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TFE3-rearranged renal cell cancer (tRCC) is a rare form of RCC that involves chromosomal translocation of the Xp11.2 TFE3 gene. Despite its early onset and poor prognosis, the molecular mechanisms of the pathogenesis of tRCC remain elusive. This study aimed to identify novel therapeutic targets for patients with primary and recurrent tRCC. We collected 19 TFE3-positive RCC tissues that were diagnosed by immunohistochemistry and subjected them to genetic characterization to examine their genomic and transcriptomic features. Tumor-specific signatures were extracted using whole exome sequencing (WES) and RNA sequencing (RNA-seq) data, and the functional consequences were analyzed in a cell line with TFE3 translocation. Both a low burden of somatic single nucleotide variants (SNVs) and a positive correlation between the number of somatic variants and age of onset were observed. Transcriptome analysis revealed that four samples (21.1%) lacked the expected fusion event and clustered with the genomic profiles of clear cell RCC (ccRCC) tissues. The fusion event also demonstrated an enrichment of upregulated genes associated with mitochondrial respiration compared with ccRCC expression profiles. Comparison of the RNA expression profile with the TFE3 ChIP-seq pattern data indicated that PPARGC1A is a metabolic regulator of the oncogenic process. Cell proliferation was reduced when PPARGC1A and its related metabolic pathways were repressed by its inhibitor SR-18292. In conclusion, we demonstrate that PPARGC1A-mediated mitochondrial respiration can be considered a potential therapeutic target in tRCC. This study identifies an uncharacterized genetic profile of an RCC subtype with unique clinical features and provides therapeutic options specific to tRCC.
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PURPOSE: The results of past studies comparing percutaneous techniques with traditional open techniques for hallux valgus are controversial. Therefore, this study aimed to compare the radiologic and clinical outcomes of percutaneous and open distal chevron osteotomies. METHODS: Seventy-one patients with mild to severe hallux valgus deformity were randomized to undergo percutaneous distal chevron osteotomy (percutaneous group, n = 36) or open distal chevron osteotomy (open group, n = 35) between October 2019 and September 2020. Radiological and clinical outcomes were assessed preoperatively and postoperatively. Outcome measures included the foot and ankle outcome score, foot functional index, visual analogue scale (VAS) scores for pain, range of motion (ROM) of the first metatarsophalangeal (MTP) joint, hallux valgus angle, intermetatarsal angle, and first metatarsal shortening. Additionally, the first metatarsal declination angle was measured to evaluate sagittal malunion. RESULTS: The mean first metatarsal declination angle decreased significantly at 12 months postoperatively in both groups (p = 0.021 and p < 0.001 in the percutaneous and open groups, respectively), and the decrement was significantly greater in the open group (p = 0.033). The mean VAS score for pain on postoperative day one was 4.2 ± 1.9 and 5.3 ± 1.7 in the percutaneous and open groups, respectively (p = 0.019). The mean ROM of the first MTP joint did not change significantly after surgery, from 72.5 ± 7.5 preoperatively to 71.0 ± 9.5 at 12 months postoperatively in the percutaneous group (p = 0.215); however, it decreased significantly from 70.6 ± 7.3 preoperatively to 63.4 ± 10.4 at 12 months postoperatively in the open group (p < 0.001). There were no significant differences between the groups regarding other clinical outcomes. CONCLUSION: The percutaneous group showed a lower immediate pain level at postoperative day 1 and better ROM of the first MTP joint at 12 months postoperatively.
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Background: A coronary artery aneurysm is a rare cardiac anomaly that may be incidentally detected on echocardiography. When associated with a coronary cameral fistula, an aneurysm can become symptomatic. We present a unique case of a giant left circumflex coronary aneurysm with a fistula to the left atrium and a large atrial septal defect causing acute heart failure in a young woman during the peripartum period. Case summary: A 32 year-old woman who presented with hypoxia after the delivery of her fourth child was found to have heart failure with severe mitral regurgitation and multiple abnormal intracardiac shunts. Echocardiography showed a large circular structure with Doppler color flow into the left atrium and between the atria. Cardiac computed tomography showed multiple dilated coronary arteries including a left circumflex coronary artery aneurysm measuring >10â cm in diameter with fistulous communication to the left atrium and a large atrial septal defect. A right heart catheterization was performed, and the patient was diagnosed with high-output heart failure. Surgical closure of the coronary cameral fistula was deferred due to the risk of worsening pressure in the coronary aneurysm, and the patient was referred for cardiac transplantation. Discussion: This case illustrates severe heart failure as a complication of a giant coronary artery aneurysm with fistulization to the left atrium and subsequent shunting through a large atrial defect. Echocardiography allows for the detection of a coronary aneurysm and shunting, and cardiac computed tomography provides detailed visualization of a coronary cameral fistula.
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Lophomonas blattarum is an anaerobic protozoan living in the intestine of cockroaches and house dust mites, with ultramicroscopic characteristics such as the presence of a parabasal body, axial filament, and absence of mitochondria. More than 200 cases of Lophomonas infection of the respiratory tract have been reported worldwide. However, the current diagnosis of such infection depends only on light microscopic morphological findings from respiratory secretions. In this study, we attempted to provide more robust evidence of protozoal infection in an immunocompromised patient with atypical pneumonia, positive for Lophomonas-like protozoal cell forms. A direct search of bronchoalveolar lavage fluid via polymerase chain reaction (PCR), transmission electron microscopy (TEM), and metagenomic next-generation sequencing did not prove the presence of protozoal infection. PCR results were not validated with sufficient rigor, while de novo assembly and taxonomic classification results did not confirm the presence of an unidentified pathogen. The TEM results implied that such protozoal forms in light microscopy are actually non-detached ciliated epithelial cells. After ruling out infectious causes, the patient's final diagnosis was drug-induced pneumonitis. These findings underscore the lack of validation in the previously utilized diagnostic methods, and more evidence in the presence of L. blattarum is required to further prove its pathogenicity.
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Pneumopatias Parasitárias , Parabasalídeos , Pneumonia por Mycoplasma , Infecções por Protozoários , Humanos , Pneumopatias Parasitárias/diagnóstico , Infecções por Protozoários/diagnóstico , Líquido da Lavagem BroncoalveolarRESUMO
The development of a chemically attenuated, replication-incompetent virus vaccine can provide protection against diseases caused by DNA viruses. In this study, we have developed a method to produce live-attenuated, replication-defective viruses using centanamycin (CM), a chemical compound that alkylates the A-T-rich minor groove of the DNA and thereby blocks DNA replication. We tested the efficacy of CM to produce live-attenuated, replication-defective human cytomegalovirus, mouse cytomegalovirus, and herpes simplex virus-2 (HSV-2), suggesting a broad application for generating live-attenuated, replication-defective DNA viruses. Mass spectrometry analysis showed that CM alkylate viral DNA at the adenine-N3 position. Moreover, mice immunization with CM-attenuated mouse cytomegalovirus (MCMV) produced a robust immune response and reduced the viral load in immunized animals against challenges with live, wild-type MCMV. Our study offers a unifying and attractive therapeutic opportunity that chemically attenuated live DNA viruses can be readily developed as new frontline vaccines.
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Herpesvirus Humano 2 , Imunização , Animais , Camundongos , Humanos , Herpesvirus Humano 2/genética , Vacinação , DNA , Desenvolvimento de VacinasRESUMO
Chalcones (1,3-diphenyl-2-propen-1-ones) either natural or synthetic have a plethora of biological properties including antileishmanial activities, but their development as drugs is hampered by their largely unknown mechanisms of action. We demonstrate herein that our previously described benzochalcone fluorogenic probe (HAB) could be imaged by fluorescence microscopy in live Leishmania amazonensis promastigotes where it targeted the parasite acidocalcisomes, lysosomes and the mitochondrion. As in the live zebrafish model, HAB formed yellow-emitting fluorescent complexes when associated with biological targets in Leishmania. Further, we used HAB as a reversible probe to study the binding of a portfolio of diverse chalcones and analogues in live promastigotes, using a combination of competitive flow cytometry analysis and cell microscopy. This pharmacological evaluation suggested that the binding of HAB in promastigotes was representative of chalcone pharmacology in Leishmania, with certain exogenous chalcones exhibiting competitive inhibition (ca. 20-30%) towards HAB whereas non-chalconic inhibitors showed weak capacity (ca. 3-5%) to block the probe intracellular binding. However, this methodology was restricted by the strong toxicity of several competing chalcones at high concentration, in conjunction with the limited sensitivity of the HAB fluorophore. This advocates for further optimization of this undirect target detection strategy using pharmacophore-derived reversible fluorescent probes.
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Antiprotozoários , Chalcona , Chalconas , Leishmania , Animais , Antiprotozoários/química , Antiprotozoários/farmacologia , Sítios de Ligação , Chalcona/farmacologia , Chalconas/química , Chalconas/farmacologia , Corantes Fluorescentes , Peixe-ZebraRESUMO
BACKGROUND AND AIMS: In order to provide high-quality care, providers need to understand their patients' goals and concerns. This study aims to identify and predict the goals and concerns prioritised by patients with inflammatory bowel disease [IBD] in the outpatient setting. METHODS: Mixed-methods analysis was performed to identify the types, frequencies, and predictors of IBD patients' goals and concerns using 4873 surveys collected over 2016-2019 at 25 gastroenterology clinics across the USA participating in the Crohn's & Colitis Foundation's IBD Qorus Learning Health System. RESULTS: Patients with IBD most often prioritised goals and concerns related to symptoms/disease activity [50%] and clinical course/management [20%], whereas psychosocial/quality of life [12%] and medication [6%] concerns were less frequent. Females (odds ratio [OR] 22.1, 95% confidence interval [CI] 5.3-91.5) and patients in clinical remission [OR 2.2, 95% CI 1.2-4.1] were more likely to prioritise family planning. Patients >60 years old [OR 3.1, 95% CI 1.5-6.5] and patients with active disease [OR 3.2, 95% CI 1.4-7.6] were more often concerned about travelling. Smokers were more often concerned about nutrition [OR 4.2, 95% CI 1.9-9.2]. Surgery was more often a concern of patients with perianal Crohn's disease [OR 2.1, 95% CI 1.2-3.5], active disease [OR 1.9, 95% CI 1.1-3.4], and those with recent hospitalisations [OR 2.5, 95% CI 1.2-5.4]. CONCLUSIONS: IBD patients prioritised the remission of physical symptoms as treatment goals and they were less frequently concerned about medications and their side effects. Patients' demographics, IBD characteristics, and health care utilisation patterns can predict specific types of concerns/goals.
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Colite Ulcerativa , Colite , Doenças Inflamatórias Intestinais , Feminino , Objetivos , Humanos , Doenças Inflamatórias Intestinais/terapia , Pessoa de Meia-Idade , Qualidade de VidaRESUMO
Five X-HxIP (Hx-amides) 6a-e, in which the N-terminus p-anisyl moiety is modified, were designed and synthesised with the purpose of optimising DNA binding, improving cellular uptake/nuclear penetration, and enhancing the modulation of the topoisomerase IIα (TOP2A) gene expression. The modifications include a fluorophenyl group and other heterocycles bearing different molecular shapes, size, and polarity. Like their parent compound HxIP 3, all five X-HxIP analogues bind preferentially to their cognate sequence 5'-TACGAT-3', which is found embedded on the 5' flank of the inverted CCAAT box-2 (ICB2) site in the TOP2A gene promoter, and inhibit protein complex binding. Interestingly, the 4-pyridyl analog 6a exhibits greater binding affinity for the target DNA sequence and abolishes the protein:ICB2 interaction in vitro, at a lower concentration, compared to the prototypical compound HxIP 3. Analogues 6b-e, display improved DNA sequence specificity, but reduced binding affinity for the cognate sequence, relative to the unmodified HxIP 3, with polyamides 6b and 6e being the most sequence selective. However, unlike 3 and 6b, 6a was unable to enter cells, access the nucleus and thereby affect TOP2A gene expression in confluent human lung cancer cells. These results show that while DNA binding affinity and sequence selectivity are important, consideration of cellular uptake and concentration in the nucleus are critical when exerting biological activity is the desired outcome. By characterising the DNA binding, cellular uptake and gene regulatory properties of these small molecules, we can elucidate the determinants of the elicited biological activity, which can be impacted by even small structural modifications in the polyamide molecular design.
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Amidas/farmacologia , DNA Topoisomerases Tipo II/genética , DNA de Neoplasias/efeitos dos fármacos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Amidas/síntese química , Amidas/química , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Relação Estrutura-AtividadeRESUMO
Recent data have demonstrated >80% 1-year survival probability after liver transplantation (LT) for patients with severe acute-on-chronic liver failure (ACLF). However, longterm outcomes and complications are still unknown for this population. Our aim was to compare longterm patient and graft survival among patients transplanted across all grades of ACLF. We analyzed the United Network for Organ Sharing database for the years 2004-2017. Patients with ACLF were identified using the European Association for the Study of the Liver-Chronic Liver Failure criteria. Kaplan-Meier and Cox regression methods were used to determine patient and graft survival and associated predictors of mortality in adjusted models. A total of 56,801 patients underwent transplantation of which 31,024 (54.6%) had no ACLF, 8757 (15.4%) had ACLF grade 1, 9039 (15.9%) had ACLF grade 2, and 7891 (14.1%) had ACLF grade 3. The 5-year patient survival after LT was lower in the ACLF grade 3 patients compared with the other groups (67.7%; P < 0.001), although after year 1, the percentage decrease in survival was similar among all groups. Infection was the primary cause of death among all patient groups in the first year. Infection was the primary cause of death among all patient groups in the first year. After the first year, infection was the main cause of death in patients transplanted with ACLF grade 1 (32.1%), ACLF grade 2 (33.9%), and ACLF grade 3 (37.6%), whereas malignancy was the predominant cause of death in those transplanted with no ACLF (28.5%). In conclusion, patients transplanted with ACLF grade 3 had lower 5-year survival as compared with patients with ACLF grades 0-2, but mortality rates were not significantly different after the first year following LT. Graft survival was excellent across all ACLF groups.
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Insuficiência Hepática Crônica Agudizada , Transplante de Fígado , Insuficiência Hepática Crônica Agudizada/diagnóstico , Insuficiência Hepática Crônica Agudizada/cirurgia , Bases de Dados Factuais , Sobrevivência de Enxerto , Humanos , Transplante de Fígado/efeitos adversos , Prognóstico , Estudos RetrospectivosRESUMO
Leigh syndrome (LS), the most common childhood mitochondrial disorder, has characteristic clinical and neuroradiologic features. Mutations in more than 75 genes have been identified in both the mitochondrial and nuclear genome, implicating a high degree of genetic heterogeneity in LS. To profile these genetic signatures and understand the pathophysiology of LS, we recruited 64 patients from 62 families who were clinically diagnosed with LS at Seoul National University Children's Hospital. Mitochondrial genetic analysis followed by whole-exome sequencing was performed on 61 patients. Pathogenic variants in mitochondrial DNA were identified in 18 families and nuclear DNA mutations in 22. The following 17 genes analyzed in 40 families were found to have genetic complexity: MTATP6, MTND1, MTND3, MTND5, MTND6, MTTK, NDUFS1, NDUFV1, NDUFAF6, SURF1, SLC19A3, ECHS1, PNPT1, IARS2, NARS2, VPS13D, and NAXE. Two treatable cases had biotin-thiamine responsive basal ganglia disease, and another three were identified as having defects in the newly recognized genes (VPS13D or NAXE). Variants in the nuclear genes that encoded mitochondrial aminoacyl tRNA synthetases were present in 27.3% of cases. Our findings expand the genetic and clinical spectrum of LS, showing genetic heterogeneity and highlighting treatable cases and those with novel genetic causes.
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Heterogeneidade Genética , Doença de Leigh/genética , Doenças Mitocondriais/genética , Proteínas/genética , Racemases e Epimerases/genética , Adolescente , Aminoacil-tRNA Sintetases/genética , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , DNA Mitocondrial/genética , Feminino , Testes Genéticos , Humanos , Lactente , Doença de Leigh/patologia , Masculino , Mitocôndrias/genética , Mitocôndrias/patologia , Doenças Mitocondriais/patologia , Mutação/genética , Linhagem , Sequenciamento do ExomaRESUMO
A substantial portion of Mendelian disease patients suffers from genetic variants that are inherited in a recessive manner. A precise understanding of pathogenic recessive variants in a population would assist in pre-screening births of such patients. However, a systematic understanding of the contribution of recessive variants to Mendelian diseases is still lacking. Therefore, genetic diagnosis and variant discovery of 553 undiagnosed Korean patients with complex neurodevelopmental problems (KND for Korean NeuroDevelopmental cohort) were performed using whole exome sequencing of patients and their parents. Disease-causing variants, including newly discovered variants, were identified in 57.5% of the probands of the KND cohort. Among the patients with the previous reported pathogenic variants, 35.1% inherited these variants in a recessive manner. Genes that cause recessive disorders in our cohort tend to be less constrained by loss-of-function variants and were enriched in lipid metabolism and mitochondrial functions. This observation was applied to an estimation that approximately 1 in 17 healthy Korean individuals carry at least one of these pathogenic variants that develop severe neurodevelopmental problems in a recessive manner. Furthermore, the feasibility of these genes for carrier screening was evaluated. Our results will serve as a foundation for recessive variant screening to reduce occurrences of rare Mendelian disease patients. Additionally, our results highlight the utility and necessity of whole exome sequencing-based diagnostics for improving patient care in a country with a centralized medical system.
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Genes Recessivos/genética , Triagem de Portadores Genéticos/métodos , Transtornos do Neurodesenvolvimento/genética , Adolescente , Criança , Pré-Escolar , Estudos de Viabilidade , Feminino , Triagem de Portadores Genéticos/estatística & dados numéricos , Humanos , Lactente , Recém-Nascido , Masculino , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/epidemiologia , República da Coreia/epidemiologia , Sequenciamento do Exoma , Adulto JovemRESUMO
Parkinson's disease (PD) is neurodegenerative movement disorder characterized by degeneration of midbrain-type dopamine (mDA) neurons in the substantia nigra (SN). The RNA-binding protein Lin28 plays a role in neuronal stem cell development and neuronal differentiation. In this study, we reveal that Lin28 conditional knockout (cKO) mice show degeneration of mDA neurons in the SN, as well as PD-related behavioral deficits. We identify a loss-of-function variant of LIN28A (R192G substitution) in two early-onset PD patients. Using an isogenic human embryonic stem cell (hESC)/human induced pluripotent stem cell (hiPSC)-based disease model, we find that the Lin28 R192G variant leads to developmental defects and PD-related phenotypes in mDA neuronal cells that can be rescued by expression of wild-type Lin28A. Cell transplantation experiments in PD model rats show that correction of the LIN28A variant in the donor patient (pt)-hiPSCs leads to improved behavioral phenotypes. Our data link LIN28A to PD pathogenesis and suggest future personalized medicine targeting this variant in patients.
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Doença de Parkinson/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/fisiologia , Substância Negra/metabolismo , Animais , Comportamento Animal , Transplante de Células , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/fisiologia , Células-Tronco Embrionárias/fisiologia , Edição de Genes , Predisposição Genética para Doença , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Camundongos , Camundongos Knockout , Mutação , Células-Tronco Neurais/fisiologia , Células-Tronco Neurais/transplante , Doença de Parkinson/genética , Ratos , Transplante de Células-TroncoRESUMO
Aim: This study investigated a coordinated strategy of revitalizing bone allograft with circulating multipotent stromal cells (MSCs). Materials & methods: After chemotactic and releasing assessments, stromal cell-derived factor 1 and platelet-derived growth factor BB in copolymers were coated on the bone allograft (AlloS-P). Allograft coated with copolymers alone (Allo), as controls, or AlloS-P was implanted into the femur of athymic mice, which received intravenous injections of human MSCs or saline at weeks 1, 2 and 3. Results: At week 8, the total callus volume (both cartilaginous and bony callus) around the allograft was the largest in the AlloS-P + MSC group (p < 0.05). Conclusion: Coating bone allograft with stromal cell-derived factor 1 and platelet-derived growth factor BB and intravenous injections of MSCs improved allograft incorporation.
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Transplante Ósseo , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Células-Tronco Multipotentes/citologia , Osteogênese , Células Estromais/citologia , Cicatrização , Administração Intravenosa , Aloenxertos , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos NusRESUMO
The regulation of osteogenesis is important for bone formation and fracture healing. Despite advances in understanding the molecular mechanisms of osteogenesis, crucial modulators in this process are not well-characterized. Here we demonstrate that suppression of signal transducer and activator of transcription 5A (STAT5A) activates distal-less homeobox 5 (DLX5) in human bone marrow-derived stromal cells (hBMSCs) and enhances osteogenesis in vitro and in vivo. We show that STAT5A negatively regulates expression of Dlx5 in vitro and that STAT5A deletion results in increased trabecular and cortical bone mass and bone mineral density in mice. Additionally, STAT5A deletion prevents age-related bone loss. In a murine fracture model, STAT5A deletion was found to significantly enhance bone remodeling by stimulating the formation of a fracture callus. Our findings indicate that STAT5A inhibition enhances bone formation by promoting osteogenesis of BMSCs.
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Fraturas Ósseas/genética , Proteínas de Homeodomínio/genética , Osteoblastos/metabolismo , Osteogênese/genética , Osteoporose/genética , Fator de Transcrição STAT5/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Densidade Óssea/genética , Diferenciação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Fêmur/lesões , Fêmur/metabolismo , Consolidação da Fratura/genética , Fraturas Ósseas/metabolismo , Fraturas Ósseas/patologia , Fraturas Ósseas/terapia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Humanos , Sialoproteína de Ligação à Integrina/genética , Sialoproteína de Ligação à Integrina/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Osteoblastos/citologia , Osteoporose/metabolismo , Osteoporose/patologia , Osteoporose/prevenção & controle , Cultura Primária de Células , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT5/antagonistas & inibidores , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismoRESUMO
DNA minor groove binding polyamides have been extensively developed to control abnormal gene expression. The establishment of novel, inherently fluorescent 2-(p-anisyl)benzimidazole (Hx) amides has provided an alternative path for studying DNA binding in cells by direct observation of cell localization. Because of the 2:1 antiparallel stacking homodimer binding mode of these molecules to DNA, modification of Hx amides to 2-(p-anisyl)-4-azabenzimidazole (AzaHx) amides has successfully extended the DNA-recognition repertoire from central CG [recognized by Hx-I (I=N-methylimidazole)] to central GC [recognized by AzaHx-P (P=N-methylpyrrole)] recognition. For potential targeting of two consecutive GG bases, modification of the AzaHx moiety to 2- and 3-pyridyl-aza-benzimidazole (Pyr-AzaHx) moieties was explored. The newly designed molecules are also small-sized, fluorescent amides with the Pyr-AzaHx moiety connected to two conventional five-membered heterocycles. Complementary biophysical methods were performed to investigate the DNA-binding properties of these molecules. The results showed that neither 3-Pyr-AzaHx nor 2-Pyr-AzaHx was able to mimic I-I=N-methylimidazole-N-methylimidazole to target GG dinucleotides specifically. Rather, 3-Pyr-AzaHx was found to function like AzaHx, f-I (f=formamide), or P-I as an antiparallel stacked dimer. 3-Pyr-AzaHx-PI (2) binds 5'-ACGCGT'-3' with improved binding affinity and high sequence specificity in comparison to its parent molecule AzaHx-PI (1). However, 2-Pyr-AzaHx is detrimental to DNA binding because of an unfavorable steric clash upon stacking in the minor groove.
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Benzimidazóis/química , DNA/química , Corantes Fluorescentes/química , Nylons/química , Pirróis/química , Sequência de Bases , Benzimidazóis/metabolismo , Sítios de Ligação , Dicroísmo Circular , DNA/metabolismo , Corantes Fluorescentes/metabolismo , Conformação de Ácido Nucleico , Nylons/metabolismo , Pirróis/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
OBJECTIVE: The aim of this study was to compare the effects of combined virtual monoenergetic extrapolation (VME) of dual-energy computed tomography data and iterative metal artifact reduction (iMAR) at higher photon energies on low- and high-density metal artifacts and overall image quality of the ankle arthroplasty implants with iMAR, weighted filtered back projection (WFBP), and WFBP-based VME. MATERIALS AND METHODS: Total ankle arthroplasty implants in 6 human cadaver ankles served as surrogates for arthroplasty implants. All specimens underwent computed tomography with a 2 × 192-slice dual-source computed tomography scanner at tube voltages of 80 and tin-filtered 150 kVp to produce mixed 120 kVp equivalent polychromatic and virtual monoenergetic extrapolated images at 150 and 190 keV (VME 150 and VME 190, respectively). By implementing the WFBP and iMAR reconstruction algorithms on polychromatic, VME 150 and VME 190 data, 6 image datasets were created: WFBP-Polychromatic, iMAR-Polychromatic, WFBP-VME 150, WFBP-VME 190, iMAR-VME 150, and iMAR-VME 190. High-density and low-density artifacts were separately quantified with a threshold-based computer algorithm. After anonymization and randomization, 2 observers independently ranked the datasets for overall image quality. Repeated measures analysis of variance, Friedman, and Cohen weighted κ tests were applied for statistical analysis. A conservative P value of less than 0.001 was considered statistically significant. RESULTS: iMAR-VME 190 keV and iMAR-VME 150 keV created the least amount of high-density artifacts (all P < 0.001), whereas iMAR-Polychromatic was the most effective method to mitigate low-density streaks (P < 0.001). For low- and high-density artifacts, polychromatic iMAR acquisition was superior to WFBP-VME 150 keV and WFBP-VME 190 keV (all P < 0.001). On sharp kernel reconstructions, readers ranked the overall image quality of iMAR-Polychromatic images highest (all P < 0.001). Similarly, on soft tissue kernel reconstructions, readers ranked iMAR-Polychromatic images highest with a statistically significant difference over other techniques (all P < 0.001), except for iMAR-VME 150 keV (P = 0.356). CONCLUSIONS: In computed tomography imaging of ankle arthroplasty implants, iMAR reconstruction results in fewer metal artifacts and better image quality than WFBP reconstruction for both polychromatic and virtual monoenergetic data. The combination of iMAR and VME at higher photon energies results in mixed effects on implant-induced metal artifacts, including decreased high-density and increased low-density artifacts, which in combination does not improve image quality over iMAR reconstruction of the polychromatic data. Our results suggest that, for ankle arthroplasty implants, the highest image quality is obtained by iMAR reconstruction of the polychromatic data without the need to implement VME at high-energy levels.
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Artroplastia de Substituição do Tornozelo/instrumentação , Artefatos , Processamento de Imagem Assistida por Computador/métodos , Metais , Próteses e Implantes , Tomografia Computadorizada por Raios X/métodos , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Articulação do Tornozelo/diagnóstico por imagem , Cadáver , Feminino , Humanos , Interpretação de Imagem Assistida por Computador , Masculino , FótonsRESUMO
Objectives: Blood stage malaria parasites attenuated with seco-cyclopropyl pyrrolo indole (CPI) analogues induce robust immunity in mice to homologous and heterologous malaria parasites and are being considered for the development of a human vaccine. However, it is not understood how attenuated parasites induce immunity. We showed that following vaccination, parasite DNA persisted in blood for several months, raising the possibility that ongoing immune stimulation may be critical. However, parasites were not seen microscopically beyond 24 h postvaccination. We aimed to provide a mechanistic understanding of immune induction. Methods: Mice were vaccinated with chemically attenuated Plasmodium chabaudi parasites. PCR and adoptive transfer studies were used to determine the presence of parasites and antigen in vivo. In other experiments, Plasmodium falciparum parasitised red blood cells were attenuated in vitro and RNA and antigen expression studied. Results: We show that blood transferred from vaccinated mice into naïve mice activates T cells and induces complete protective immunity in the recipient mice strongly suggesting that there is persistence of parasite antigen postvaccination. This is supported by the presence of parasite RNA in vaccinated mice and both RNA and antigen expression in P. falciparum cultures treated with CPI drugs in vitro. In addition, drugs that block parasite growth also prevent the induction of immunity in vaccinated mice, indicating that some growth of attenuated parasites is required for immune induction. Conclusions: Attenuated parasites persist at submicroscopic levels in the blood of mice postvaccination with the ability to activate T cells and induce ongoing protective immune responses.
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HxTfA 4 is a fluorescent analog of a potent cytotoxic and antimalarial agent, TfA 3, which is currently being investigated for the development of an antimalarial vaccine, PlasProtect®. HxTfA contains a p-anisylbenzimidazole or Hx moiety, which is endowed with a blue emission upon excitation at 318â¯nm; thus enabling it to be used as a surrogate for probing the cellular fate of TfA using confocal microscopy, and addressing the question of nuclear localization. HxTfA exhibits similar selectivity to TfA for A-tract sequences of DNA, alkylating adenine-N3, albeit at 10-fold higher concentrations. It also possesses in vitro cytotoxicity against A549 human lung carcinoma cells and Plasmodium falciparum. Confocal microscopy studies showed for the first time that HxTfA, and by inference TfA, entered A549 cells and localized in the nucleus to exert its biological activity. At biologically relevant concentrations, HxTfA elicits DNA damage response as evidenced by a marked increase in the levels of γH2AX observed by confocal microscopy and immunoblotting studies, and ultimately induces apoptosis.
Assuntos
Antimaláricos/farmacologia , Benzimidazóis/farmacologia , Núcleo Celular/metabolismo , DNA/química , Corantes Fluorescentes/farmacologia , Indóis/farmacologia , Células A549 , Alquilantes/síntese química , Alquilantes/metabolismo , Alquilantes/farmacologia , Alquilantes/toxicidade , Antimaláricos/síntese química , Antimaláricos/metabolismo , Antimaláricos/toxicidade , Apoptose/efeitos dos fármacos , Sequência de Bases , Benzimidazóis/síntese química , Benzimidazóis/metabolismo , Benzimidazóis/toxicidade , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Humanos , Indóis/síntese química , Indóis/metabolismo , Indóis/toxicidade , Plasmodium falciparum/efeitos dos fármacosRESUMO
BACKGROUND: GM1 gangliosidosis is a rare lysosomal storage disorder caused by GLB1 mutations. Because of its extreme rarity and symptoms that overlap with other neurodegenerative diseases, its diagnosis is sometimes challenging, especially in the late infantile form with less severe phenotype. We aim to expand the clinical and genetic spectrum of late infantile GM1 gangliosidosis. METHODS: We confirmed a diagnosis of GM1 gangliosidosis based on GLB1 mutations and/or the deficiency of ß-galactosidase activity. We identified the first two cases by whole-exome sequencing, and then the other six cases by direct sequencing of GLB1 with enzyme analysis. RESULTS: All eight patients presented with developmental delay or regression during late infancy and later developed epilepsy, mostly intractable generalized tonic seizures. No clinical signs of storage disorders were noted except for skeletal abnormalities. Interestingly, we found aspartate transaminase (AST) elevations alone with normal alanine transaminase (ALT) levels in all patients. The recurrent mutation, p.D448V in GLB1, accounted for 50.0% of total alleles in our cohort. CONCLUSIONS: With a high index of clinical suspicion, skeletal survey and AST level would be important for early diagnosis of GM1 gangliosidosis. In addition, we would highlight the clinical usefulness of whole-exome sequencing in the diagnosis of non-classical presentation of ultra-rare neurodegenerative disease in children.