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Core (C, N, and O 1s regions) and valence band electronic structures of bulk uracil and 5-fluoro-, -chloro-, and -iodouracils were investigated using X-ray photoemission spectroscopy and comprehensively compared with those of 5-bromouracil measured under the same experimental conditions before. The halogenation of uracil shifted the core peaks of the 5-position carbons toward the higher binding energy side and reduced the ionization potentials depending on the type of halogen. Theoretical calculations supported these results. The alterations of electronic properties induced by the halogenation would result in the characteristic properties of 5-halouracils.
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BACKGROUND: While rapid population ageing is occurring worldwide, its speed is especially prominent in Asian countries. In Asia, cultural diversity might significantly affect care burden; however, few studies have investigated the differences in care burden, and mental, physical and social conditions among Asian countries. This study aimed to clarify the situations of and differences in family caregivers (FCs) of older persons in Japan and Thailand, and evaluate the factors associated with care burden in both countries. METHODS: A cross-national survey of 217 in-home FCs was conducted in Japan and Thailand and mainly included items regarding care burden and psychosocial conditions. Differences between the two countries were obtained, and the factors associated with care burden were analysed by multiple regression analyses. RESULTS: The FCs' and care receivers' mean ages (64.8 ± 11.1 and 84.8 ± 8.6 years, respectively) in Japan were significantly higher than the mean ages (49.1 ± 13.3 and 77.1 ± 9.5 years) in Thailand. In Japan, FCs had more severe care burden, loneliness, and stress compared to those in Thailand. In Thailand, FCs had more social connections and informal support than those in Japan. The factors associated with care burden were different in each country; thus, severe stress, low emotional care preparation, and low willingness to continue care at home were significantly associated with severe care burden in Japan, whereas poor relationships with care receivers, few social connections, low confidence in providing care, low emotional care preparation, and lack of informal support were significantly associated with severe care burden in Thailand. CONCLUSIONS: Despite there being a long-term care insurance system in Japan, which is absent in Thailand, care burden and psychosocial conditions of FCs might be worse in Japan. There was a clear difference in the factors associated with care burden between Japan and Thailand.
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Cuidadores , Humanos , Tailândia , Masculino , Cuidadores/psicologia , Cuidadores/estatística & dados numéricos , Japão , Feminino , Idoso , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Inquéritos e Questionários , Comparação Transcultural , Apoio Social , Estresse Psicológico/epidemiologia , Estresse Psicológico/psicologia , Estudos Transversais , Sobrecarga do Cuidador/psicologia , Solidão/psicologia , Adulto , Família/psicologiaRESUMO
To understand the mechanism underlying the high radio-sensitisation of living cells possessing brominated genomic DNA, X-ray photoelectron spectroscopy (XPS) using synchrotron X-rays with energies of 2000 or 2500 eV was used to study brominated and nonbrominated nucleobases, nucleosides and nucleotides. The bromine atom significantly reduced the energy gap between the valence and conduction states, although the core level states were not greatly affected. This finding was supported by quantum chemical calculation for the nucleobases and nucleosides. Our findings strongly indicate that the energy gaps between the valence and conduction levels of the molecules are significantly reduced by bromination. Furthermore, the brominated molecules are more likely to produce inelastic scattering low energy electrons upon exposure to 2000 or 3000 eV X-rays. This modification of electronic properties around the brominated group may both facilitate electron transfer to the brominated site in DNA and increase the probability of reaction with low energy electrons. These processes can induce DNA damage, presumably resulting in debromination of the uracil moiety and a subsequent cytotoxic effect.
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Bromo , DNA , DNA/química , Uracila/química , Dano ao DNARESUMO
Detection of CpG methylation levels holds immense potential for application in medical diagnosis of various diseases. In this study, we report the development of a recombinase polymerase amplification (RPA)-based CpG methylation level sensing system on G-quadruplex (G4) and intercalated motif (i-motif)-forming regions, which are stabilized by CpG methylation. This detection system is based on the principle that DNA polymerase is stalled at the methylated G4 and i-motif-forming region, which results in a decrease in the initial elongation efficiency of RPA. This reduction in turn affects the onset of amplification depending on the extent of CpG methylation; therefore, the methylation level is quantified by RPA. We demonstrate that the onset of amplification was delayed by CpG methylation when PCR products containing the vascular endothelial growth factor (VEGF) G4 and i-motif-forming region were used as the template. Furthermore, onset of amplification was delayed with the increase in CpG methylation of the VEGF region on genomic DNA. These results demonstrate that the sensing system is capable of directly detecting the methylation level at a constant temperature (39 °C) within 30 min without performing bisulfite conversion or affinity capture of methylated DNA.
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Quadruplex G , Recombinases , Ilhas de CpG , DNA/genética , DNA/metabolismo , Metilação de DNA , Recombinases/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: An increase in the incidence of pediatric complicated appendicitis (CA) during the COVID-19 pandemic has been reported in many countries. We investigated how the pandemic has affected Japan. METHODS: We retrospectively reviewed children of ≤ 15 years old treated for acute appendicitis across 5 medical centers during the pandemic period (January to October in 2020), with the pre-pandemic period (January to October in 2017 to 2019) evaluated as a historical control. The incidence of CA and disease characteristics were then compared between the periods. RESULTS: The total number of patients was 55 in 2020 and 192 in 2017-2019. In all centers, the incidence of acute pediatric CA in the pandemic period significantly increased compared to the pre-pandemic period (18.2% vs. 32.7%, p = 0.02). On limiting our evaluation to the 3 institutions with reductions in patient numbers, the incidence of CA increased (16.3% vs. 37.9%, p = 0.01), and the duration of pre-operative symptoms was prolonged (1.3% vs. 1.7 days, p = 0.03). There were no significant differences in the age, sex, white blood cell count, C-reactive protein, or body temperature. No cases were diagnosed with SARS-CoV-2. CONCLUSIONS: The incidence of acute pediatric CA increased during the pandemic period. This may be related to an extended duration of symptoms due to individuals fearing contracting COVID-19 while visiting a hospital.
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Apendicite , COVID-19 , Humanos , Criança , Adolescente , Apendicite/epidemiologia , Apendicite/cirurgia , COVID-19/epidemiologia , Pandemias , Estudos Retrospectivos , SARS-CoV-2 , Doença Aguda , ApendicectomiaRESUMO
BACKGROUND: The aim of this study was to evaluate the efficacy of adhesive strapping (AS) for umbilical hernia (UH) in infants. METHODS: A retrospective review of 30 mature infants without any treatment for UH between January 2006 and December 2008 and 87 infants (42 mature and 45 premature) who were treated with AS between January 2010 and December 2014 was conducted. The rate and age of closure of UH were compared between the groups and the complications and limitations of AS were evaluated. RESULTS: In the observation-only group, 24 UHs spontaneously closed at a mean age of 13.5 months, but a protruding umbilicus with redundant skin was present in three of 27 infants. Three infants had persistent UH, so a total of six infants, including the three with redundant skin and three with persistent UH, underwent surgery at the mean age of 30.0 months. In the AS group, 72 UHs were cured at a mean age of 4.3 months, which was significantly earlier than in the observation-only group (P < 0.01). Eleven infants did not continue AS because of skin irritation or discomfort. Four infants underwent repair due to persistent UH, but not for redundant skin of the umbilicus. CONCLUSIONS: Adhesive strapping is effective for promoting the early resolution of UH in infants and preventing the forming of redundant skin, which can reduce the need for surgical intervention. To achieve better results with AS, we recommend applying this technique before the UH grows large.
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Hérnia Umbilical , Adesivos , Pré-Escolar , Hérnia Umbilical/cirurgia , Humanos , Lactente , Estudos Retrospectivos , UmbigoRESUMO
PURPOSE: To elucidate the natural history of asymptomatic hydroceles (AHs) in children with conservative management and to discuss management strategies for AHs in children. METHODS: We retrospectively reviewed 113 children with AHs who were followed without immediate surgery. Patients were divided into four groups according to age at presentation; group 1: ≤ 1 month, group 2: 1-12 months, group 3: 12-24 months, and group 4: > 24 months. Ages at spontaneous resolution or surgery were reviewed. To assess the effect of AH on the testis, testicular size before and after conservative management was compared in 11 cases. RESULTS: In groups 1, 2, 3, and 4, spontaneous resolution occurred in 94.3%, 75.0%, 65.0%, and 33.3%, and the average time to resolution from presentation was 5, 5, 17, and 9 months, respectively. 41 patients underwent surgery at a mean age of 4.2 years. The testicular size did not differ significantly between before and after conservative management (14.4 vs. 14.5 mm, p = 0.483). CONCLUSION: About one-third of children over 2 years of age achieve spontaneous resolution. Hydroceles with conservative management may not affect the testicular size. Thus, initial conservative management before surgery is recommend for AHs in children, even over 2 years of age.
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Doenças Assintomáticas , Tratamento Conservador/métodos , Hidrocele Testicular/terapia , Criança , Pré-Escolar , Humanos , Masculino , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Abnormal DNA methylations such as hypermethylation on tumor suppressor genes and global hypomethylation have been recognized as hallmarks of cancer. Previously, we reported a bioluminescence resonance energy transfer (BRET)-based global DNA methylation level assay using a methyl-CpG-binding domain-fused firefly luciferase (MBD-Fluc) and unmethylated CpG-binding domain-fused firefly luciferase (CXXC-Fluc). The BRET signal between MBD-Fluc and BOBO-3 DNA intercalating dye depends on the methylated CpG contents, whereas the BRET signal between CXXC-Fluc and BOBO-3 depends on the unmethylated CpG contents. Therefore, the global DNA methylation level can be quantified using the BRET assay. However, these assays must be performed separately, because the same luciferase fuses to both MBD and CXXC. In this study, we developed a one-step quantification assay of global DNA methylation based on a multicolor BRET assay using MBD-Fluc and CXXC-fused Oplophorus luciferase (CXXC-Oluc). We demonstrated that MBD-Fluc and CXXC-Oluc simultaneously excite BOBO-3 and BOBO-1 DNA intercalating dyes on genomic DNA, respectively. Moreover, the BRET signals produced from MBD-Fluc and CXXC-Oluc depended on the methylation status of the CpG contents. These results demonstrate that global DNA methylation can be quantified by this multicolor BRET assay in a single tube. Graphical abstract á .
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Metilação de DNA , Ilhas de CpG , Transferência de Energia , Células HeLa , Humanos , Medições Luminescentes/métodos , Fatores de Transcrição/genéticaRESUMO
An enhancer located upstream of the transcriptional start site of Ccnb1ip1 containing two GGA-rich regions and a 14-GGA repeat (GGA)14 region has been previously identified. Three copies of four GGA repeats in the c-myb promoter that form a tetrad:heptad:heptad:tetrad (T:H:H:T) dimerized G-quadruplex (G4) structure reportedly functions as both a transcriptional repressor and activator. Here, the secondary structures of the two GGA-rich and (GGA)14 regions were analyzed using circular dichroism spectral analysis, which indicated that the two GGA-rich DNAs formed parallel-type G4 structures, whereas (GGA)14 DNA formed the T:H:H:T dimerized G4 structure. Reporter assays demonstrated that individual regions did not show enhancer activity; however, the deletion of the (GGA)14 region resulted in 1.5-fold higher enhancer activity than that of the whole enhancer. These results indicate that the (GGA)14 region that forms the T:H:H:T dimerized G4 structure functions as a negative regulator of the Ccnb1ip1 enhancer.
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Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Elementos Facilitadores Genéticos , Quadruplex G , Sequências Repetitivas de Ácido Nucleico , Animais , Dicroísmo Circular , Humanos , Camundongos , Células NIH 3T3 , Ubiquitina-Proteína LigasesRESUMO
AIM: Prolapsed ovary (PO) in an inguinal hernia (IH) may cause torsion and infarction; however, the management of IH with PO in very low birthweight (VLBW) infants during neonatal intensive care unit (NICU) hospitalisation remains inconsistent. METHODS: The medical records of 47 IHs in 30 VLBW infants during NICU hospitalisation between 2008 and 2017 were reviewed retrospectively. RESULTS: Of the 47 IHs, PO was diagnosed in 18 (38.3%). Post-natal age and body weight at diagnosis of IH with PO were 71 (44-172) days and 2120 (1305-2965) g, respectively. Seventeen IHs with PO underwent surgery for correction electively just before discharge from the NICU at the age and body weight of 94 (51-187) days and 2645 (2340-2945) g, respectively. Therefore, the time interval between diagnosis and surgery was 15 (7-90) days. There was no torsion of ovary before surgery and no postoperative complications, including apnoea and recurrence. CONCLUSIONS: The high incidence of IH with PO in VLBW infants during NICU hospitalisation was confirmed in this study. Under careful observation, elective IH repair just before discharge from the NICU could be one acceptable option for the management of IH with PO in VLBW infants during NICU hospitalisation to prevent torsion and infarction of ovary.
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Hérnia Inguinal/complicações , Hérnia Inguinal/cirurgia , Recém-Nascido de muito Baixo Peso , Ovário/irrigação sanguínea , Feminino , Hérnia Inguinal/epidemiologia , Hospitalização , Humanos , Lactente , Recém-Nascido , Infarto/epidemiologia , Unidades de Terapia Intensiva Neonatal , Masculino , Torção Ovariana/epidemiologia , Ovário/anormalidadesRESUMO
PURPOSE: Treatment for high-risk neuroblastoma is still challenging. The purpose of the present study was to determine whether thalidomide suppresses etoposide-induced NF-κB activation and thus potentiates apoptosis in murine neuroblastoma. METHODS: A murine neuroblastoma cell line, C1300, and A/J mice were used in this study. We evaluated NF-κB activation after using etoposide with or without thalidomide by quantitative analysis of NF-κB by ELISA and by Western blot analysis of IκB phosphorylation in vitro and in vivo. Induction of apoptosis was evaluated by Western blot analysis of the apoptotic signals caspase-3, 8, and 9 in vitro and by TUNEL assays in vivo. We also evaluated the efficacy of the combination of etoposide and thalidomide by assessing tumor growth and mouse survival in vivo. RESULTS: Etoposide activated NF-κB in C1300 cells. This activation was suppressed by thalidomide and IκB was re-upregulated. The apoptotic signals were enhanced by the combination of thalidomide and etoposide compared with etoposide alone in vitro, which was consistent with TUNEL assays. The combination of etoposide and thalidomide also slowed tumor growth and mouse survival. CONCLUSION: Thalidomide potentiates etoposide-induced apoptosis in murine neuroblastoma by suppressing NF-κB.
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Apoptose/efeitos dos fármacos , Etoposídeo/farmacologia , Regulação Neoplásica da Expressão Gênica , NF-kappa B/genética , Neoplasias Experimentais , Neuroblastoma/genética , Talidomida/intoxicação , Animais , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Camundongos , NF-kappa B/biossíntese , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosforilação , Transdução de Sinais , Regulação para Cima/efeitos dos fármacosRESUMO
DNA methylation plays an important role in the regulation of gene expression. In normal cells, transposable elements that constitute approximately 45% of the human genome are highly methylated to silence their expression. In cancer cells, transposable elements are hypomethylated; therefore, global DNA methylation level is considered as a biomarker for cancer diagnostics. In this study, a homogeneous assay for measuring global DNA methylation level based on bioluminescence resonance energy transfer (BRET) was developed using methyl-CpG binding domain (MBD)-fused luciferase. In this assay, the MBD-luciferase recognizes methylated CpG, thus, BRET between the luciferase and fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the DNA methylation level of the target DNA. Moreover, the BRET signal was correlated with the LINE1 DNA methylation level on human genomic DNA, as determined by the bisulfite method. These results indicate that the global DNA methylation level of human genomic DNA could be detected simply by measuring the BRET signal.
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Técnicas Biossensoriais/métodos , Metilação de DNA , Transferência de Energia , Medições Luminescentes/métodos , Ilhas de CpG , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Luciferases/química , Luciferases/metabolismo , Modelos Moleculares , Neoplasias/diagnóstico , Neoplasias/genética , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
BACKGROUND: This study reviewed the medical records of very low-birthweight infants (VLBWI) followed up to adolescence, with emphasis on inguinal hernia (IH), to discuss the ideal time for IH repair in VLBWI. METHODS: Medical records from 274 VLBWI treated in the neonatal intensive care unit (NICU) between 1994 and 1999 were collected retrospectively. Outpatient data after NICU discharge were included. Additionally, a follow-up study was undertaken via questionnaire sent to the families of VLBWI in 2011. RESULTS: During NICU hospitalization, IH was diagnosed in 39 of 274 VLBWI, and two developed incarceration. Thirty-eight VLBWI were discharged with known hernia, and elective repair was performed in 19. Three developed incarceration before elective repair at the mean age of 316 days. In the remaining 18 cases of IH, spontaneous regression occurred at a mean age of 180 days and repair was not performed. New IH appeared in 25 VLBWI after NICU discharge. CONCLUSIONS: The high incidence of IH in VLBWI was confirmed during follow up to adolescence. The rate of incarceration was low and the incidence of spontaneous regression was high. Observation up to 6 months without surgery, with the expectation of spontaneous regression, is one option to avoid unnecessary surgery, but repair should be performed before 10 months to reduce the risk of incarceration. Further large, prospective, and randomized controlled studies with a long follow up are necessary to validate the present results and to define the ideal time for IH repair in VLBWI.
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Hérnia Inguinal/cirurgia , Recém-Nascido de muito Baixo Peso , Adolescente , Criança , Pré-Escolar , Seguimentos , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Estudos ProspectivosRESUMO
BACKGROUND/PURPOSE: Laparoscopic percutaneous extraperitoneal closure (LPEC) for pediatric inguinal hernia is a simple technique in which a purse-string suture made of nonabsorbable material is placed extraperitoneally around the hernia orifice by a special suture needle (Lapaherclosure™). Concerns have been raised about the extensive learning curve for both attending surgeons and residents to master this technique. This study assesses the difference in learning curves for the safe performance of LPEC by attending surgeons and residents. METHODS: A retrospective analysis was performed on the surgical charts of 409 consecutive patients (175 girls, 234 boys) who had undergone LPEC for inguinal hernia repair from December 2005 to December 2011 at Jikei University Hospital. The number of operation needed by attending surgeons and residents to reach the appropriate operation time was analyzed by the Mann-Whitney U test. RESULTS: LPEC was performed by three attending surgeons and four residents who had not previously performed LPEC. The standard operation time for LPEC by attending surgeons who have performed more than 100 LPEC cases safely is 30 min. In our study, the attending surgeons needed a mean of 12 operations (range, 10-16) to reach 30 min for LPEC. Three residents needed a mean of 31 operations (range, 27-33) to reach 30 min for LPEC. The fourth resident could not perform LPEC in 30 min or less. The difference between the number of operations needed by the attending surgeons and the residents to perform LPEC safely was statistically significant (P < 0.05). The overall incidence of contralateral patent processus vaginalis was 47.9 %. CONCLUSIONS: Our learning curve analysis showed that whereas attending surgeons needed a mean of 12 operations to perform LPEC repairs safely in 30 min or less, residents needed more than 30 operations to safely perform LPEC repairs without supervision.
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Competência Clínica/estatística & dados numéricos , Hérnia Inguinal/cirurgia , Laparoscopia/métodos , Curva de Aprendizado , Técnicas de Sutura , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Estudos Retrospectivos , Resultado do TratamentoRESUMO
PURPOSE: To clarify the radiosensitization mechanism masking the Auger effect of the cells possessing brominated DNA, the electronic properties of DNA-related molecules containing Br were investigated by X-ray spectroscopy and specific heat measurement. MATERIALS AND METHODS: X-ray absorption near-edge structure (XANES) and X-ray photoemission spectroscopy (XPS) were used to measure the electronic properties of the nucleotides with and without Br. We determined the specific heat of 5-bromouracil crystals with thymine as a reference molecule at low temperatures of 3-48 K to calculate the microscopic state numbers. RESULTS: Obtained XANES and XPS spectra indicated that both the lowest unoccupied molecular orbital (LUMO) and the core-levels were not affected by the Br incorporation. The state numbers of 5-bromouracil calculated from the specific heats obtained around 25 K was about 1.5 times larger than that for thymine below 20 K, although the numbers were almost the same below 5 K. DISCUSSION: Our results suggest that the Br atom may not contribute substantially to the LUMO and core-level electronic states of the molecule, but rather to the microscopic states related to the excitation of lattice vibrations, which may be involved in valence electronic states.
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Bromo , Timina , Bromouracila , DNARESUMO
Introduction: No studies have directly compared postoperative umbilical port site (UPS)-related complications between transumbilical and periumbilical incisions (TUI and PUI) after laparoscopic surgery in children. Patients and Methods: We conducted a retrospective review of 324 children of ≤15 years of age who underwent laparoscopic repair (LR) for inguinal hernia, via either a TUI or PUI, between 2010 and 2020. UPS-related complications, such as wound infection and umbilical deformity, were compared between TUI and PUI. We also investigated the risk factors for the development of UPS-related complications after LR. Results: TUIs and PUIs were used for 228 and 96 children, respectively. The incidence rates of postoperative wound infection and umbilical deformity in the PUI group were higher in comparison to the TUI group; although not to a statistically significant extent (7.3% versus 5.3%, P = .451, 6.3% versus 4.8%, P = .593). One case of incisional hernia at the UPS was seen in the TUI group. In the univariate analysis, sex, age, weight for age Z-score, nutrition status, insertion and closure time, total operative time, American Society of Anesthesiologists score, blood loss, and comorbidities were not significantly associated with the development of UPS-related complications after LR. Conclusion: The incidence of UPS-related complications in TUI and PUI was not significantly different. Due to the low incidence of UPS-related complications, it was difficult to draw conclusions regarding contributing factors; however, meticulous care should be taken to avoid UPS-related complications when closing the UPS (Ethical approval No. 2019-24).
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Hérnia Incisional , Laparoscopia , Criança , Humanos , Hérnia Incisional/cirurgia , Laparoscopia/efeitos adversos , Complicações Pós-Operatórias/etiologia , Estudos Retrospectivos , Infecção da Ferida Cirúrgica/epidemiologia , Infecção da Ferida Cirúrgica/etiologia , Umbigo/cirurgiaRESUMO
We report a 13-year-old boy who had massive intra-abdominal bleeding without a history of bleeding episodes or traumatic cause of bleeding. The patient underwent surgical treatment because bleeding was not controlled after treatment with tranexamic acid and transfusions including fresh-frozen plasma. Bleeding was traced to the lower left lobe of the liver. The mother's side of the family had a history of bleeding episodes in the boy's grandfather, great uncle, and son of a great aunt. A low level of plasma factor VIII coagulant activity (22%) led to a diagnosis of mild hemophilia A. Compared with severe hemophilia, mild hemophilia is more difficult to diagnose because bleeding episodes are less frequent. Most cases are found after incidental trauma or uncontrolled surgery-related bleeding, there is rarely a family history of hemophilia and activated partial thromboplastin time is normal or slightly prolonged. However, bleeding episodes in mild hemophilia may result in excessive, sometimes life-threatening hemorrhage and require early diagnosis and replacement treatment with adequate amounts of factor VIII, as in severe hemophilia.
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Hemofilia A/complicações , Hemofilia A/diagnóstico , Hemorragia/etiologia , Hepatopatias/etiologia , Adolescente , Fator VIII/administração & dosagem , Hemofilia A/classificação , Hemorragia/cirurgia , Hemorragia/terapia , Humanos , Hepatopatias/cirurgia , Hepatopatias/terapia , Masculino , Cuidados Pós-Operatórios , Resultado do TratamentoRESUMO
Global hypomethylation of genomic DNA is associated with genomic instability and carcinogenic processes. The loss of DNA methylation has been reported in several cancers; therefore, global methylation levels have been considered as biomarkers for cancer diagnosis. Bisulfite conversion analysis has been widely used as the gold standard method for quantification of DNA methylation levels. However, this method requires cumbersome and time-consuming steps. To quantify global DNA methylation levels in homogeneous solutions, we exemplify a sensing system based on bioluminescence resonance energy transfer (BRET) using methyl-CpG binding domain (MBD)-fused firefly luciferase (MBD-FLuc) and unmethyl-CpG binding domain (CXXC)-fused firefly luciferase (CXXC-FLuc). MBD-FLuc and CXXC-FLuc bind to methylated and unmethylated CpGs, respectively, in the genomic DNA to excite BOBO-3, an intercalating dye on genomic DNA. These BOBO-3 emission intensities depend on the methylated and unmethylated CpG content. The global DNA methylation levels can be quantified from the BOBO-3 emission intensities. Moreover, we introduce a multicolor BRET assay using MBD-FLuc and CXXC-fused Oplophorus luciferase (CXXC-OLuc) for the simultaneous quantification of methylated and unmethylated CpG content in genomic DNA. CXXC-OLuc excites the BOBO-1 DNA-intercalating dye depending on the unmethylated CpG content. Thus, the emission intensities of BOBO-1 and BOBO-3 excited by CXXC-OLuc and MBD-FLuc, respectively, can be simultaneously measured, thereby enabling the determination of global DNA methylation level in a single step. Here, we describe the detailed protocols for the expression of MBD-FLuc, CXXC-FLuc, and CXXC-OLuc in Escherichia coli and determine the global DNA methylation levels using these BRET assays.
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Metilação de DNA , Proteínas de Ligação a DNA , Ilhas de CpG , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transferência de Energia , Luciferases/metabolismo , Luciferases de Vaga-Lume/genéticaRESUMO
INTRODUCTION: Considerable debate exists regarding the benefits of laparoscopic percutaneous extraperitoneal closure (LPEC) over conventional open repair (OR). We assessed the outcomes and feasibility of LPEC compared to OR for pediatric inguinal hernia (IH). METHODS: We retrospectively analyzed 570 children who underwent LPEC or OR. Parents decided the operative method after obtaining informed consent. Patient characteristics, operative time, complications and contralateral metachronous IH (CMIH) were compared between the groups. RESULTS: A total of 329 children underwent LPEC and 241 underwent OR. There was no significant difference in the incidence of recurrence or testicular ascent between the LPEC and OR groups (0.3% vs 0.4%, P = 0.825, 0.3% vs 0.8%, P = 0.391, respectively). No testicular atrophy was recognized in either group. One patient with postoperative chronic inguinal pain was recognized in each group. There was no surgical site infection (SSI) in the OR group; however, the LPEC group more frequently demonstrated umbilical port site (UPS)-related complications, such as incisional hernia, minor deformity, granuloma formation, cellulitis and superficial SSI. Ten (4.1%) developed CMIH in OR; in contrast, no case of CMIH was experienced after LPEC (P < 0.001). CONCLUSION: In conclusion, both LPEC and OR are feasible in the management of pediatric IH, because of their high success rates and low risk of complications. LPEC could be the superior procedure with respect to the prevention of CMIH. However, to maximize the merits of LPEC over OR, it is important to reduce UPS-related complications in LPEC. A longer follow-up is needed to assess male fertility in patients who receive LPEC.
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Hérnia Inguinal , Laparoscopia , Criança , Hérnia Inguinal/cirurgia , Herniorrafia/métodos , Humanos , Lactente , Laparoscopia/métodos , Masculino , Estudos Retrospectivos , Medição de Risco , Resultado do TratamentoRESUMO
The adsorption of atomic hydrogen on hexagonal boron nitride (h-BN) is studied using two element-specific spectroscopies, i.e., near-edge x-ray absorption fine structure (NEXAFS) spectroscopy and x-ray photoelectron spectroscopy (XPS). B K-edge NEXAFS spectra show a clear change in the energy region of the π* band before and after reaction with atomic deuterium. On the other hand, N K-edge NEXAFS spectra show only a little change. B 1s XPS spectra show a distinct component at the low binding energy side of a main component, while N 1s XPS spectra show peak broadening at the high binding energy side. These experimental results are analyzed by the discrete variational Xα method with a core-hole effect and are explained by a model in which hydrogen atoms are preferentially adsorbed on the B sites of h-BN. Based on the experimental and theoretical results, we propose a site-selective property of BN material on adsorption of atomic hydrogen.