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Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
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Azepinas/farmacologia , Encéfalo/patologia , Proteínas de Ciclo Celular/metabolismo , Interneurônios/patologia , Proteína 2 de Ligação a Metil-CpG/fisiologia , Síndrome de Rett/patologia , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacos , Triazóis/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Feminino , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Embrionárias Humanas/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Fenótipo , Síndrome de Rett/tratamento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Fatores de Transcrição/genéticaRESUMO
Meiosis is essential for generating genetic diversity and sexual spores, but the regulation of meiosis and ascosporogenesis is not clear in filamentous fungi, in which dikaryotic and diploid cells formed inside fruiting bodies are not free living and independent of pheromones or pheromone receptors. In this study, Gia1, a non-pheromone GPCR (G protein-coupled receptor) with sexual-specific expression in Fusarium graminearum, is found to be essential for ascosporogenesis. The gia1 mutant was normal in perithecium development, crozier formation, and karyogamy but failed to undergo meiosis, which could be partially rescued by a dominant active mutation in GPA1 and activation of the Gpmk1 pathway. GIA1 orthologs have conserved functions in regulating meiosis and ascosporogenesis in Sordariomycetes. GIA1 has a paralog, GIP1, in F. graminearum and other Hypocreales species which is essential for perithecium formation. GIP1 differed from GIA1 in expression profiles and downstream signaling during sexual reproduction. Whereas the C-terminal tail and IR3 were important for intracellular signaling, the N-terminal region and EL3 of Gia1 were responsible for recognizing its ligand, which is likely a protein enriched in developing perithecia, particularly in the gia1 mutant. Taken together, these results showed that GIA1 encodes a non-pheromone GPCR that regulates the entry into meiosis and ascosporogenesis via the downstream Gpmk1 MAP kinase pathway in F. graminearum and other filamentous ascomycetes.
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Ascomicetos , Fusarium , Triticum/microbiologia , Feromônios/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Ascomicetos/genética , Ascomicetos/metabolismo , Meiose/genética , Esporos FúngicosRESUMO
Fusarium head blight (FHB), caused by the fungal pathogen Fusarium graminearum, is a destructive disease worldwide. Ascospores are the primary inoculum of F. graminearum, and sexual reproduction is a critical step in its infection cycle. In this study, we characterized the functions of FgCsn12. Although the ortholog of FgCsn12 in budding yeast was reported to have a direct interaction with Csn5, which served as the core subunit of the COP9 signalosome, the interaction between FgCsn12 and FgCsn5 was not detected through the yeast two-hybrid assay. The deletion of FgCSN12 resulted in slight defects in the growth rate, conidial morphology, and pathogenicity. Instead of forming four-celled, uninucleate ascospores, the Fgcsn12 deletion mutant produced oval ascospores with only one or two cells and was significantly defective in ascospore discharge. The 3'UTR of FgCsn12 was dispensable for vegetative growth but essential for sexual reproductive functions. Compared with those of the wild type, 1204 genes and 2240 genes were up- and downregulated over twofold, respectively, in the Fgcsn12 mutant. Taken together, FgCsn12 demonstrated an important function in the regulation of ascosporogenesis in F. graminearum.
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Fusarium , Regiões 3' não Traduzidas , Proteínas Fúngicas/genética , Doenças das Plantas/microbiologia , Esporos Fúngicos/genética , Triticum/genética , Triticum/microbiologiaRESUMO
Using consumer depth cameras at close range yields a higher surface resolution of the object, but this makes more serious noises. This form of noise tends to be located at or on the edge of the realistic surface over a large area, which is an obstacle for real-time applications that do not rely on point cloud post-processing. In order to fill this gap, by analyzing the noise region based on position and shape, we proposed a composite filtering system for using consumer depth cameras at close range. The system consists of three main modules that are used to eliminate different types of noise areas. Taking the human hand depth image as an example, the proposed filtering system can eliminate most of the noise areas. All algorithms in the system are not based on window smoothing and are accelerated by the GPU. By using Kinect v2 and SR300, a large number of contrast experiments show that the system can get good results and has extremely high real-time performance, which can be used as a pre-step for real-time human-computer interaction, real-time 3D reconstruction, and further filtering.
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BACKGROUND: The effects of short-term high fat diets on physiology are elusive and the molecular changes following fat overconsumption remain largely unknown. In this study, we aimed to evaluate exercise capacity in mice fed with a high fat diet (HFD) for 3 days and investigate the molecular mechanisms in the early response to high-fat feeding. METHODS: Exercise capacity was assessed by weight-loaded swimming test in mice fed a control diet (10 kcal% fat) or a HFD (60 kcal% fat) for 3 days. Global gene expression of ten important tissues (brain, heart, liver, spleen, lung, kidney, stomach, duodenum, skeletal muscle and blood) was analyzed using RNA Sequencing. RESULTS: A HFD for just 3 days can induce 71% decrease of exercise performance prior to substantial weight gain (P <0.01). Principle component analysis revealed that differential gene expression patterns existed in the ten tissues. Out of which, the brain, spleen and lung were demonstrated to have more pronounced transcriptional changes than other tissues. Biological process analysis for differentially expressed genes in the brain, spleen and lung showed that dysregulation of peripheral and central immune response had been implicated in the early stage of HFD exposure. Neurotransmission related genes and circulatory system process related genes were significantly down-regulated in the brain and lung, respectively. CONCLUSIONS: Our findings provide new insights for the deleterious effects of high-fat feeding, especially revealing that the lung maybe as a new important target attacked by short-term high-fat feeding.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Condicionamento Físico Animal/fisiologia , Transcriptoma , Animais , Sangue/metabolismo , Peso Corporal , Encéfalo/fisiologia , Pulmão/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Baço/fisiologiaRESUMO
PURPOSE: To compare the efficacy of swim-up and DGC in improving sperm deformity and DNA fragmentation and to determine which method is better in teratozoospermic patients requiring artificial reproduction. METHODS: The present study compared the effects of swim-up and density gradient centrifugation (DGC), the two most commonly used semen preparation methods, on sperm deformity rate and DNA fragmentation index (DFI) in semen samples from teratozoospermic patients. RESULTS: The results demonstrated that both swim-up and DGC yielded a significantly lower sperm deformity rate and DFI in comparison to unprocessed whole semen, with DGC having more favorable results. Sperm deformity rate in unprocessed whole semen samples was significantly lower in the 20-29 age group than in the 40-49 age group, but no significant difference was observed in DFI between different age groups. There was no significant correlation between sperm deformity rate and DFI. CONCLUSIONS: Our findings suggest that enrichment of sperm with normal morphology and intact DNA in teratozoospermic patients could be achieved by both DGC and swim-up procedures, and that DGC is a better method.
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Fragmentação do DNA , Infertilidade Masculina/genética , Técnicas de Reprodução Assistida , Análise do Sêmen/métodos , Espermatozoides/anormalidades , Adulto , Fatores Etários , Centrifugação com Gradiente de Concentração , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
A catalytic hairpin self-assembly (CHA) amplification method was developed for CAP detection based on cross-shaped DNA and UiO-66. MOF was used to quench the fluorescent signal of FAM labeled DNA. Cross-shaped DNA with four fluorophore group (FAM) was utilized to enhance the fluorescent intensity. CAP could open hairpin structure of H-apt and induce CHA reaction. The product of CHA hybridized with cross-shaped DNA, resulting its leaving from the surface of UiO-66 and recovery of fluorescent signal. The limit of detection (LOD) was low to 0.87 pM. This method had been successfully applied for the detection of CAP in actual samples. Importantly, the high sensitivity was attributed to the great amplification efficiency of CHA, strong fluorescent intensity of cross-shaped DNA structure and great fluorescent quenched efficiency of UiO-66.
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Técnicas Biossensoriais , DNA Catalítico , Estruturas Metalorgânicas , Ácidos Ftálicos , Cloranfenicol , DNA/química , Espectrometria de Fluorescência/métodos , Limite de Detecção , Técnicas Biossensoriais/métodos , DNA Catalítico/químicaRESUMO
[This corrects the article DOI: 10.3389/fnmol.2023.1052288.].
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The neurobiological mechanism of post-traumatic stress disorder (PTSD) is poorly understood. The inhibition of GABA neurons, especially in the amygdala, is crucial for the precise regulation of the consolidation, expression, and extinction of fear conditioning. The GABAergic system is involved in the pathophysiological process of PTSD, with several studies demonstrating that the function of the GABAergic system decreases in PTSD patients. This paper reviews the preclinical and clinical studies, neuroimaging techniques, and pharmacological studies of the GABAergic system in PTSD and summarizes the role of the GABAergic system in PTSD. Understanding the role of the GABAergic system in PTSD and searching for new drug targets will be helpful in the treatment of PTSD.
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The theory of engrams, proposed several years ago, is highly crucial to understanding the progress of memory. Although it significantly contributes to identifying new treatments for cognitive disorders, it is limited by a lack of technology. Several scientists have attempted to validate this theory but failed. With the increasing availability of activity-dependent tools, several researchers have found traces of engram cells. Activity-dependent tools are based on the mechanisms underlying neuronal activity and use a combination of emerging molecular biological and genetic technology. Scientists have used these tools to tag and manipulate engram neurons and identified numerous internal connections between engram neurons and memory. In this review, we provide the background, principles, and selected examples of applications of existing activity-dependent tools. Using a combination of traditional definitions and concepts of engram cells, we discuss the applications and limitations of these tools and propose certain developmental directions to further explore the functions of engram cells.
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AIMS: N6-methyladenosine (m6A), the most abundant and conserved epigenetic modification of mRNA, participates in various physiological and pathological processes. However, the roles of m6A modification in liver lipid metabolism have yet to be understood entirely. We aimed to investigate the roles of the m6A "writer" protein methyltransferase-like 3 (Mettl3) in liver lipid metabolism and the underlying mechanisms. MAIN METHODS: We assessed the expression of Mettl3 in liver tissues of diabetes (db/db) mice, obese (ob/ob) mice, high saturated fat-, cholesterol-, and fructose-induced non-alcoholic fatty liver disease (NAFLD) mice, and alcohol abuse and alcoholism (NIAAA) mice by quantitative reverse-transcriptase PCR (qRT-PCR). Hepatocyte-specific Mettl3 knockout mice were used to evaluate the effects of Mettl3 deficiency in mouse liver. The molecular mechanisms underlying the roles of Mettl3 deletion in liver lipid metabolism were explored by multi-omics joint analysis of public data from the Gene Expression Omnibus database and further validated by qRT-PCR and Western blot. KEY FINDINGS: Significantly decreased Mettl3 expression was associated with NAFLD progression. Hepatocyte-specific knockout of Mettl3 resulted in significant lipid accumulation in the liver, increased serum total cholesterol levels, and progressive liver damage in mice. Mechanistically, loss of Mettl3 significantly downregulated the expression levels of multiple m6A-modified mRNAs related to lipid metabolism, including Adh7, Cpt1a, and Cyp7a1, further promoting lipid metabolism disorders and liver injury in mice. SIGNIFICANCE: In summary, our findings demonstrate that the expression alteration of genes related to lipid metabolism by Mettl3-mediated m6A modification contributes to the development of NAFLD.
Assuntos
Transtornos do Metabolismo dos Lipídeos , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Metiltransferases/genética , Metiltransferases/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Metabolismo dos Lipídeos/genética , Expressão GênicaRESUMO
In this study, we analyzed the status and research trends of the GABAergic system in depression from 2004 to 2020 to provide a reference for further research. The Web of Science database was used as the data source and 1,658 publishments were included. Using two visualization analysis software, CiteSpace and VOSviewer, we analyzed the publishing years, countries, institutions, authors, journals, categories, keywords, and research frontiers in depression. The publishments revealed an upward trend from 2004 to 2020; the most prolific country and institutions were the United States and INSERM, respectively. The journal of Neuroscience was the most published and cited journal. The most relevant category was neurosciences. The hot topics in this field were GABAergic research in Gaba(a) receptor; the research frontier was depressive model. These analysis results provide a new perspective for researchers to conduct studies on related topics in the future and guidance for scientists to identify potential collaborators and research cooperation institutions.
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Millipede assassin bugs are a diverse group of specialized millipede predators. However, the feeding behavior of Ectrichodiinae remains poorly known, especially how the mouthpart structures relate to various functions in feeding. In this study, fine morphology of the mouthparts and feeding performance of Haematoloecha nigrorufa (Stål, 1867) was observed and described in detail for the first time. The triangular labrum is divided by a conspicuous transverse membrane into a strongly sclerotized basilabrum and a less sclerotized distilabrum. Fifteen types of sensilla are distributed on the mouthparts. Each mandibular stylet has an expanded spatulate apex and about 150 approximately transverse ridges on the external middle side; these help in penetrating the ventral trunk area and the intersegmental membranes of millipede prey. The right maxilla is tapered. On the internal surface are a row dorsal short bristles near the apex and a row of ventral bristles preapically. A longitudinal row of long lamellate structures extend proximate for a considerable distance, lie entirely within the food canal, and bear several short spines and short bristles. There is no obvious difference between males and females in the distribution, number, and types of sensilla on mouthparts. The adult feeding process involves several steps, including searching and capturing prey, paralyzing prey, a resting phase, and a feeding phase. The evolution of the mouthpart morphology and the putative functional significance of their sensilla are discussed, providing insight into the structure and function of the mouthparts adapted for millipede feeding.
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The endothelial cell (EC) barrier disruption has been implicated in vascular leakage and pulmonary edema. Many reports have shown that the EC barrier dysfunction is regulated by the sphingosine-1-phophate (S1P)/S1P receptor-1 (S1PR1) axis. Identifying downstream effectors for the S1P/S1PR1 axis in pulmonary vasculature has been limited by mixed populations in vitro cultures that do not retain physiological EC phenotype and complex of tedious proteomics. In this study, we used a combination of in vivo biotinylation and liquid chromatograph tandem mass spectrometry on three mouse models of S1pr1 expression, namely normal, knockout (KO) and high, to identify EC membrane proteins whose cell-surface expression is S1pr1-dependent. EC-specific KO of S1pr1 caused severe pulmonary vascular disruption and reduction of many membrane proteins on ECs. Using the MaxQuant software we were able to identify novel membrane targets of S1pr1, for instance, Cd105 and Plvap, by comparison with their membrane expressions among the three EC model systems. Moreover, regulation of Cd105 and Plvap by S1pr1 were validated with Western blot and immunostaining in vivo and in vitro. Our data suggest that S1pr1 dictates cell-surface localization of several apical membrane proteins in ECs. Our results are insightful for development of novel therapeutics to specifically target EC barrier function.
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Membrana Celular/metabolismo , Endoglina/metabolismo , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Modelos Biológicos , Receptores de Esfingosina-1-Fosfato/metabolismo , Animais , Linhagem Celular , Membrana Celular/genética , Endoglina/genética , Células Endoteliais/citologia , Camundongos , Camundongos Knockout , Receptores de Esfingosina-1-Fosfato/genéticaRESUMO
Long-term treatment with high-dose Interferon-alpha (IFN-α) has resulted in depression in 30-50% of the patients. Paeoniflorin may ameliorate the IFN-α-induced depression; however, the underlying mechanism is less studied. Here, we investigated the prophylactic antidepressant and anti-neuroinflammatory effects of paeoniflorin on the behaviors and specific emotion-related regions of the brain in mice with IFN-α-induced depression. A series of behavior assessments were conducted to identify the depressive state after subcutaneously IFN-α injections and with or without intragastrically paeoniflorin administration in C57BL/6J mice. Levels of many inflammatory-related cytokines in serum, mPFC, vHi and amygdala were determined by cytokine array analysis. Furthermore, microglia and astrocyte activation in these three regions were evaluated by immunohistochemistry. We found that the mice which were subcutaneously injected IFN-α 15×106 IU/kg for 4 successive weeks to mimic an IFN-α-induced depression model had distinct inflammatory changes in the amygdala. Interestingly, 4-week 20 mg/kg or 40 mg/kg paeoniflorin pretreatments reversed the depressive-like behaviors and the abnormal inflammatory cytokine levels in the serum, mPFC, vHi and amygdala. These cytokines were not limited to the commonly reported IL-6, IL-1ß and TNF-α, but also IL-9, IL-10, IL-12, and MCP-1. Besides, the increased density of microglia in IFN-α-treated mice was reversed by paeoniflorin in these three brain areas. Taken together, our data suggest that paeoniflorin can reverse the long-term, high-dose IFN-α-induced depressive-like behaviors that were associated with local distinct neuroinflammation in the mPFC, vHi and particularly the amygdala. Paeoniflorin might have a preventive therapeutic potential in IFN-α-induced depression.
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Anti-Inflamatórios/farmacologia , Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Citocinas/metabolismo , Depressão/prevenção & controle , Encefalite/prevenção & controle , Glucosídeos/farmacologia , Mediadores da Inflamação/metabolismo , Interferon-alfa , Monoterpenos/farmacologia , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Tonsila do Cerebelo/fisiopatologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Citocinas/sangue , Depressão/induzido quimicamente , Depressão/metabolismo , Depressão/psicologia , Modelos Animais de Doenças , Encefalite/induzido quimicamente , Encefalite/metabolismo , Encefalite/fisiopatologia , Preferências Alimentares/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Mediadores da Inflamação/sangue , Interferon alfa-2 , Masculino , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Atividade Motora/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiopatologia , Proteínas Recombinantes , Natação , Fatores de TempoRESUMO
OBJECTIVE: Ovarian mature cystic teratomas with malignant transformation are rare, and squamous cell carcinoma is the most common pathological entity. Among these malignant transformations, urothelial cell carcinoma is rare. CASE REPORT: We report a woman presenting with a huge pelvic cystic mass, favoring a right ovarian mature cystic teratoma with malignant transformation, based on magnetic resonance imaging, who was successfully treated with surgery. CONCLUSION: The final pathology confirmed concomitant malignant transformation of urothelial carcinoma.
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Carcinoma de Células de Transição/patologia , Transformação Celular Neoplásica/patologia , Neoplasias Primárias Múltiplas/patologia , Neoplasias Ovarianas/patologia , Teratoma/patologia , Neoplasias da Bexiga Urinária/patologia , Carcinoma de Células de Transição/etiologia , Carcinoma de Células de Transição/cirurgia , Feminino , Seguimentos , Humanos , Achados Incidentais , Laparotomia/métodos , Imageamento por Ressonância Magnética/métodos , Pessoa de Meia-Idade , Neoplasias Primárias Múltiplas/diagnóstico , Neoplasias Primárias Múltiplas/cirurgia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/etiologia , Neoplasias Ovarianas/cirurgia , Ovariectomia/métodos , Pós-Menopausa , Medição de Risco , Teratoma/diagnóstico , Resultado do Tratamento , Neoplasias da Bexiga Urinária/etiologia , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
BACKGROUND: The objective of this study was to investigate the influence of Er,Cr:YSGG laser irradiated at different powers on the micromorphology and the bonding property of non-carious sclerotic dentin to resin composites. METHODS: Two hundred bovine incisors characterized by non-carious sclerotic dentin were selected, and the seventy-two teeth of which for surface morphological analysis were divided into nine groups according to various treatments (A: the control group, B: only treated with the adhesive Adper Easy One, C: diamond bur polishing followed by Adper Easy One, D-I: Er,Cr:YSGG laser irradiating at 1W, 2W, 3W, 4W, 5W, 6W output power, respectively, followed by Adper Easy One). The surface roughness values were measured by the non-contact three-dimensional morphology scanner, then the surface micromorphologies of surfaces in all groups were assessed by scanning electron microscopy (SEM); meanwhile, Image Pro-Plus 6.0 software was used to measure the relative percentage of open tubules on SEM images. The rest, one hundred twenty-eight teeth for bond strength test, were divided into eight groups according to the different treatments (A: only treated with the adhesive Adper Easy One, B: diamond bur polishing followed by the above adhesive, C-H: Er,Cr:YSGG laser irradiating at 1 W, 2 W, 3 W, 4 W, 5 W, 6 W output power, respectively, followed by the above adhesive), and each group was subsequently divided into two subgroups according to whether aging is performed (immediately tested and after thermocycling). Micro-shear bond strength test was used to evaluate the bond strength. RESULTS: The 4W laser group showed the highest roughness value (30.84±1.93µm), which was statistically higher than the control group and the diamond bur groups (p<0.05). The mean percentages ((27.8±1.8)%, (28.0±2.2)%, (30.0±1.9)%) of open tubules area in the 4W, 5W, 6W group were higher than other groups (p<0.05). The 4W laser group showed the highest micro-shear bond strength not only in immediately tested (17.60±2.55 PMa) but after thermocycling (14.35±2.08MPa). CONCLUSION: The Er,Cr:YSGG laser at 4W power can effectively improve the bonding property between non-carious sclerotic dentin and resin composites by increasing the roughness and mean percentage area of open tubules.
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Dentina/patologia , Lasers de Estado Sólido , Cura Luminosa de Adesivos Dentários/métodos , Resinas Sintéticas , Animais , Bovinos , Dentina/efeitos da radiação , Esclerose , Resistência ao Cisalhamento , Propriedades de SuperfícieRESUMO
Setting up breeding programs for transgenic mouse strains require to distinguish homozygous from the heterozygous transgenic animals. The combinational use of the fluorescence reporter transgene and small animal in-vivo imaging system might allow us to rapidly and visually determine the transgenic mice homozygous for transgene(s) by the in vivo fluorescence imaging. RLG, RCLG or Rm17LG transgenic mice ubiquitously express red fluorescent protein (RFP). To identify homozygous RLG transgenic mice, whole-body fluorescence imaging for all of newborn F2-generation littermates produced by mating of RFP-positive heterozygous transgenic mice (F1-generation) derived from the same transgenic founder was performed. Subsequently, the immediate data analysis of the in vivo fluorescence imaging was carried out, which greatly facilitated us to rapidly and readily distinguish RLG transgenic individual(s) with strong fluorescence from the rest of F2-generation littermates, followed by further determining this/these RLG individual(s) showing strong fluorescence to be homozygous, as strongly confirmed by mouse mating. Additionally, homozygous RCLG or Rm17LG transgenic mice were also rapidly and precisely distinguished by the above-mentioned optical approach. This approach allowed us within the shortest time period to obtain 10, 8 and 2 transgenic mice homozygous for RLG, RCLG and Rm17LG transgene, respectively, as verified by mouse mating, indicating the practicality and reliability of this optical method. Taken together, our findings fully demonstrate that the in vivo fluorescence imaging offers a visual, rapid and reliable alternative method to the traditional approaches (i.e., mouse mating and real-time quantitative PCR) in identifying homozygous transgenic mice harboring fluorescence reporter transgene under the control of a ubiquitous promoter in the situation mentioned in this study.
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Homozigoto , Camundongos Transgênicos/genética , Imagem Óptica/métodos , Animais , Cruzamento/métodos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos TestesRESUMO
Hepatic expression profiling has revealed miRNA changes in liver diseases, while hepatic miR-155 expression was increased in murine non-alcoholic fatty liver disease, suggesting that miR-155 might regulate the biological process of lipid metabolism. To illustrate the effects of miR-155 gain of function in transgenic mouse liver on lipid metabolism, transgenic mice (i.e., Rm155LG mice) for the conditional overexpression of mouse miR-155 transgene mediated by Cre/lox P system were firstly generated around the world in this study. Rm155LG mice were further crossed to Alb-Cre mice to realize the liver-specific overexpression of miR-155 transgene in Rm155LG/Alb-Cre double transgenic mice which showed the unaltered body weight, liver weight, epididymal fat pad weight and gross morphology and appearance of liver. Furthermore, liver-specific overexpression of miR-155 transgene resulted in significantly reduced levels of serum total cholesterol, triglycerides (TG) and high-density lipoprotein (HDL), as well as remarkably decreased contents of hepatic lipid, TG, HDL and free fatty acid in Rm155LG/Alb-Cre transgenic mice. More importantly, microarray data revealed a general downward trend in the expression profile of hepatic genes with functions typically associated with fatty acid, cholesterol and triglyceride metabolism, which is likely at least partially responsible for serum cholesterol and triglyceride lowering observed in Rm155LG/Alb-Cre mice. In this study, we demonstrated that hepatic overexpression of miR-155 alleviated nonalcoholic fatty liver induced by a high-fat diet. Additionally, carboxylesterase 3/triacylglycerol hydrolase (Ces3/TGH) was identified as a direct miR-155 target gene that is potentially responsible for the partial liver phenotypes observed in Rm155LG/Alb-Cre mice. Taken together, these data from miR-155 gain of function study suggest, for what we believe is the first time, the altered lipid metabolism and provide new insights into the metabolic state of the liver in Rm155LG/Alb-Cre mice.