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Achaete-scute family bHLH transcription factor 2 (ASCL2) is highly expressed in hepatoblastoma (HB) tissues, but its role remains unclear. Thus, biological changes in the HB cell line HepG2 in response to induced ASCL2 expression were assessed. ASCL2 expression was induced in HepG2 cells using the Tet-On 3G system, which includes doxycycline. Cell viability, proliferation activity, mobility, and stemness were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, migration, invasion, and sphere-formation assays. Quantitative reverse-transcription polymerase chain reaction was used to assess the expression of markers for proliferation (CCND1 and MYC), epithelial-mesenchymal transition (EMT; SNAI1, TWIST1, and ZEB1), mesenchymal-epithelial transition (CDH1), and stemness (KLF4, POU5F1, and SOX9). Compared with the non-induced HepG2 cells, cells with induced ASCL2 expression showed significant increases in viability, colony number, migration area (%), and sphere number on days 7, 14, 8, and 7, respectively, and invasion area (%) after 90 h. Furthermore, induction of ASCL2 expression significantly upregulated CCND1, MYC, POU5F1, SOX9, and KLF4 expression on days 2, 2, 3, 3, and 5, respectively, and increased the ratios of SNAI1, TWIST1, and ZEB1 to CDH1 on day 5. ASCL2 promoted the formation of malignant phenotypes in HepG2 cells, which may be correlated with the upregulation of the Wnt signaling pathway-, EMT-, and stemness-related genes. ASCL2 activation may therefore be involved in the progression of HB.
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Hepatoblastoma , Neoplasias Hepáticas , Humanos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hepatoblastoma/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Transição Epitelial-Mesenquimal/genética , Neoplasias Hepáticas/genéticaRESUMO
PURPOSES: STAT1 is a transduction and transcriptional regulator that functions within the classical JAK/STAT pathway. In addition to chronic mucocutaneous candidiasis, bacterial infections are a common occurrence in patients with STAT1 gain-of-function (GOF) mutations. These patients often exhibit skewing of B cell subsets; however, the impact of STAT1-GOF mutations on B cell-mediated humoral immunity remains largely unexplored. It is also unclear whether these patients with IgG within normal range require regular intravenous immunoglobulin (IVIG) therapy. METHODS: Eleven patients (harboring nine different STAT1-GOF mutations) were enrolled. Reporter assays and immunoblot analyses were performed to confirm STAT1 mutations. Flow cytometry, deep sequencing, ELISA, and ELISpot were conducted to assess the impact of STAT1-GOF on humoral immunity. RESULTS: All patients exhibited increased levels of phospho-STAT1 and total STAT1 protein, with two patients carrying novel mutations. In vitro assays showed that these two novel mutations were GOF mutations. Three patients with normal total IgG levels received regular IVIG infusions, resulting in effective control of bacterial infections. Four cases showed impaired affinity and specificity of pertussis toxin-specific antibodies, accompanied by reduced generation of class-switched memory B cells. Patients also had a disrupted immunoglobulin heavy chain (IGH) repertoire, coupled with a marked reduction in the somatic hypermutation frequency of switched Ig transcripts. CONCLUSION: STAT1-GOF mutations disrupt B cell compartments and skew IGH characteristics, resulting in impaired affinity and antigen-specificity of antibodies and recurrent bacterial infections. Regular IVIG therapy can control these infections in patients, even those with normal total IgG levels.
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Linfócitos B , Infecções Bacterianas , Mutação com Ganho de Função , Imunoglobulinas Intravenosas , Fator de Transcrição STAT1 , Humanos , Fator de Transcrição STAT1/genética , Infecções Bacterianas/imunologia , Infecções Bacterianas/genética , Feminino , Masculino , Criança , Imunoglobulinas Intravenosas/uso terapêutico , Linfócitos B/imunologia , Adulto , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Pré-Escolar , Adolescente , Adulto Jovem , Imunidade HumoralRESUMO
Patients with chromosome 18q deletion syndrome generally experience hypogammaglobulinemia. Herein, we describe two patients with chromosome 18q deletion syndrome who presented with late-onset combined immune deficiency (LOCID), which has not been previously reported. Patient 1 was a 29-year-old male with 18q deletion syndrome, who was being managed for severe motor and intellectual disabilities at the Yamabiko Medical Welfare Center for 26 years. Although the patient had few infections, he developed Pneumocystis pneumonia at the age of 28. Patient 2, a 48-year-old female with intellectual disability and congenital malformations, was referred to Tokyo Medical and Dental University Hospital with abnormal bilateral lung shadows detected on her chest radiography. Computed tomography showed multiple lymphadenopathies and pneumonia. A lymph node biopsy of the inguinal region revealed granulomatous lymphadenitis, and a chromosomal examination revealed 18q deletion. Array-based genomic hybridization analysis revealed deletion at 18q21.32-q22.3 for patient 1 and at 18q21.33-qter for patient 2. Immune status work-up of the two patients revealed panhypogammaglobulinemia, decreased number of memory B cells and naïve CD4+ and/or CD8+ cells, reduced response on the carboxyfluorescein diacetate succinimidyl ester T-cell division test, and low levels of T-cell receptor recombination excision circles and Ig κ-deleting recombination excision circles. Consequently, both patients were diagnosed with LOCID. Although patients with 18q deletion syndrome generally experience humoral immunodeficiency, the disease can be further complicated by cell-mediated immunodeficiency, causing combined immunodeficiency. Therefore, patients with 18q deletion syndrome should be regularly tested for cellular/humoral immunocompetence.
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Deleção Cromossômica , Transtornos Cromossômicos , Cromossomos Humanos Par 18 , Humanos , Masculino , Feminino , Cromossomos Humanos Par 18/genética , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Adulto , Pessoa de Meia-Idade , Idade de Início , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/diagnóstico , Imunodeficiência Combinada Severa/complicações , Deficiência Intelectual/genética , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/etiologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/complicaçõesRESUMO
Autosomal recessive CARD9 deficiency can underly deep and superficial fungal diseases. We identified two Japanese patients, suffering from superficial and invasive Candida albicans diseases, carrying biallelic variants of CARD9. Both patients, in addition to another Japanese and two Korean patients who were previously reported, carried the c.820dup CARD9 variant, either in the homozygous (two patients) or heterozygous (three patients) state. The other CARD9 alleles were c.104G > A, c.1534C > T and c.1558del. The c.820dup CARD9 variant has thus been reported, in the homozygous or heterozygous state, in patients originating from China, Japan, or South Korea. The Japanese, Korean, and Chinese patients share a 10 Kb haplotype encompassing the c.820dup CARD9 variant. This variant thus originates from a common ancestor, estimated to have lived less than 4,000 years ago. While phaeohyphomycosis caused by Phialophora spp. was common in the Chinese patients, none of the five patients in our study displayed Phialophora spp.-induced disease. This difference between Chinese and our patients probably results from environmental factors. (161/250).
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Proteínas Adaptadoras de Sinalização CARD , Efeito Fundador , Humanos , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/deficiência , Masculino , Feminino , Candidíase Mucocutânea Crônica/genética , Candidíase Mucocutânea Crônica/diagnóstico , Haplótipos , Mutação/genética , Ásia Oriental , Alelos , Candida albicans/genética , Adulto , Linhagem , Povo Asiático/genéticaRESUMO
PURPOSE: Transcription factor 3 (TCF3) encodes 2 transcription factors generated by alternative splicing, E12 and E47, which contribute to early lymphocyte differentiation. In humans, autosomal dominant (AD) E47 transcription factor deficiency is an inborn error of immunity characterized by B-cell deficiency and agammaglobulinemia. Only the recurrent de novo p.E555K pathogenic variant has been associated with this disease and acts via a dominant-negative (DN) mechanism. In this study, we describe the first Asian patient with agammaglobulinemia caused by the TCF3 p.E555K variant and provide insights into the structure and function of this variant. METHODS: TCF3 variant was identified by inborn errors of immunity-related gene panel sequencing. The variant E555K was characterized by alanine scanning of the E47 basic region and comprehensive mutational analysis focused on position 555. RESULTS: The patient was a 25-year-old male with B-cell deficiency, agammaglobulinemia, and mild facial dysmorphic features. We confirmed the diagnosis of AD E47 transcription factor deficiency by identifying a heterozygous missense variant, c.1663 G>A; p.E555K, in TCF3. Alanine scanning of the E47 basic region revealed the structural importance of position 555. Comprehensive mutational analysis focused on position 555 showed that only the glutamate-to-lysine substitution had a strong DN effect. 3D modeling demonstrated that this variant not only abolished hydrogen bonds involved in proteinâDNA interactions, but also inverted the charge on the surface of the E47 protein. CONCLUSIONS: Our study reveals the causative mutation hotspot in the TCF3 DN variant and highlights the weak negative selection associated with the TCF3 gene.
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Agamaglobulinemia , Humanos , Masculino , Adulto , Agamaglobulinemia/genética , Agamaglobulinemia/diagnóstico , Linfócitos B/imunologia , Genes Dominantes , Análise Mutacional de DNA , Mutação de Sentido Incorreto/genética , Mutação/genética , Linhagem , Predisposição Genética para Doença , Modelos Moleculares , Fatores de Transcrição Hélice-Alça-Hélice BásicosRESUMO
PURPOSE: Heterozygous STAT1 Gain-of-Function (GOF) mutations are the most common cause of chronic mucocutaneous candidiasis (CMC) among Inborn Errors of Immunity. Clinically, these mutations manifest as a broad spectrum of immune dysregulation, including autoimmune diseases, vascular disorders, and malignancies. The pathogenic mechanisms of immune dysregulation and its impact on immune cells are not yet fully understood. In treatment, JAK inhibitors have shown therapeutic effectiveness in some patients. METHODS: We analyzed clinical presentations, cellular phenotypes, and functional impacts in five Taiwanese patients with STAT1 GOF. RESULTS: We identified two novel GOF mutations in 5 patients from 2 Taiwanese families, presenting with symptoms of CMC, late-onset rosacea, and autoimmunity. The enhanced phosphorylation and delayed dephosphorylation were displayed by the patients' cells. There are alterations in both innate and adaptive immune cells, including expansion of CD38+HLADR +CD8+ T cells, a skewed activated Tfh cells toward Th1, reduction of memory, marginal zone and anergic B cells, all main functional dendritic cell lineages, and a reduction in classical monocyte. Baricitinib showed therapeutic effectiveness without side effects. CONCLUSION: Our study provides the first comprehensive clinical and molecular characteristics in STAT1 GOF patient in Taiwan and highlights the dysregulated T and B cells subsets which may hinge the autoimmunity in STAT1 GOF patients. It also demonstrated the therapeutic safety and efficacy of baricitinib in pediatric patient. Further research is needed to delineate how the aberrant STAT1 signaling lead to the changes in cellular populations as well as to better link to the clinical manifestations of the disease.
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Candidíase Mucocutânea Crônica , Mutação com Ganho de Função , Imunofenotipagem , Pirazóis , Fator de Transcrição STAT1 , Humanos , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Candidíase Mucocutânea Crônica/genética , Candidíase Mucocutânea Crônica/diagnóstico , Candidíase Mucocutânea Crônica/terapia , Masculino , Feminino , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Azetidinas/uso terapêutico , Purinas/uso terapêutico , Criança , Adolescente , Taiwan , AdultoRESUMO
PURPOSE: We aimed to describe the clinical, immunological, and genetic features of patients with DOCK8 deficiency (DOCK8-Def) in a tertiary care center for children. METHODS: Retrospective chart review of patients' clinical, immunological, and genetic characteristics with DOCK8-Def. Genetic analysis was performed with targeted- or whole-exome sequencing; we also assessed DOCK8 protein expression and a lymphoproliferation assay and analyzed survival by the Kaplan-Meier method. RESULTS: We described 11 patients from 8 unrelated kindreds. The median age at symptoms' onset was 10 months (range 1-54 months). The median follow-up time was 53.4 months (4.8-118.8). All patients presented eczema and recurrent sinopulmonary and cutaneous infections. Besides those symptoms, the most frequent manifestations were bronchiectases (8/11), food allergies (6/11), and severe infections (6/11). Infrequent characteristics were detection of CMV in bronchial lavage, C. parvum-driven sclerosing cholangitis, Takayasu vasculitis, neurological syndromes, pulmonary tuberculosis, and lymphomatoid granulomatosis. CONCLUSION: DOCK8-Def has a broad spectrum of manifestations, including allergy, autoimmunity, inflammation, infection, and cancer. The hallmark of this inborn error of immunity is IEI-associated eczema with eosinophilia and increased IgE. Here, we report six new mutations causing human DOCK8 deficiency and symptoms previously unrecognized to occur in DOCK8-Def. Therefore, an early diagnosis of DOCK8-Def is essential to facilitate an adequate treatment such as HSCT.
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Eczema , Hipersensibilidade , Síndrome de Job , Criança , Humanos , Lactente , Pré-Escolar , Estudos Retrospectivos , Síndrome de Job/genética , Eczema/epidemiologia , Eczema/genética , Mutação , Fatores de Troca do Nucleotídeo Guanina/genéticaRESUMO
In this study, the atypical swelling gelation of chitin physical hydrogels was investigated. Just by tuning the amount of the N-acetylation reagent, the degree of acetylation varied and mouldable chitin hydrogels with a wide variety of gel concentrations (0.2-6.4 wt%) were obtained. In response to the gel concentration, the mechanical properties ranged from swollen soft gels to shrunken rigid gels (compressive moduli of 4-310 kPa). The thus-prepared chitin hydrogels, which were composed of only chitin and water, exhibited high transparency and integrity. The swelling gelation of chitin physical hydrogels was achieved owing to both the positive charges of the amino groups inducing the osmotic pressure and the toughness of the crystalline nanofibrous network structure of the chitin hydrogels that endured the large volume change. These previously unnoticed advantageous aspects of chitin have pioneered a novel area of swellable physical gels that has been exclusive to chemical gels so far.
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BACKGROUND: Although randomized controlled trials (RCTs) have shown that calcitonin gene-related peptide (CGRP)-targeted monoclonal antibodies (CGRP mAbs) are an efficacious and safe therapeutic modality for migraine prevention, their clinical benefits have not been well validated in Japanese patients in the real-world setting. The present study aimed to evaluate the real-world efficacy and safety of galcanezumab, fremanezumab, and erenumab in Japanese patients with migraine. METHODS: This observational retrospective cohort study was conducted at two headache centers in Japan. Patients with migraine who had experienced treatment failure with at least one traditional oral migraine preventive agent were treated with a CGRP mAb de novo. The primary efficacy endpoints were the changes from baseline in monthly migraine days (MMDs) and Headache Impact Test-6 (HIT-6) score after 3 dosing intervals (V3). We explored whether demographic and clinical characteristics predicted therapeutic outcomes at V3. RESULTS: Sixty-eight patients who completed three doses of a CGRP mAb (85.3% female [58/68], mean age: 46.2 ± 13.1 years) were included in the analysis. There were 19 patients with chronic migraine. The baseline MMDs were 13.4 ± 6.0. After 3 doses, the MMDs significantly decreased to 7.4 ± 5.5 (p < 0.0001), and the 50% response rate was 50.0%. HIT-6 score was significantly reduced from 66.7 ± 5.4 to 56.2 ± 8.7 after 3 doses (P = 0.0001). There was a positive correlation between the changes in MMDs and HIT-6 score from baseline after 2 doses (p = 0.0189). Those who achieved a ≥ 50% therapeutic response after the first and second doses were significantly more likely to do so at V3 (crude odds ratio: 3.474 [95% CI: 1.037 to 10.4], p = 0.0467). The most frequent adverse event was constipation (7.4%). None of the adverse events were serious, and there was no need for treatment discontinuation. CONCLUSIONS: This real-world study demonstrated that CGRP mAbs conferred Japanese patients with efficacious and safe migraine prevention, and an initial positive therapeutic response was predictive of subsequent favorable outcomes. Concomitant measurement of MMDs and HIT-6 score was useful in evaluating the efficacy of CGRP mAbs in migraine prevention.
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Peptídeo Relacionado com Gene de Calcitonina , Transtornos de Enxaqueca , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos de Coortes , Cefaleia/tratamento farmacológico , Japão/epidemiologia , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/prevenção & controleRESUMO
Blinatumomab is a CD3/CD19-directed bispecific T-cell engager used to treat relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Although blinatumomab has shown efficacy, it can cause serious adverse events, including cytokine release syndrome and neurological events. Among the neurological events, encephalopathy is rare, and knowledge is lacking. Herein, we present a pediatric case of blinatumomab-associated encephalopathy that initially presented with refractory convulsions and later developed into a cerebral infarction. The patient experienced prolonged paralysis and increased brain damage.
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Spatial distribution of water-soluble molecules and ions in living organisms is still challenging to assess. Energy-dispersive X-ray spectroscopy (EDS) via cryogenic scanning electron microscopy (cryo-SEM) is one of the promising methods to study them without loss of dissolved contents. High-resolution cryo-SEM-EDS has challenges in sample preparation, including cross-section exposure and sample drift/charging due to insulative surrounding water. The former becomes problematic for large and inseparable organisms, such as benthic foraminifera, a unicellular eukaryote playing significant roles in marine ecosystems, which often exceed the size limit for the most reliable high-pressure freezing. Here we show graphite oxide dispersed in sucrose solution as a good glue to freeze, expose cross-section by cryo-ultramicrotome, and analyze elemental distribution owing to the glue's high viscosity, adhesion force, and electron conductivity. To demonstrate the effectiveness and applicability of the glue for cryo-SEM-EDS, deep-sea foraminifer Uvigerina akitaensis was sampled during a cruise and plunge frozen directly on the research vessel, where the liquid nitrogen supply is limited. The microstructures were preserved as faithfully in cryo-SEM images as those with the conventional resin-substituted transmission electron micrograph. We found elements colocalized within the cytoplasm originating from water-soluble compounds that can be lost with conventional dehydrative fixation.
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Adesivos , Microscopia Crioeletrônica , Foraminíferos , Congelamento , Espectrometria por Raios X , Foraminíferos/ultraestrutura , Microscopia Crioeletrônica/métodos , Adesivos/química , Espectrometria por Raios X/métodos , Microscopia Eletrônica de Varredura/métodosRESUMO
While SARS-CoV-2 infection causes a mild disease in most children, SARS-CoV-2 infection may be lethal in a few of them. In the defense against SARS-CoV-2, type I interferons are key players, and several studies have identified a defective or neutralized interferon response as the cause of overwhelming viral infection. However, inappropriate, untimely, or excessive interferon production may also be detrimental to the host. Here, we describe two patients with STAT1 gain-of-function (GOF), a known type I interferonopathy, who died of COVID-19. Whole-exome sequencing and interferon-gamma-activated sequence (GAS) and interferon-sensitive responsive element (ISRE) reporter assay were performed to identify and characterize STAT1 variants. Patient 1 developed hemophagocytic lymphohistiocytosis (HLH) in the context of COVID-19 infection and died in less than a week at the age of 4 years. Patient 2 developed a high fever, cough, and hypoxemia and succumbed to COVID-19 pneumonia at the age of 5 years. Two heterozygous missense variants, p.E563Q and p.K344E, in STAT1 were identified. Functional validation by reporter assay and immunoblot confirmed that both variants are gain-of-function (GOF). GOF variants transiently expressing cells exhibited enhanced upregulation of downstream genes, including ISG15, MX1, and OAS1, in response to IFN-α stimulation. A catastrophic course with HLH or acute respiratory failure is thought to be associated with inappropriate immunoregulatory mechanisms to handle SARS-CoV-2 in STAT1 GOF. While most patients with inborn errors of immunity who developed COVID-19 seem to handle it well, these cases suggest that patients with STAT1-GOF might be at risk of developing fatal complications due to SARS-CoV-2.
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COVID-19 , Interferon Tipo I , Criança , Pré-Escolar , Humanos , COVID-19/genética , Mutação com Ganho de Função , Interferon-alfa/genética , SARS-CoV-2/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismoRESUMO
PURPOSE: Inborn errors of the IL-17A/F-responsive pathway lead to chronic mucocutaneous candidiasis (CMC) as a predominant clinical phenotype, without other significant clinical manifestations apart from mucocutaneous staphylococcal diseases. Among inborn errors affecting IL-17-dependent immunity, autosomal recessive (AR) IL-17RC deficiency is a rare disease with only three kindreds described to date. The lack of an in vitro functional evaluation system of IL17RC variants renders its diagnosis difficult. We sought to characterize a 7-year-old Japanese girl with CMC carrying a novel homozygous duplication variant of IL17RC and establish a simple in vitro system to evaluate the impact of this variant. METHODS: Flow cytometry, qPCR, RNA-sequencing, and immunoblotting were conducted, and an IL17RC-knockout cell line was established for functional evaluation. RESULTS: The patient presented with oral and mucocutaneous candidiasis without staphylococcal diseases since the age of 3 months. Genetic analysis showed that the novel duplication variant (Chr3: 9,971,476-9,971,606 dup (+131bp)) involving exon 13 of IL17RC results in a premature stop codon (p.D457Afs*16 or p.D457Afs*17). Our functional evaluation system revealed this duplication to be loss-of-function and enabled discrimination between loss-of-function and neutral IL17RC variants. The lack of response to IL-17A by the patient's SV40-immortalized fibroblasts was restored by introducing WT-IL17RC, suggesting that the genotype identified is responsible for her clinical phenotype. CONCLUSIONS: The clinical and cellular phenotype of the current case of AR IL-17RC deficiency supports a previous report on this rare disorder. Our newly established evaluation system will be useful for the diagnosis of AR IL-17RC deficiency, providing accurate validation of unknown IL17RC variants.
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Candidíase Mucocutânea Crônica , Candidíase , Feminino , Humanos , Lactente , Criança , Candidíase Mucocutânea Crônica/diagnóstico , Candidíase Mucocutânea Crônica/genética , Interleucina-17/genética , Candidíase/genética , Fibroblastos/metabolismo , Sequência de BasesRESUMO
Gold (Au), as one of the most precious metal resources that is used for both industrial products and private ornaments, is a global investment target, and mining companies are making huge investments to discover new Au deposits. Here, we report in situ Au adsorption in an acidic hot spring by a unique adsorption sheet made from blue-green algae with a high preferential adsorption ability for Au. The results of in situ Au adsorption experiments conducted for various reaction times ranging from 0.2 h to 7 months showed that a maximum Au concentration of 30 ppm was adsorbed onto the blue-green algal sheet after a reaction time of 7 months. The Au concentration in the hot spring water was below the detection limit (< 1 ppt); therefore, Au was enriched by preferential adsorption onto the blue-green algal sheet by a factor of more than ~ 3 × 107. Thus, our gold recovery method has a high potential to recover Au even from an Au-poor solution such as hot spring water or mine wastewater with a low impact on the environment.
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Ouro , Fontes Termais , Adsorção , Ácidos , ÁguaRESUMO
Introduction: The Act on the Safety of Regenerative Medicine enforced in Japan in 2014, regulates the manufacture of cellular processed products. However, with regards to the manufacturing facilities at medical institutions, only the submission of necessary documents is required for a license, and the need for third-party inspection has been highlighted. Remote activities are becoming more prominent with the spread of the Severe Acute Respiratory Syndrome Coronavirus 2 infection; therefore, the current assessment of compliance with structural facility standards was conducted remotely. Methods: The entire process, including start-up meetings, preparation of the survey schedule, submission and review of preliminary materials, audits, and reporting of results, was conducted via e-mail and web conferencing systems. The survey was conducted remotely, to minimize the risk of contamination of the cell processing facility (CPF) and reduce the burden on surveyors, while contributing to the establishment of suitable structural facilities by identifying and highlighting the areas or items that were considered to be non-compliant with the regulations. The series of audits were completed in ten weeks, with a period of six weeks between the start-up meeting and the audit implementation. The audit was completed in approximately 3 h on the day of the inspection. Results: The audit results were delivered in the report, with four items requiring improvement and several other recommended items listed as non-conformities. Conclusions: We believe that this remote method allows the effective inspection of regenerative medicine manufacturing facilities and assessment of more cell culture processing facilities than the current in-person audit method, with limited human resources.
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Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as â¼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression.
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Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Humanos , Replicação do DNA , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Duplicação Gênica , Origem de Replicação/genética , Quebras de DNA de Cadeia Dupla , Sistemas CRISPR-Cas/genéticaRESUMO
RATIONALE: Since the precise mechanisms of posttraumatic stress disorder (PTSD) remain unknown, effective treatment interventions have not yet been established. Impaired extinction of fear memory (EFM) is one of the core symptoms of PTSD and is associated with stress-induced epigenetic change in gene expression. OBJECTIVES: In this study, we examined whether the involvement of histone H3 lysine 9 dimethylation (H3K9me2) in EFM is mediated through brain-derived neurotrophic factor (BDNF) expression in the hippocampus, and whether BIX01294, a selective G9a and GLP histone methyltransferase inhibitor, could be treatment for impaired EFM in an animal model of PTSD. METHODS: The single prolonged stress (SPS) paradigm was used to model PTSD. We measured BDNF mRNA levels by RT-PCR, and H3K9me2 levels in the BDNF gene promoters by chromatin immunoprecipitation-qPCR. After undergoing contextual fear conditioning and hippocampal injection of BIX01294, male rats were subjected to extinction training and extinction testing and their freezing times and BDNF mRNA levels were measured. RESULTS: Compared to sham rats, SPS rats showed decreased BDNF mRNA levels 2 h after extinction training, no significant changes in levels of global H3K9me2 prior to extinction training, and increased levels of H3K9me2 in BDNF gene promoter IV, but not in BDNF gene promoter I. Administration of BIX01294 ameliorated the decrease in BDNF mRNA levels 2 h after extinction training and subsequently alleviated impaired EFM in extinction tests in SPS rats. CONCLUSION: We conclude that reduced hippocampal levels of BDNF mRNA due to increase in H3K9me2 levels may play a role in PTSD-associated EFM impairment, and BIX01294 could be a PTSD treatment option.
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Severe congenital neutropenia (SCN) is characterized by chronic neutropenia with recurrent infections from early infancy and a predisposition to myelodysplastic syndrome/acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for patients with SCN who develop myelodysplastic syndrome/AML. We report an 8-year-old girl with SCN carrying an ELANE mutation that had been refractory to granulocyte colony-stimulating factor. The patient experienced recurrent infections and then developed AML. The counts of leukemic blasts that harbored both CSF3R and RUNX1 mutations spontaneously decreased with antimicrobial therapy, leading to partial remission. After AML recurrence, HSCT was successfully performed using modified chemotherapy and a conditioning regimen. Serial donor lymphocyte infusions against mixed chimerism induced complete donor chimerism over 4 years without any infections or AML relapse. This case suggests the importance of carefully managing neutropenia-related infections, leukemia progression, and HSCT in patients with SCN developing AML.