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1.
Nagoya J Med Sci ; 86(2): 223-236, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38962411

RESUMO

Cleft palate is the most common facial birth defect worldwide. It is caused by environmental factors or genetic mutations. Environmental factors such as pharmaceutical exposure in women are known to induce cleft palate. The aim of the present study was to investigate the protective effect of Sasa veitchii extract against medicine-induced inhibition of proliferation of human embryonic palatal mesenchymal cells. We demonstrated that all-trans-retinoic acid inhibited human embryonic palatal mesenchymal cell proliferation in a dose-dependent manner, whereas dexamethasone treatment had no effect on cell proliferation. Cotreatment with Sasa veitchii extract repressed all-trans-retinoic acid-induced toxicity in human embryonic palatal mesenchymal cells. We found that cotreatment with Sasa veitchii extract protected all-trans-retinoic acid-induced cyclin D1 downregulation in human embryonic palatal mesenchymal cells. Furthermore, Sasa veitchii extract suppressed all-trans-retinoic acid-induced miR-4680-3p expression. Additionally, the expression levels of the genes that function downstream of the target genes ( ERBB2 and JADE1 ) of miR-4680-3p in signaling pathways were enhanced by cotreatment with Sasa veitchii extract and all-trans-retinoic acid compared to all-trans-retinoic acid treatment. These results suggest that Sasa veitchii extract suppresses all-trans-retinoic acid-induced inhibition of cell proliferation via modulation of miR-4680-3p expression.


Assuntos
Proliferação de Células , Fissura Palatina , Palato , Extratos Vegetais , Tretinoína , Humanos , Tretinoína/farmacologia , Proliferação de Células/efeitos dos fármacos , Palato/efeitos dos fármacos , Palato/embriologia , Palato/citologia , Extratos Vegetais/farmacologia , MicroRNAs/metabolismo , MicroRNAs/genética , MicroRNAs/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina D1/genética , Células Cultivadas , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
Biochem Biophys Res Commun ; 725: 150266, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-38878759

RESUMO

Cisplatin (CDDP) is a platinum-based anticancer drug widely prescribed for its effectiveness in treating various forms of cancer. However, its major side effect is nephrotoxicity. Although several methods have been developed to mitigate CDDP-induced nephrotoxicity, an optimal approach has yet to be established. This study aimed to investigate the "chronotoxicity" of CDDP as a potential strategy to reduce its side effects. Male ICR mice were treated with CDDP (20 mg/kg, intraperitoneal injection, one shot) at zeitgeber time (ZT) 2 or ZT14 (light or dark phase). After 72 h, we collected plasma and kidney and evaluated several markers. We found that body weight change between ZT2 and ZT14 by CDDP was comparable. In contrast, many toxicological factors, such as plasma blood urine nitrogen, plasma creatinine, renal oxidative stress (malondialdehyde), DNA damage (γH2AX), acute kidney injury biomarker (KIM-1), and inflammation (Tnfα), were significantly induced at ZT14 compared to than that of ZT2. Our present data suggested that chronotoxicology might provide beneficial information on the importance of administration timings for toxic evaluations and unacceptable side effects.


Assuntos
Antineoplásicos , Ritmo Circadiano , Cisplatino , Rim , Camundongos Endogâmicos ICR , Animais , Cisplatino/toxicidade , Masculino , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Antineoplásicos/toxicidade , Antineoplásicos/efeitos adversos , Camundongos , Ritmo Circadiano/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Nefropatias/induzido quimicamente , Nefropatias/metabolismo , Nefropatias/patologia
3.
J Toxicol Sci ; 49(1): 1-8, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38191190

RESUMO

Cleft palate (CP) is one of the most common birth defects and is caused by a combination of genetic and/or environmental factors. Environmental factors such as pharmaceutical exposure in pregnant women are known to induce CP. Recently, microRNA (miRNA) was found to be affected by environmental factors. The aim of the present study was to investigate the involvement of miRNA against phenytoin (PHE)-induced inhibition of proliferation in human embryonic palatal mesenchymal (HEPM) cells. We demonstrated that PHE inhibited HEPM cell proliferation in a dose-dependent manner. We found that treatment with PHE downregulated cyclin-D1 and cyclin-E expressions in HEPM cells. Furthermore, PHE increased miR-4680-3p expression and decreased two downstream genes (ERBB2 and JADE1). Importantly, an miR-4680-3p-specific inhibitor restored HEPM cell proliferation and altered expression of ERBB2 and JADE1 in cells treated with PHE. These results suggest that PHE suppresses cell proliferation via modulation of miR-4680-3p expression.


Assuntos
MicroRNAs , Fenitoína , Gravidez , Humanos , Feminino , Fenitoína/toxicidade , MicroRNAs/genética , Proliferação de Células , Palato
4.
Nat Commun ; 15(1): 821, 2024 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-38280850

RESUMO

Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. Here, we perform single-cell multiome sequencing and profile chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study charts epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.


Assuntos
Fissura Palatina , Camundongos , Animais , Fissura Palatina/genética , Multiômica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento
5.
Biochem Biophys Res Commun ; 696: 149516, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38241808

RESUMO

Cleft palate (CP) is one of the most common congenital diseases, and is accompanied by a complicated etiology. Medical exposure in women is among one of the reasons leading to CP. Recently, it has been reported that microRNA (miRNA) plays a crucial role in palate formation and the disruption of miRNA that influence the development of CP. Although association with pharmaceuticals and miRNAs were suggested, it has remained largely unknow. The aim of the current investigation is to elucidate upon the miRNA associated with the inhibition of phenobarbital (PB)-induced cell proliferation in human embryonic palatal mesenchymal (HEPM) cells. We showed that PB inhibited HEPM cell viability in a dose-dependent manner. We demonstrated that PB treatment suppressed cyclin-D1 expression in HEPM cells. Furthermore, PB upregulated let-7c-5p expression and downregulated the expression of two downstream genes (BACH1 and PAX3). Finally, we demonstrated that the let-7c-5p inhibitor alleviated PB-induced inhibition of cell proliferation and altered BACH1 and PAX3 expression levels. These results suggest that PB suppresses cell viability by modulating let-7c-5p expression.


Assuntos
Fissura Palatina , Células-Tronco Mesenquimais , MicroRNAs , Humanos , Feminino , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proliferação de Células/genética
6.
Cell Signal ; 113: 110980, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37981065

RESUMO

Previous studies have demonstrated that extracellular vesicles (EVs) from dental pulp stem cells (DPSCs), which release abundant hepatocyte growth factor (HGF) and transforming growth factor-ß1 (TGF-ß1), contribute to the pathogenesis of Sjögren's syndrome (SS). However, depending on the condition of DPSCs, this effect is often not achieved. In this study, we established induced pluripotent stem (iPS) cells highly capable of releasing HGF and TGF-ß1 and iPS cells barely capable of releasing them, and administered each EV to SS model mice to see if there was a difference in therapeutic effect. EVs were collected from each iPS cell and their characteristics and shapes were examined. When they were administered to SS model mice, the EVs from iPS cells with higher concentrations of HGF and TGF-ß1 showed significantly reduced inflammatory cell infiltration in salivary gland tissues, increased saliva volume, and decreased anti-SS-A and anti-SS-B antibodies. A comprehensive search of microRNA arrays for differences among those EVs revealed that EVs from iPS cells with higher concentrations of HGF and TGF-ß1 contained more of the let-7 family. Thereafter, we examined the expression of toll-like receptors (TLRs), which are said to be regulated by the let-7 family, by qPCR, and found decreased TLR4 expression. Focusing on MAPK, a downstream signaling pathway, we examined cytokine concentrations in mouse macrophage culture supernatants and Western blotting of murine splenic tissues and found higher concentrations of anti-inflammatory cytokines in the EVs-treated group and decreased TLR4, NF-κB and phosphorylation (p)-p-38 MAPK expression by Western blotting. Alternatively, p-Smad2/3 was upregulated in the EVs-treated group. Our findings suggest that the let-7 family in EVs may suppress the expression of TLR4 and NF-κB, which may be involved in the suppression of MAPK-mediated pro-inflammatory cytokine production.


Assuntos
Vesículas Extracelulares , Células-Tronco Pluripotentes Induzidas , Síndrome de Sjogren , Animais , Camundongos , Vesículas Extracelulares/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Imunidade Inata , Células-Tronco Pluripotentes Induzidas/metabolismo , NF-kappa B/metabolismo , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/patologia , Receptor 4 Toll-Like/metabolismo , Fator de Crescimento Transformador beta1
7.
Artigo em Inglês | MEDLINE | ID: mdl-38092388

RESUMO

BACKGROUND: This study aimed to investigate diurnal variations in copper-induced hepatic toxicity and the molecular mechanisms underlying this chronotoxicity. METHODS: Male C57BL/6J mice were intraperitoneally injected with copper chloride (CuCl2) at zeitgeber time 2 (ZT2) or 14 (ZT14), twice per week for 5 or 8 weeks. Seventy-two hours after the final CuCl2 injection, the mice were euthanized, and plasma samples were collected. The livers and kidneys were collected and weighed. In vitro experiments were performed to assess cell viability and fluctuations in clock gene expression levels in Hepa1-6 cells after CuCl2 treatment. We examined copper homeostasis- and apoptosis-related genes under clock genes overexpression. RESULTS: Repeated CuCl2 administration for 8 weeks resulted in more severe toxicity at ZT14 compared to ZT2. CuCl2 administration at ZT14 elevated plasma aspartate aminotransferase, hepatic tumor necrosis factor-α, and interleukin-6 for 5 weeks, whereas the toxic effects of CuCl2 administration at ZT2 were weaker. Moreover, CuCl2 treatment inhibited Hepa1-6 cell viability in a dose-dependent manner. We observed increased expression of three clock genes (Ciart, Cry2, and Per1) after CuCl2 treatment. Among them, overexpression of Cry2 and Per1 accelerated CuCl2-induced inhibition of Hepa1-6 cell viability. Moreover, we found that the overexpression of Cry2 and Per1 regulates cleaved caspase-3 by modulating the copper transporter genes ATP7B and CTR1. CONCLUSION: These results suggest that CuCl2-induced diurnal toxicity is associated with Cry2 and Per1 expression through the regulation of copper transporter genes in mice.


Assuntos
Cobre , Fatores de Transcrição , Masculino , Camundongos , Animais , Cobre/toxicidade , Cobre/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Fígado/metabolismo , Ritmo Circadiano , Criptocromos/genética , Criptocromos/metabolismo , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo
8.
Biol Pharm Bull ; 46(6): 824-829, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37258148

RESUMO

Circadian rhythms are endogenous oscillators that regulate 24 h behavioral and physiological processes. Our previous investigation demonstrated that bromobenzene metabolite (4-bromocatechol: 4-BrCA) exhibited chronotoxicity (i.e., the nephrotoxicity induced by 4-BrCA was observed during the dark phase, while not observed at light phase in mice). However, the molecular mechanism is still unknown. The aim of the present study is to investigate the cellular molecule(s) involved in the 4-BrCA-induced nephrotoxicity using mouse renal cortex tubular cell lines (MuRTE61 cells). We found that 4-BrCA showed dose dependent (0.01-1 mM) cell proliferation defect in MuRTE61 cells. By treating with 0.03 mM 4-BrCA, we demonstrated that major clock genes (Bmal1, Clock, Cry1, Cry2, Per1, and Per2) were significantly downregulated. Interestingly, the expression levels of two genes, Bmal1 and Clock, continued to decrease after 3 h of treatment with 4-BrCA, while Cry1, Per1, and Per2 were unchanged until 24 h of treatment. Moreover, BMAL1 and CLOCK levels are higher at light phase. We speculated that BMAL1 and CLOCK might function defensively against 4-BrCA-induced nephrotoxicity since the expression levels of Bmal1 and Clock were rapidly decreased. Finally, overexpression of Bmal1 and Clock restored 4-BrCA-induced cell proliferation defect in MuRTE61 cells. Taken together, our results suggest that Bmal1 and Clock have protective roles against 4-BrCA-induced nephrotoxicity.


Assuntos
Fatores de Transcrição ARNTL , Bromobenzenos , Camundongos , Animais , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Ritmo Circadiano/genética , Regulação da Expressão Gênica
9.
Biomed Res ; 44(2): 73-80, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37005285

RESUMO

A cleft lip, with or without a cleft palate, is a common birth defect caused by environmental factors or genetic mutations. Environmental factors, such as pharmaceutical exposure in pregnant women, are known to induce cleft lip, with or without cleft palate in the child. This study aimed to investigate the protective effect of Sasa veitchii extract (SE) on phenytoin-induced inhibition of cell proliferation in human lip mesenchymal cells (KD cells) and human embryonic palatal mesenchymal cells (HEPM cells). We demonstrated that cell proliferation was inhibited by phenytoin in a dose-dependent manner in both KD and HEPM cells. Co-treatment with SE restored phenytoin-induced toxicity in KD cells but did not protect HEPM cells against phenytoin-induced toxicity. Several microRNAs (miR-27b, miR-133b, miR-205, miR-497-5p, and miR-655-3p) is reported to associate with cell proliferation in KD cells. We measured the seven kinds of microRNAs (miR27b-3p, miR-27b-5p, miR-133b, miR-205-3p, miR-205-5p, miR-497-5p, and miR-655-3p) and found that SE suppressed miR-27b-5p induced by phenytoin in KD cells. Furthermore, co-treatment with SE enhanced the expression of miR-27b-5p downstream genes (PAX9, RARA, and SUMO1). These results suggest that SE protects phenytoin-induced cell proliferation inhibition by modulating miR-27b-5p.


Assuntos
Fenda Labial , Fissura Palatina , MicroRNAs , Sasa , Gravidez , Criança , Humanos , Feminino , Fenitoína/farmacologia , Sasa/genética , Sasa/metabolismo , Fissura Palatina/induzido quimicamente , Fissura Palatina/genética , Fenda Labial/genética , MicroRNAs/genética , Proliferação de Células/genética
10.
Biochim Biophys Acta Gen Subj ; 1867(5): 130331, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36804277

RESUMO

This study determined the effect of brefeldin A (BFA) on the free N-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type free N-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolic endo-ß-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.


Assuntos
Glicosídeo Hidrolases , Polissacarídeos , Humanos , Brefeldina A/farmacologia , Células Hep G2 , Polissacarídeos/metabolismo , Manosil-Glicoproteína Endo-beta-N-Acetilglucosaminidase
11.
J Toxicol Sci ; 47(12): 547-553, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36450499

RESUMO

Zinc (Zn) is one of the most essential trace elements in the body and an integral part of many enzyme systems. Zn deficiency is characterized by growth retardation, loss of appetite, and impaired immune function. In contrast, Zn overdoses can be associated with liver, kidney, and stomach damage. We focused on the "chronotoxicity," or the relationship between injection time and severity of chemical toxicity. The aim of this study was to investigate the chronotoxicity of Zn and the in vivo factors involved. Seven-week-old male ICR mice were administered Zn at six different time points per day (zeitgeber time [ZT]: ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22). Mortality was monitored for 7-days after administration. The mice were tolerant to Zn administered at ZT2 and ZT6, and were highly sensitive at ZT14 and ZT18. Furthermore, when mice were administered a non-lethal dose of Zn, the levels of hepatic injury indicators (AST and ALT) were much higher at ZT14 than at ZT2. To explore the mechanism of Zn-induced diurnal hepatotoxicity, we performed an in vitro experiment, focusing on the clock genes. We found that Zn downregulated the expression levels of several clock genes, neuronal PAS domain protein 2 (Npas2) and Peroid2 (Per2), in Hepa1-6 cells. Interestingly, overexpression of both Npas2 and Per2 restored Zn-induced toxicity in Hepa1-6 cells. Since NPAS2 and PER2 are known to modulate the hepatic injury induced by carbon tetrachrolide or acetaminophen, our results suggest that Zn-induced diurnal toxicity may be associated with modulation of Npas2 and Per2 gene expression.


Assuntos
Overdose de Drogas , Zinco , Masculino , Camundongos , Animais , Camundongos Endogâmicos ICR , Zinco/toxicidade , Camundongos Endogâmicos , Fígado , Proteínas do Tecido Nervoso , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas Circadianas Period
12.
J Bone Miner Metab ; 40(5): 839-852, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35947192

RESUMO

INTRODUCTION: Osteoblasts require substantial amounts of energy to synthesize the bone matrix and coordinate skeleton mineralization. This study analyzed the effects of mitochondrial dysfunction on bone formation, nano-organization of collagen and apatite, and the resultant mechanical function in mouse limbs. MATERIALS AND METHODS: Limb mesenchyme-specific Tfam knockout (Tfamf/f;Prx1-Cre: Tfam-cKO) mice were analyzed morphologically and histologically, and gene expressions in the limb bones were assessed by in situ hybridization, qPCR, and RNA sequencing (RNA-seq). Moreover, we analyzed the mitochondrial function of osteoblasts in Tfam-cKO mice using mitochondrial membrane potential assay and transmission electron microscopy (TEM). We investigated the pathogenesis of spontaneous bone fractures using immunohistochemical analysis, TEM, birefringence analyzer, microbeam X-ray diffractometer and nanoindentation. RESULTS: Forelimbs in Tfam-cKO mice were significantly shortened from birth, and spontaneous fractures occurred after birth, resulting in severe limb deformities. Histological and RNA-seq analyses showed that bone hypoplasia with a decrease in matrix mineralization was apparent, and the expression of type I collagen and osteocalcin was decreased in osteoblasts of Tfam-cKO mice, although Runx2 expression was unchanged. Decreased type I collagen deposition and mineralization in the matrix of limb bones in Tfam-cKO mice were associated with marked mitochondrial dysfunction. Tfam-cKO mice bone showed a significantly lower Young's modulus and hardness due to poor apatite orientation which is resulted from decreased osteocalcin expression. CONCLUSION: Mice with limb mesenchyme-specific Tfam deletions exhibited spontaneous limb bone fractures, resulting in severe limb deformities. Bone fragility was caused by poor apatite orientation owing to impaired osteoblast differentiation and maturation.


Assuntos
Fraturas Espontâneas , Animais , Apatitas , Colágeno Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fraturas Espontâneas/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Integrases , Mesoderma/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/metabolismo , Osteocalcina/metabolismo
13.
Biochim Biophys Acta Gen Subj ; 1866(9): 130168, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35594965

RESUMO

Swainsonine (SWA), a potent inhibitor of class II α-mannosidases, is present in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants. The mechanisms underlying SWA-induced animal poisoning are not fully understood. In this study, we analyzed the alterations that occur in N- and free N-glycomic upon addition of SWA to HepG2 cells to understand better SWA-induced glycomic alterations. After SWA addition, we observed the appearance of SWA-specific glycomic alterations, such as unique fucosylated hybrid-type and fucosylated M5 (M5F) N-glycans, and a remarkable increase in all classes of Gn1 FNGs. Further analysis of the context of these glycomic alterations showed that (fucosylated) hybrid type N-glycans were not the precursors of these Gn1 FNGs and vice versa. Time course analysis revealed the dynamic nature of glycomic alterations upon exposure of SWA and suggested that accumulation of free N-glycans occurred earlier than that of hybrid-type N-glycans. Hybrid-type N-glycans, of which most were uniquely core fucosylated, tended to increase slowly over time, as was observed for M5F N-glycans. Inhibition of swainsonine-induced unique fucosylation of hybrid N-glycans and M5 by coaddition of 2-fluorofucose caused significant increases in paucimannose- and fucosylated paucimannose-type N-glycans, as well as paucimannose-type free N-glycans. The results not only revealed the gross glycomic alterations in HepG2 cells induced by swainsonine, but also provide information on the global interrelationships between glycomic alterations.


Assuntos
Glicômica , Swainsonina , Animais , Glicosilação , Células Hep G2 , Humanos , Polissacarídeos , Swainsonina/toxicidade
14.
Cell Mol Life Sci ; 79(6): 307, 2022 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-35593968

RESUMO

Sjögren's syndrome (SjS) is a chronic autoimmune disease characterized by immune cell infiltration of the exocrine glands, mainly the salivary and lacrimal glands. Despite recent advances in the clinical and mechanistic characterization of the disease, its etiology remains largely unknown. Here, we report that mice with a deficiency for either Atg7 or Atg3, which are enzymes involved in the ubiquitin modification pathway, in the salivary glands exhibit a SjS-like phenotype, characterized by immune cell infiltration with autoantibody detection, acinar cell death, and dry mouth. Prior to the onset of the SjS-like phenotype in these null mice, we detected an accumulation of secretory vesicles in the acinar cells of the salivary glands and found that GATE16, an uncharacterized autophagy-related molecule activated by ATG7 (E1-like enzyme) and ATG3 (E2-like enzyme), was highly expressed in these cells. Notably, GATE16 was activated by isoproterenol, an exocytosis inducer, and localized on the secretory vesicles in the acinar cells of the salivary glands. Failure to activate GATE16 was correlated with exocytosis defects in the acinar cells of the salivary glands in Atg7 and Atg3 cKO mice. Taken together, our results show that GATE16 activation regulated by the autophagic machinery is crucial for exocytosis and that defects in this pathway cause SjS.


Assuntos
Doenças Autoimunes , Síndrome de Sjogren , Animais , Autoanticorpos/metabolismo , Modelos Animais de Doenças , Exocitose , Camundongos , Glândulas Salivares , Síndrome de Sjogren/genética , Síndrome de Sjogren/metabolismo
15.
Front Genet ; 13: 788259, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35401675

RESUMO

Amelogenesis imperfecta is a congenital disorder within a heterogeneous group of conditions characterized by enamel hypoplasia. Patients suffer from early tooth loss, social embarrassment, eating difficulties, and pain due to an abnormally thin, soft, fragile, and discolored enamel with poor aesthetics and functionality. The etiology of amelogenesis imperfecta is complicated by genetic interactions. To identify mouse amelogenesis imperfecta-related genes (mAIGenes) and their respective phenotypes, we conducted a systematic literature review and database search and found and curated 70 mAIGenes across all of the databases. Our pathway enrichment analysis indicated that these genes were enriched in tooth development-associated pathways, forming four distinct groups. To explore how these genes are regulated and affect the phenotype, we predicted microRNA (miRNA)-gene interaction pairs using our bioinformatics pipeline. Our miRNA regulatory network analysis pinpointed that miR-16-5p, miR-27b-3p, and miR-23a/b-3p were hub miRNAs. The function of these hub miRNAs was evaluated through ameloblast differentiation assays with/without the candidate miRNA mimics using cultured mouse ameloblast cells. Our results revealed that overexpression of miR-16-5p and miR-27b-3p, but not miR-23a/b-3p, significantly inhibited ameloblast differentiation through regulation of mAIGenes. Thus, our study shows that miR-16-5p and miR-27b-3p are candidate pathogenic miRNAs for amelogenesis imperfecta.

16.
Development ; 149(9)2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35420127

RESUMO

The etiology of cleft lip with or without cleft palate (CL/P), a common congenital birth defect, is complex, with genetic and epigenetic, as well as environmental, contributing factors. Recent studies suggest that fetal development is affected by maternal conditions through microRNAs (miRNAs), a group of short noncoding RNAs. Here, we show that miR-129-5p and miR-340-5p suppress cell proliferation in both primary mouse embryonic palatal mesenchymal cells and O9-1 cells, a neural crest cell line, through the regulation of Sox5 and Trp53 by miR-129-5p, and the regulation of Chd7, Fign and Tgfbr1 by miR-340-5p. Notably, miR-340-5p, but not miR-129-5p, was upregulated following all-trans retinoic acid (atRA; tretinoin) administration, and a miR-340-5p inhibitor rescued the cleft palate (CP) phenotype in 47% of atRA-induced CP mice. We have previously reported that a miR-124-3p inhibitor can also partially rescue the CP phenotype in atRA-induced CP mouse model. In this study, we found that a cocktail of miR-124-3p and miR-340-5p inhibitors rescued atRA-induced CP with almost complete penetrance. Taken together, our results suggest that normalization of pathological miRNA expression can be a preventive intervention for CP.


Assuntos
Fenda Labial , Fissura Palatina , MicroRNAs , Animais , Proliferação de Células/genética , Fenda Labial/induzido quimicamente , Fenda Labial/genética , Fenda Labial/patologia , Fissura Palatina/induzido quimicamente , Fissura Palatina/genética , Fissura Palatina/patologia , Camundongos , MicroRNAs/metabolismo , Tretinoína/farmacologia
17.
Acta Radiol Open ; 11(2): 20584601221077074, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35273810

RESUMO

On conventional magnetic resonance imaging (MRI), hemangioblastomas typically appear as mural nodules with an adjacent surrounding cyst or a solid mass in the cerebellum. However, hemangioblastomas sometimes cannot be reliably distinguished using this imaging technique from other tumors, especially pilocytic astrocytomas and metastatic tumors, because of their similar imaging findings and locations. Herein, we report three cases of cerebellar hemangioblastomas and review their findings on conventional and advanced MRI, including diffusion-weighted imaging (DWI), dynamic susceptibility-weighted contrast-enhanced perfusion-weighted imaging (DSC-PWI), and magnetic resonance spectroscopy (MRS). Solid contrast-enhanced lesions of hemangioblastomas showed increased apparent diffusion coefficient values on DWI, increased relative cerebral blood volume ratio on DSC-PWI, and high lipid/lactate peak on MRS. Therefore, advanced MRI techniques can be helpful in understanding the pathological and metabolic changes of hemangioblastomas and may be useful for their characterization.

18.
Arch Orthop Trauma Surg ; 142(4): 607-613, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33226460

RESUMO

PURPOSE: To investigate whether tibial tubercle fracture affected clinical outcomes and bony union in medial opening wedge high tibial osteotomy with distal tibial tubercle osteotomy (DTO) and to determine the anatomical risk factors for tibial tubercle fracture. MATERIALS AND METHODS: All patients who underwent DTO were retrospectively reviewed, and 104 successive patients were included. The Knee Society Score and complications including tibial tubercle fracture were recorded. On radiographs and computed tomography scans, the length, thickness, width, height, and bony union of the osteotomized tibial tubercle and the posterior tibial slope were statistically analysed. RESULTS: Fracture of the tibial tubercle occurred intraoperatively in 11 patients (10.6%) and in the postoperative period in 1 (1.0%). The case of postoperative fracture showed non-union. There was no significant difference in the Knee Society Score between the non-fracture and fracture groups. There were significant differences in the posterior tibial slope and the height of the tibial tubercle between the groups (p < 0.0001 for each comparison). The logistic regression analysis showed that the height of the tibial tubercle was associated with a higher risk of the fracture of the tibial tubercle (p < 0.01; OR, 1.548; 95% CI, 1.149-2.085). However, there were no significant differences in the bony union rate of the tibial tubercle at 6 months after surgery between the groups. CONCLUSIONS: Tibial tubercle fracture did not affect the clinical outcome and bony union in spite of the relatively high occurrence rate. Anatomical risk factors for the fractures was a lower tibial tubercle position. LEVEL OF EVIDENCE: Level IV.


Assuntos
Osteoartrite do Joelho , Fraturas da Tíbia , Humanos , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/cirurgia , Osteoartrite do Joelho/cirurgia , Osteotomia/efeitos adversos , Osteotomia/métodos , Estudos Retrospectivos , Tíbia/cirurgia , Fraturas da Tíbia/diagnóstico por imagem , Fraturas da Tíbia/etiologia , Fraturas da Tíbia/cirurgia
19.
Pharmaceutics ; 13(12)2021 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-34959378

RESUMO

(1) Background: T cells are important target cells, since they exert direct cytotoxic effects on infected/malignant cells, and affect the regulatory functions of other immune cells in a target antigen-specific manner. One of the current approaches for modifying the function of T cells is gene transfection by viral vectors. However, the insertion of the exogenous DNA molecules into the genome is attended by the risk of mutagenesis, especially when a transposon-based gene cassette is used. Based on this scenario, the transient expression of proteins by an in vitro-transcribed messenger RNA (IVT-mRNA) has become a subject of interest. The use of lipid nanoparticles (LNPs) for the transfection of IVT-mRNA is one of the more promising strategies for introducing exogenous genes. In this study, we report on the development of LNPs with transfection efficiencies that are comparable to that for electroporation in a T cell line (Jurkat cells). (2) Methods: Transfection efficiency was improved by optimizing the phospholipids and polyethylene glycol (PEG)-conjugated lipid components. (3) Results: Modification of the lipid composition resulted in the 221-fold increase in luciferase activity compared to a previously optimized formulation. Such a high transfection activity was due to the efficient uptake by clathrin/dynamin-dependent endocytosis and the relatively efficient escape into the cytoplasm at an early stage of endocytosis.

20.
Int J Mol Sci ; 22(22)2021 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-34830336

RESUMO

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of Slc24a2 expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.


Assuntos
Fissura Palatina/genética , Dexametasona/farmacologia , Glucocorticoides/farmacologia , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Animais , Antagomirs/genética , Antagomirs/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fissura Palatina/induzido quimicamente , Fissura Palatina/metabolismo , Fissura Palatina/patologia , Modelos Animais de Doenças , Embrião de Mamíferos , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/classificação , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Cultura Primária de Células , Transdução de Sinais , Trocador de Sódio e Cálcio/genética , Trocador de Sódio e Cálcio/metabolismo
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