the control group (P < 0.05). In contrast, cell apoptosis was markedly increased. Increased expression levels of GFAP and decreased Nestin were observed in the embryonic brain tissues in Ara-C induced NTDs. The level of ß-catenin was also decreased on both GD11.5 and GD13.5. These results were confirmed in vitro using mouse Sv129 embryonic stem cells (mESC). Ara-C at a dose comparable to the environment level (0.05 nM) had cytotoxicity. Impaired Wnt/ß-catenin signaling pathway is involved in Ara-C exposure induced imbalance between cell proliferation, apoptosis, and differentiation, which might contribute to Ara-C-induced occurrence of NTDs. Our data indicated the environmental concentration of Ara-C had cytotoxicity and that maternal exposure to Ara-C induced NTDs. These results might provide more information to understand the environmental toxic impact of Ara-C on neurodevelopment.
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PURPOSE: To investigate the pathogenesis of the hedgehog (Hh) signaling pathway activated by inflammation in the development of chronic rhinosinusitis with nasal polyps (CRSwNP). METHODS: The 82 people including CRSwNP patients (case group) and nasal septal deviation patients (control group) were recruited. The samples in the case group were collected and classified into two groups: mucosal tissue of nasal polyps (NP group) and mucosal tissue adjacent to nasal polyps (NM group), the samples were collected from the control group as CM group. Clinical characteristics were assessed. Hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining were performed to detect eosinophils (EOS), the expression of the key genes of the pathway and epithelial-mesenchymal transition (EMT) markers in the samples. RESULTS: There were significant differences in the nasal obstruction visual analog scale (VAS) score, rhinorrhea VAS score, percentage of blood EOS, blood EOS absolute counts and tissue EOS counts in the case group compared with the control group (P < 0.05). The EOS level and expression levels of PTCH1, SMO, Gli1, Gli2, Ki67 and vimentin were higher in NP group than in the other two groups (P < 0.05). E-cadherin expression was decreased in NP group (P < 0.05). A positive correlation between PTCH1 expression and CRSwNP Lund-Mackay score in NP group. CONCLUSIONS: Our results indicated that the activation of Hh signaling pathway might promote cell proliferation and EMT occurrence, ultimately leading to the development of CRSwNP, which might provide a new target for treatment.
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Pólipos Nasales , Rinitis , Sinusitis , Humanos , Proteínas Hedgehog , Pólipos Nasales/patología , Rinitis/patología , Transición Epitelial-Mesenquimal/fisiología , Sinusitis/patología , Proliferación Celular , Transducción de Señal , Enfermedad CrónicaRESUMEN
Neural tube defects (NTDs) are complex congenital malformations resulting from failure of neural tube closure during embryogenesis, which is affected by the interaction of genetic and environmental factors. It is well known that folate deficiency increases the incidence of NTDs; however, the underlying mechanism remains unclear. Folate deficiency not only causes DNA hypomethylation, but also blocks the synthesis of 2'-deoxythymidine-5'-monophosphate (dTMP) and increases uracil misincorporation, resulting in genomic instabilities such as base mismatch, DNA breakage, and even chromosome aberration. DNA repair pathways are essential for ensuring normal DNA synthesis, genomic stability and integrity during embryonic neural development. Genomic instability or lack of DNA repair has been implicated in risk of development of NTDs. Here, we reviewed the relationship between folate deficiency, DNA repair pathways and NTDs so as to reveal the role and significance of DNA repair system in the pathogenesis of NTDs and better understand the pathogenesis of NTDs.
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Deficiencia de Ácido Fólico , Defectos del Tubo Neural , Humanos , Ácido Fólico/metabolismo , Defectos del Tubo Neural/genética , Deficiencia de Ácido Fólico/metabolismo , Reparación del ADN/genética , ADN , Inestabilidad GenómicaRESUMEN
Chemotherapeutic agents such as methotrexate (MTX), raltitrexed (RTX), 5-fluorouracil (5-FU), hydroxyurea (HU), and retinoic acid (RA), and valproic acid (VPA), an antiepileptic drug, all can cause malformations in the developing central nervous system (CNS), such as neural tube defects (NTDs). However, the common pathogenic mechanisms remain unclear. This study aimed to explore the mechanisms of NTDs caused by MTX, RTX, 5-FU, HU, RA, and VPA (MRFHRV), based on network pharmacology and molecular biology experiments. The MRFHRV targets were integrated with disease targets, to find the potential molecules related to MRFHRV-induced NTDs. Protein-protein interaction analysis and molecular docking were performed to analyze these common targets. Utilizing the kyoto encyclopedia of genes and genomes (KEGG) signaling pathways, we analyzed and searched the possible causative pathogenic mechanisms by crucial targets and the signaling pathway. Results showed that MRFHRV induced NTDs through several key targets (including TP53, MAPK1, HSP90AA1, ESR1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN) and multiple signaling pathways such as PI3K/Akt pathway, suggesting that abnormal proliferation and differentiation could be critical pathogenic contributors in NTDs induced by MRFHRV. These results were further validated by CCK8 assay in mouse embryonic stem cells and GFAP staining in embryonic brain tissue. This study indicated that chemotherapeutic and antiepileptic agents induced NTDs might through predicted targets TP53, MAPK1, GRB2, HDAC1, EGFR, PIK3CA, RXRA, and FYN and multiple signaling pathways. More caution was required for the clinical administration for women with childbearing potential and pregnant.
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Antineoplásicos , Defectos del Tubo Neural , Animales , Femenino , Ratones , Embarazo , Anticonvulsivantes/efectos adversos , Fosfatidilinositol 3-Quinasa Clase I , Receptores ErbB , Fluorouracilo/efectos adversos , Hidroxiurea/efectos adversos , Simulación del Acoplamiento Molecular , Farmacología en Red , Defectos del Tubo Neural/inducido químicamente , Fosfatidilinositol 3-Quinasas , Tretinoina/efectos adversos , Ácido Valproico/efectos adversos , Metotrexato/efectos adversos , Antineoplásicos/efectos adversosRESUMEN
Neural-tube defects (NTDs) are one type of the most serious birth defects. Studies have shown that inositol deficiency is closely related to the occurrence of NTDs. Bone morphogenetic protein (BMP)-mediated Smad signaling pathways have been implicated in neurogenesis and neural-tube closure. However, the role of the BMP/Smad pathway in inositol-deficiency-induced NTDs remains unclear. Inositol-deficiency models in C57 mice and mouse neural stem cells (mNSCs) were induced with Li2CO3 treatment or inositol withdrawal. The role of the BMP/Smad pathway in the regulation of cell proliferation and the development of NTDs was determined utilizing qRT-PCR, HE staining, Western blot, immunostaining, MTT assay, EdU staining, and flow cytometry. The intraperitoneal injection of Li2CO3 at Embryonic Day 7.5 induced the occurrence of NTDs. The mRNA levels of Bmp2, Bmp4, Smad1, Smad5, Smad8 and Runx2, the phosphorylation of Smad1/5/8, and the nuclear translocation of Runx2 were significantly increased in NTD embryonic brain tissues and mNSCs exposed to Li2CO3 or an inositol-free medium, which were suppressed by BMP receptor selective inhibitor LDN-193189. The Li2CO3-induced phosphorylation of Smad1/5/8 was inhibited by inositol supplementation. Cell proliferation was significantly promoted by Li2CO3 exposure or the absence of inositol in mNSCs, which was reversed by LDN-193189. These results suggest that the activation of the BMP/Smad signaling pathway might play an important role in the development of NTDs induced by maternal Li2CO3 exposure via inositol deficiency.
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Células-Madre Neurales , Defectos del Tubo Neural , Ratones , Animales , Carbonato de Litio/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Transducción de Señal/fisiología , Proteína Smad1/genética , Proteína Smad1/metabolismoRESUMEN
BACKGROUND: Lithium carbonate (Li2CO3) is widely used in the treatment of clinical-affective psychosis. Exposure to Li2CO3 during pregnancy increases the risk of neural tube defects (NTDs) in offspring, which are severe birth defects of the central nervous system. The mechanism of Li2CO3-induced NTDs remains unclear. METHODS: C57BL/6 mice were injected with different doses of Li2CO3 intraperitoneally on gestational day 7.5 (GD7.5), and embryos collected at GD11.5 and GD13.5. The mechanisms of Li2CO3 exposure-induced NTDs were determined utilizing immunohistochemistry, western blotting, EdU imaging, enzymatic method, gas chromatography-mass spectrometry (GC-MS), ELISA and HE staining. RESULTS: The NTDs incidence was 33.7% following Li2CO3 exposure. Neuroepithelial cell proliferation and phosphohistone H3 level were significantly increased in NTDs embryos, compared with control group (P < 0.01), while the expressing levels of p53 and caspase-3 were significantly decreased. IMPase and GSK-3ß activity was inhibited in Li2CO3-treated maternal and embryonic neural tissues (P < 0.01 and P < 0.05, respectively), along with decreased levels of inositol and metabolites, compared with control groups (P < 0.01). CONCLUSIONS: Lithium-induced NTDs model in C57BL/6 mice was established. Enhanced cell proliferation and decreased apoptosis following lithium exposure were closely associated with the impairment of inositol biosynthesis, which may contribute to lithium-induced NTDs. IMPACT: Impairment of inositol biosynthesis has an important role in lithium exposure-induced NTDs in mice model. Lithium-induced NTDs model on C57BL/6 mice was established. Based on this NTDs model, lithium-induced impairment of inositol biosynthesis resulted in the imbalance between cell proliferation and apoptosis, which may contribute to lithium-induced NTDs. Providing evidence to further understand the molecular mechanisms of lithium-induced NTDs and enhancing its primary prevention.
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Sistema Nervioso Central/efectos de los fármacos , Carbonato de Litio/efectos adversos , Exposición Materna , Defectos del Tubo Neural/inducido químicamente , 5'-Nucleotidasa/metabolismo , Animales , Sistema Nervioso Central/crecimiento & desarrollo , Modelos Animales de Enfermedad , Femenino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Inositol/metabolismo , Ratones , Ratones Endogámicos C57BL , EmbarazoRESUMEN
BACKGROUND Inositol is an essential nutrient for cell growth, survival and embryonic development. Myo-inositol is the predominant form in natural. To investigate the correlation between inositol metabolism and embryonic development, we assessed the metabolic characteristics of myo-inositol, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) of pregnant women in the North China (Yangquan and Weihai) and South China (Nanchang and Haikou) China. MATERIAL AND METHODS All data were collected by face-to-face interview during pregnant women health visits using a questionnaire. Plasma levels of myo-inositol, PI(4,5)P2 and PI(3,4,5)P3 from 89 randomly collected pregnant women were detected by gas chromatography-mass spectrometry and enzyme linked immunosorbent assay. RESULTS A total of 400 pregnant women were included in this survey. The plasma levels of myo-inositol and PI(4,5)P2 in the North China group of pregnant women were significantly higher than that in the South China group (P<0.01). The birth weight of fetuses in the North China group was heavier than that in the South China group (P<0.01). The birth length of fetuses in Yangquan was the longest among the 4 cities (P<0.01). The incidence rate of birth defects was 3.05% in the North China group, and 0.0% in the South China group. In bivariate linear correlation analysis, the body weight correlated with myo-inositol (r=0.5044, P<0.0001), PI(4,5)P2 (r=0.5950, P<0.0001) and PI(3,4,5)P3 (r=0.4710, P<0.0001), the body length was correlated with PI(4,5)P2 (r=0.3114, P=0.0035) and PI(3,4,5)P3 (r=0.2638, P<0.0130). CONCLUSIONS The plasma levels of myo-inositol and PI(4,5)P2 in pregnant women had significant difference between the North and the South of China, which might be correlated with fetal development and birth defects.
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Anomalías Congénitas/epidemiología , Desarrollo Fetal/fisiología , Inositol/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Adulto , China/epidemiología , Anomalías Congénitas/metabolismo , Femenino , Geografía , Humanos , Incidencia , Recién Nacido , Inositol/sangre , Fosfatidilinositol 4,5-Difosfato/sangre , Fosfatos de Fosfatidilinositol/sangre , Fosfatos de Fosfatidilinositol/metabolismo , EmbarazoRESUMEN
Human Mesenchymal Stem Cells (MSCs) especially human umbilical cord MSCs is the novel regenerative cell resource for regenerative therapy. However, the biological underpinning of MSCs in neuroprotections requires deep understanding. Exosomes is an important biological factor due to its multiple types of contents with various biological function. In current study, we collected the exosome from umbilical cord mesenchymal stem cells (hUC-MSCs) and tested the neuroprotective effects to brain stress. Proteomic analysis indicates significant enriched protein components display the functions in metabolic regulation. We then injected the exosome (MSC-Ex) to adult mice by i.v injection. On physiological level, treatment of MSC-Ex increased the adiponectin level in peripheral central nervous system (CNS). Moreover, MSC-Ex significantly accelerated the differentiation of adult neural stem cells but did not benefit the related cognitive behavior. We then created acute brain disorder model with STZ intra-hippocampal injection. Compared with STZ group, treatment of MSC-Ex improved cognitive function. Moreover, MSC-Ex promotes hippocampal neurogenesis that was suppressed by STZ injection. In conclusion, hUC-MSCs derived exosome would exert the neural regenerative effects associating with its metabolism regulatory capacity.
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Exosomas/metabolismo , Hipocampo/metabolismo , Trasplante de Células Madre Mesenquimatosas/métodos , Estrés Psicológico/metabolismo , Estrés Psicológico/terapia , Cordón Umbilical/metabolismo , Animales , Células Cultivadas , Exosomas/química , Exosomas/trasplante , Femenino , Hipocampo/química , Humanos , Masculino , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Neurogénesis/fisiología , Embarazo , Cordón Umbilical/química , Cordón Umbilical/trasplanteRESUMEN
BACKGROUND Maternal folate deficiency-mediated metabolic disruption is considered to be associated with the risk of intrauterine growth retardation (IUGR), but the exact mechanism remains unclear. The retrotransposon long interspersed nucleotide element-1 (LINE-1), which can induce birth defects via RNA intermediates, plays crucial roles during embryonic development. We investigated potential relationships between maternal folate and DNA methylation, and possible roles of LINE-1 in IUGR. MATERIAL AND METHODS The IUGR model was established by feeding female mice 1 of 3 diets - control diet (CD), folate-deficient diet for 2 weeks (FD2w), and folate-deficient diet for 4 weeks (FD4w) - prior to mating. Maternal serum folate, 5-methyltetrahydrofolate (5-MeTHF), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) concentrations and global DNA methylation were assessed by LC/MS/MS method. LINE-1 methylation levels in fetuses were examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LINE-1 expression levels were validated by real-time PCR. RESULTS Maternal folate deficiency caused plasma folate and 5-MeTHF levels to decrease and SAH level to increase in the FD4w group. Compared with the CD group, methylation levels of genomic DNA and LINE-1 decreased significantly in placenta and fetal tissues from the FD4w group. Expression of LINE-1 open reading frame 1 (ORF1) protein was elevated in fetal liver tissues. Furthermore, a strong correlation was found between methylation and disrupted one-carbon metabolism, implying that dietary folate plays important roles during embryogenesis. CONCLUSIONS Maternal dietary folate deficiency impaired one-carbon metabolism, leading to global DNA and LINE-1 hypomethylation, and then increased retrotransposition in fetuses, which can lead to IUGR.
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Retardo del Crecimiento Fetal/genética , Deficiencia de Ácido Fólico/genética , Deficiencia de Ácido Fólico/metabolismo , Animales , Metilación de ADN/genética , Modelos Animales de Enfermedad , Femenino , Retardo del Crecimiento Fetal/metabolismo , Feto/metabolismo , Ácido Fólico/sangre , Ácido Fólico/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , Elementos de Nucleótido Esparcido Largo/fisiología , Masculino , Intercambio Materno-Fetal/fisiología , Ratones , Ratones Endogámicos C57BL , Placenta/metabolismo , Embarazo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismo , Tetrahidrofolatos/metabolismoRESUMEN
BACKGROUND DNA Base Excision Repair Gene-DNA LigaseIII (LIG3) is an important repair gene in the repair pathway and plays an important role in maintaining the integrity of mitochondria. Rs1052536 and rs3135967 polymorphisms of the gene are associated with lung cancer, keratoconus, and Fuchs endothelial corneal dystrophy. There is no previously published report on the relationship between the polymorphisms and neural tube defects (NTDs). MATERIAL AND METHODS Mass ARRAY iPLEX was used to determine the distribution of the polymorphisms in the case group of 108 NTD pregnant women and a control group of 233 normal healthy pregnant women to examine the relevance of their polymorphisms and NTD occurrence. RESULTS The homozygotes of rs1052536 TT were associated with an increased risk for NTDs than CC (P=0.014, OR=2.31, 95%CI [1.17-4.54]), and variants of rs1052536 T were associated with an increased risk of NTDs (P=0.024, OR=1.50, 95%CI [1.06-2.13]). The stratified analysis showed that TT genotype of rs1052536 increased the risk of anencephaly (P=0.016, OR=2.69, 95%CI [1.18-6.10]) and the T allele significantly increased the risk of cranial NTDs (P=0.033, OR=1.56, 95%CI [1.04-2.35]). CONCLUSIONS Rs1052536 in LIG3 gene might be a potential genetic risk factor in a high-risk area of NTDs in China.
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ADN Ligasa (ATP)/genética , Defectos del Tubo Neural/genética , Proteínas de Unión a Poli-ADP-Ribosa/genética , Adulto , Alelos , Anencefalia/genética , Estudios de Casos y Controles , China/epidemiología , ADN Ligasa (ATP)/fisiología , Reparación del ADN/genética , Femenino , Frecuencia de los Genes/genética , Predisposición Genética a la Enfermedad , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/fisiología , Polimorfismo de Nucleótido Simple/genética , Embarazo , Factores de Riesgo , Adulto JovenRESUMEN
BACKGROUND The INPP5E gene encodes for the inositol polyphosphate-5-phosphatase (INPP5E) 72 kDa protein that regulates the phosphoinositide signaling pathway and other cellular activities, but the functional role of this gene in embryonic neurodevelopment and neural tube defect (NTD) remains unclear. The aim of this study was to use a mouse model of NTD to investigate the expression levels of the INPP5E gene during neural development and the occurrence of NTD. MATERIAL AND METHODS In an established NTD mouse model, stereoscopy was used to look for morphological defects. Transcription and expression levels of the INPP5E gene in neural tissues were detected using real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blotting in the NTD mouse embryos and compared with control mouse embryos. RESULTS The expression levels of the INPP5E gene decreased as embryonic development progressed in the neural tissue of control mice embryos, but showed no obvious trend in the neural tissues of the NTD mouse embryos. The expression levels of the INPP5E gene in NTD mouse embryos were significantly lower compared with control embryos, at the time of neural tube closure (gestational day 11.5). CONCLUSIONS The INPP5E gene regulates the process of embryonic neural development. Abnormal levels of expression of the INPP5E gene may contribute to NTDs. Increased knowledge of the expression pattern of the INPP5E gene may lead to an advanced understanding of the molecular mechanism of embryonic neurodevelopment and identify more specific directions to explore potential treatments for NTDs associated with abnormalities in INPP5E gene expression levels.
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Defectos del Tubo Neural/genética , Monoéster Fosfórico Hidrolasas/genética , Animales , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Defectos del Tubo Neural/metabolismo , Neurogénesis , Monoéster Fosfórico Hidrolasas/metabolismo , EmbarazoRESUMEN
The causal metabolic pathway and the underlying mechanism between folate deficiency and neural tube defects (NTDs) remain obscure. Thymidylate (dTMP) is catalyzed by thymidylate synthase (TS) using the folate-derived one-carbon unit as the sole methyl donor. This study aims to examine the role of dTMP biosynthesis in the development of neural tube in mice by inhibition of TS via a specific inhibitor, raltitrexed (RTX). Pregnant mice were intraperitoneally injected with various doses of RTX on gestational day 7.5, and embryos were examined for the presence of NTDs on gestational day 11.5. TS activity and changes of dUMP and dTMP levels were measured following RTX treatment at the optimal dose. DNA damage was determined by detection of phosphorylated replication protein A2 (RPA2) and γ-H2AX in embryos with NTDs induced by RTX. Besides, apoptosis and proliferation were also analyzed in RTX-treated embryos with NTDs. We found that NTDs were highly occurred by the treatment of RTX at the optimal dose of 11.5 mg/kg b/w. RTX treatment significantly inhibited TS activity. Meanwhile, dTMP was decreased associated with the accumulation of dUMP in RTX-treated embryos. Phosphorylated RPA2 and γ-H2AX were significantly increased in RTX-treated embryos with NTDs compared to control. More apoptosis and decreased proliferation were also found in embryos with NTDs induced by RTX. These results indicate that impairment of dTMP biosynthesis caused by RTX led to the development of NTDs in mice. DNA damage and imbalance between apoptosis and proliferation may be potential mechanisms.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Daño del ADN , Embrión de Mamíferos/efectos de los fármacos , Defectos del Tubo Neural/metabolismo , Quinazolinas/toxicidad , Tiofenos/toxicidad , Animales , Western Blotting , Nucleótidos de Desoxiuracil/metabolismo , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Femenino , Antagonistas del Ácido Fólico/administración & dosificación , Antagonistas del Ácido Fólico/toxicidad , Edad Gestacional , Histonas/metabolismo , Inyecciones Intraperitoneales , Masculino , Ratones Endogámicos C57BL , Defectos del Tubo Neural/inducido químicamente , Defectos del Tubo Neural/genética , Fosforilación/efectos de los fármacos , Embarazo , Subunidades de Proteína/metabolismo , Quinazolinas/administración & dosificación , Proteína de Replicación A/metabolismo , Tiofenos/administración & dosificación , Timidina Monofosfato/metabolismo , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/metabolismoRESUMEN
BACKGROUND: Animal models of neural tube defects (NTDs) have indicated roles for the Fzd3 gene and the planar cell polarity signaling pathway in convergent extension. We investigated the involvement of FZD3 in genetic and epigenetic mechanisms associated with human NTDs, especially spina bifida. We explored the effects of variants spanning the FZD3 gene in NTDs and examined the role of aberrant methylation of the FZD3 promoter on gene expression in brain tissue in spina bifida. METHODS: Six FZD3 single nucleotide polymorphisms were genotyped using a MassARRAY system in tissue from 165 NTD fetuses and 152 controls. DNA methylation aberrations in the FZD3 promoter region were detected using a MassARRAY EpiTYPER (17 CpG units from -500 to -2400 bp from the transcription start site) in brain tissue from 77 spina bifida and 74 control fetuses. RESULTS: None of the six single nucleotide polymorphisms evaluated were significantly associated with spina bifida, but the mean methylation level was significantly higher in spina bifida samples (13.70%) compared with control samples (10.91%) (p = 0.001). In terms of specific sites, DNA methylation levels were significantly higher in the spina bifida samples at 14 of the 17 CpG units, which mostly included in R2 region. FZD3 mRNA expression was negatively correlated with methylation of the FZD3 promoter region, especially the R2 region (R = 0.970; p = 0.001) in HeLa cells. CONCLUSION: The results of this study suggest that DNA methylation plays an important role in FZD3 gene expression regulation and may be associated with an increased risk of spina bifida.
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Metilación de ADN , Receptores Frizzled/genética , Regulación de la Expresión Génica , Defectos del Tubo Neural/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética , Disrafia Espinal/etiología , Secuencia de Bases , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Epigénesis Genética , Femenino , Feto/metabolismo , Feto/patología , Predisposición Genética a la Enfermedad , Genotipo , Edad Gestacional , Humanos , Masculino , Datos de Secuencia Molecular , Defectos del Tubo Neural/complicaciones , Embarazo , Factores de Riesgo , Disrafia Espinal/patologíaRESUMEN
A new deoxyribonuclease (DNase), referred to as EWDNase, was isolated from earthworm tissues. The purification protocol included acetone precipitation, chromatography on CM-Sepharose, and gel electrophoresis. The overall purification was 73-fold with a recovery rate of 2.3% and a final specific activity of 2039 U/mg. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis suggested a molecular mass of 30 kD for EWDNase, with an isoelectric point of approximately 7.0. Maximum activity was detected at a pH of 5.6 and a temperature of 40°C. Addition of Mg(2+) and Ca(2+) ions promoted enzyme activity strongly, while Zn(2+) and ethylenediamine tetraacetic acid (EDTA) acted as inhibitors. Liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis indicated that there was no known matching sequence. The properties of EWDNase were sufficiently different from previously reported enzymes to suggest that it is a new enzyme requiring further confirmation and characterization.
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Desoxirribonucleasas/aislamiento & purificación , Desoxirribonucleasas/metabolismo , Oligoquetos/enzimología , Animales , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Metales/farmacología , Peso Molecular , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
Apoptosis and proliferation play important roles in embryonic development and are required for neural tube closure. The antifolate drug methotrexate (MTX) induces folate dysmetabolism by inhibition of dihydrofolate reductase and causes abnormal apoptosis and proliferation. In this study, we established an animal model of neural tube defects (NTDs) using MTX to investigate the role of apoptosis and proliferation in NTDs caused by folate deficiency. Differential gene expressions were studied by microarray and reverse transcription-polymerase chain reaction in the NTD animal model. Results showed that 30.8% of NTDs were caused by using MTX in treatment regimens. Microarray indicated that 166 genes were significantly different between the control and NTD mice, including four apoptosis-related genes (Endog, Trp53, Casp3, Bax) and three proliferation-related genes (Ptch1, Pla2g4a, Foxg1). Levels of Endog, Trp53, Casp3, Bax (fold change>1.5) were upregulated but Ptch1, Pla2g4a, Foxg1 (fold change<0.67) were downregulated (P<0.05). These results were confirmed by reverse transcription-polymerase chain reaction. TUNEL, immunohistochemical assays and Western blot were further used to detect apoptosis and proliferation in the NTD animal model. It was found that apoptosis in neuroepithelial cells was increased as determined by TUNEL (P<0.05). Expressions of caspase-3 were significantly enhanced (P<0.05) but expressions of phosphohistone H3 were greatly decreased (P<0.05). These results concluded that MTX caused a folate and folate-associated dysmetabolism, and further induced abnormal apoptosis and proliferation, which may play a critical role in the occurrence of NTDs caused by folate deficiency.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Antagonistas del Ácido Fólico/toxicidad , Metotrexato/toxicidad , Neurulación/efectos de los fármacos , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Desarrollo Embrionario/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Femenino , Ácido Fólico/metabolismo , Deficiencia de Ácido Fólico/complicaciones , Deficiencia de Ácido Fólico/embriología , Histonas/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Defectos del Tubo Neural/embriología , Embarazo , Tetrahidrofolato Deshidrogenasa/metabolismo , Regulación hacia ArribaRESUMEN
Myelomeningocele is a common congenital anomaly associated with polygenic disorders worldwide. However, the intricate molecular mechanisms underlying myelomeningocele remain elusive. To investigate whether ferroptosis and ferritinophagy contribute to the pathomechanism of myelomeningocele, differentially expressed genes (DEGs) were identified as novel biomarker and potential treatment agents. The GSE101141 dataset from Gene Expression Omnibus (GEO) was analyzed using GEO2R web tool to obtain DEGs based on |log2 fold change (FC)|≥1.5 and p < 0.05. Two datasets from the Ferroptosis Database (481 genes) and Autophagy Database (551 genes) were intersected with the DEGs from the GSE101141 dataset to identify ferroptosis- and autophagy-related DEGs using Venn diagrams. Functional and pathway enrichment, protein-protein interaction (PPI) network analyses were performed, and candidate genes were selected. Transcription factors (TFs), microRNAs (miRNAs), diseases and chemicals interacting with the candidate genes were identified. Receiver operating characteristic (ROC) curve analysis was performed to validate the diagnostic value of the candidate genes. Sixty ferroptosis-related and 74 autophagy-related DEGs were identified. These DEGs are involved in FoxO signaling pathway. Six candidate genes (EGFR, KRAS, IL1B, SIRT1, ATM, and MAPK8) were selected. miRNAs such as hsa-miR-27a-3p, hsa-miR-877-5p, and hsa-miR-892b, and TFs including P53, POU3F2, TATA are involved in regulation of candidate genes. Diseases such as schizophrenia, fibrosis, and neoplasms are the most relevant to the candidate genes. Chemicals, such as resveratrol, curcumin, and quercetin may have significant implications in the treatment of myelomeningocele. The candidate genes, especially MAPK8, also showed a high diagnostic value for myelomeningocele. These results help to shed light on the molecular mechanism of myelomeningocele and may provide new insights into diagnostic biomarker in the amniotic fluid and potential therapeutic agents of myelomeningocele.
RESUMEN
Retinal vessel segmentation is crucial for the diagnosis of ophthalmic and cardiovascular diseases. However, retinal vessels are densely and irregularly distributed, with many capillaries blending into the background, and exhibit low contrast. Moreover, the encoder-decoder-based network for retinal vessel segmentation suffers from irreversible loss of detailed features due to multiple encoding and decoding, leading to incorrect segmentation of the vessels. Meanwhile, the single-dimensional attention mechanisms possess limitations, neglecting the importance of multidimensional features. To solve these issues, in this paper, we propose a detail-enhanced attention feature fusion network (DEAF-Net) for retinal vessel segmentation. First, the detail-enhanced residual block (DERB) module is proposed to strengthen the capacity for detailed representation, ensuring that intricate features are efficiently maintained during the segmentation of delicate vessels. Second, the multidimensional collaborative attention encoder (MCAE) module is proposed to optimize the extraction of multidimensional information. Then, the dynamic decoder (DYD) module is introduced to preserve spatial information during the decoding process and reduce the information loss caused by upsampling operations. Finally, the proposed detail-enhanced feature fusion (DEFF) module composed of DERB, MCAE and DYD modules fuses feature maps from both encoding and decoding and achieves effective aggregation of multi-scale contextual information. The experiments conducted on the datasets of DRIVE, CHASEDB1, and STARE, achieving Sen of 0.8305, 0.8784, and 0.8654, and AUC of 0.9886, 0.9913, and 0.9911 on DRIVE, CHASEDB1, and STARE, respectively, demonstrate the performance of our proposed network, particularly in the segmentation of fine retinal vessels.
RESUMEN
Primary cilia are crucial for neurogenesis, and cilium-related genes are involved in the closure of neural tubes. Inositol polyphosphate-5-phosphatase (Inpp5e) was enriched in primary cilia and closely related to the occurrence of neural tube defects (NTDs). However, the role of Inpp5e in the development of NTDs is not well-known. To investigate whether Inpp5e gene is associated with the neural tube closure, we established a mouse model of NTDs by 5-fluorouracil (5-FU) exposure at gestational day 7.5 (GD7.5). The Inpp5e knockdown (Inpp5e-/-) mouse embryonic stem cells (mESCs) were produced by CRISPR/Cas9 system. The expressions of Inpp5e and other cilium-related genes including intraflagellar transport 80 (Ift80), McKusick-Kaufman syndrome (Mkks), and Kirsten rat sarcoma viral oncogene homolog (Kras) were determined, utilizing quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), western blot, PCR array, and immunofluorescence staining. The result showed that the incidence of NTDs was 37.10% (23 NTDs/62 total embryos) and significantly higher than that in the control group (P < 0.001). The neuroepithelial cells of neural tubes were obviously disarranged in NTD embryos. The mRNA and protein levels of Inpp5e, Ift80, Mkks, and Kras were significantly decreased in NTD embryonic brain tissues, compared to the control (P < 0.05). Knockdown of the Inpp5e (Inpp5e-/-) reduced the expressions of Ift80, Mkks, and Kras in mESCs. Furthermore, the levels of α-tubulin were significantly reduced in NTD embryonic neural tissue and Inpp5e-/- mESCs. These results suggested that maternal 5-FU exposure inhibited the expression of Inpp5e, which resulted in the downregulation of cilium-related genes (Ift80, Mkks, and Kras), leading to the impairment of primary cilium development, and ultimately disrupted the neural tube closure.