RESUMO
We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals.
Assuntos
Tubulina (Proteína)/metabolismo , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Encéfalo/embriologia , Encéfalo/metabolismo , Sobrevivência Celular , Criança , Deficiências do Desenvolvimento , Feminino , Humanos , Cinesinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Transporte Proteico , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMO
OBJECTIVE: To determine any relationship of diet with breast cancer risk. METHODS: The case-control study was conducted at Aga Khan University Hospital and the Karachi Institute of Radiation and Nuclear Medicine, Karachi, from February 2015 to July 2017, and comprised of cases with a confirmed diagnosis of breast cancer and healthy controls. Data was collected using the Alternate Healthy Eating Index 2010, which was modified according to the particular cultural patterns of diet in the Pakistani population. Data was analysed using SPSS 21. RESULTS: Of the 1124 subjects, 374(33.3%) were breast cancer cases and 750(66.7%) were controls. High intake of grains, both whole and refined including white rice, was associated with breast cancer (odds ratio: 2.53; 95% confidence interval: 1.69-3.79; p<0.001). There was no association of breast cancer with Alternate Healthy Eating Index 2010 score (odds ratio: 1.85; 95% confidence interval: 0.61-1.17; p=0.291). CONCLUSIONS: There was found a need for awareness of a healthy diet based on more of whole grains and brown rice replacement with refined grains and white rice, respectively. Limiting refined carbohydrate intake might be a useful public health message and may reduce breast cancer incidence in the long term.
Assuntos
Neoplasias da Mama , Dieta Saudável , Neoplasias da Mama/epidemiologia , Carboidratos , Estudos de Casos e Controles , Dieta , Feminino , Humanos , Paquistão/epidemiologia , Fatores de RiscoRESUMO
BACKGROUND: Vitamin D deficiency is becoming a serious public health problem, even in sun-drenched cities like Karachi, Pakistan. We investigated the prevalence of vitamin D deficiency and its association with sociodemographic characteristics, anthropometric measures, and lifestyle factors among premenopausal and postmenopausal women (n = 784). METHODS: Face-to-face interviews were conducted to collect information and serum concentrations of 25-hydroxyvitamin D were measured after the interviews. RESULTS: A total of 57% of women were vitamin D deficient with higher vitamin D deficiency found among premenopausal women (64.7%) compared to postmenopausal women (49%). The median serum concentrations of 25-hydroxyvitamin D (IQR) were 16.7 ng/ml (IQR 9.8-30.0). Factors associated with vitamin D deficiency were lower socioeconomic status (OR 2.00; 95% CI 1.15-3.48), younger age with highest vitamin D deficiency found in < 35 years of age group (OR 3.11; 95% CI 1.76-5.51), and winter season (OR 1.51, 95% CI 1.07-2.15) after adjusting for multiple confounders. The use of vitamin D supplement (OR 0.59, 95% CI 0.38-0.92) and vigorous exercise (OR 0.20, 95% CI 0.05-0.80) were protective against vitamin D deficiency. CONCLUSIONS: The study shows a high prevalence of vitamin D deficiency, with detrimental health effects, among younger women belonging to lower socioeconomic status and during the winter season. The use of vitamin D supplements and vigorous exercise were protective measures. Public health campaigns are needed for education and awareness about vitamin D deficiency to improve vitamin D status for younger women living in poor environments.
Assuntos
Pós-Menopausa , Deficiência de Vitamina D , Adulto , Suplementos Nutricionais , Feminino , Humanos , Paquistão/epidemiologia , Pré-Menopausa , Prevalência , Estações do Ano , Vitamina D , Deficiência de Vitamina D/epidemiologiaRESUMO
BACKGROUND: There are no studies done to evaluate the distribution of mammographic breast density and factors associated with it among Pakistani women. METHODS: Participants included 477 women, who had received either diagnostic or screening mammography at two hospitals in Karachi Pakistan. Mammographic breast density was assessed using the Breast Imaging Reporting and Data System. In person interviews were conducted using a detailed questionnaire, to assess risk factors of interest, and venous blood was collected to measure serum vitamin D level at the end of the interview. To determine the association of potential factors with mammographic breast density, multivariable polytomous logistic regression was used. RESULTS: High-density mammographic breast density (heterogeneously and dense categories) was high and found in 62.4% of women. There was a significant association of both heterogeneously dense and dense breasts with women of a younger age group < 45 years (OR 2.68, 95% CI 1.60-4.49) and (OR 4.83, 95% CI 2.54-9.16) respectively. Women with heterogeneously dense and dense breasts versus fatty and fibroglandular breasts had a higher history of benign breast disease (OR 1.90, 95% CI 1.14-3.17) and (OR 3.61, 95% CI 1.90-6.86) respectively. There was an inverse relationship between breast density and body mass index. Women with dense breasts and heterogeneously dense breasts had lower body mass index (OR 0.94 95% CI 0.90-0.99) and (OR 0.81, 95% CI 0.76-0.87) respectively. There was no association of mammographic breast density with serum vitamin D levels, diet, and breast cancer. CONCLUSIONS: The findings of a positive association of higher mammographic density with younger age and benign breast disease and a negative association between body mass index and breast density are important findings that need to be considered in developing screening guidelines for the Pakistani population.
Assuntos
Densidade da Mama , Neoplasias da Mama , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/epidemiologia , Detecção Precoce de Câncer , Feminino , Humanos , Mamografia , Pessoa de Meia-Idade , Paquistão , Fatores de RiscoRESUMO
BACKGROUND: In 2013, a task force was developed to discuss the future of the Canadian pediatric neurology workforce. The consensus was that there was no indication to reduce the number of training positions, but that the issue required continued surveillance. The current study provides a 5-year update on Canadian pediatric neurology workforce data. METHODS: Names, practice types, number of weekly outpatient clinics, and dates of certification of all physicians currently practicing pediatric neurology in Canada were obtained. International data were used to compute comparisons between countries. National data sets were used to provide information about the number of residency positions available and the number of Canadian graduates per year. Models for future projections were developed based on published projected population data and trends from the past decade. RESULTS: The number of pediatric neurologists practicing in Canada has increased 165% since 1994. During this period, wait times have not significantly shortened. There are regional discrepancies in access to child neurologists. The Canadian pediatric neurology workforce available to see outpatient consultations is proportionally less than that of USA. After accounting for retirements and emigrations, the number of child neurologists being added to the workforce each year is 4.9. This will result in an expected 10-year increase in Canadian pediatric neurologists from 151 to 200. CONCLUSIONS: Despite an increase in the number of Canadian child neurologists over the last two decades, we do not predict that there will be problems with underemployment over the next decade.
Les effectifs en neurologie pédiatrique au Canada : une mise à jour tenant compte des cinq dernières années.Contexte:En 2013, un comité de réflexion a été mis sur pied afin de discuter de l'avenir des effectifs canadiens en neurologie pédiatrique. Un consensus s'est alors dessiné : bien que rien n'indique qu'il faille réduire le nombre de places disponibles dans ce domaine de formation, cette question nécessite toutefois un suivi continu. La présente étude vise ainsi à offrir une mise à jour des données qui concernent ces effectifs en tenant compte des cinq dernières années.Méthodes:Les données suivantes ont été obtenues : noms des praticiens, types de pratique, nombre de consultations hebdomadaires en clinique externe et dates de certification de tous les médecins pratiquant actuellement la neurologie pédiatrique au Canada. Des données internationales ont également été utilisées pour effectuer des comparaisons entre divers pays. Des données au niveau national ont été rassemblées pour établir le nombre de places disponibles en résidence et le nombre de diplômés canadiens par année. Des modèles de projection ont aussi été élaborés en se fondant sur des projections de population déjà publiées et sur les tendances ayant marqué la décennie précédente.Résultats:Le nombre de neurologues pédiatriques qui pratiquent au Canada a augmenté de 165 % depuis 1994. Fait à noter, les temps d'attente n'ont pas été notablement raccourcis durant cette période. Il existe aussi des disparités régionales en matière d'accès à cette spécialité de la neurologie. Les effectifs canadiens en neurologie pédiatrique disponibles pour des consultations en clinique externe demeurent proportionnellement moins importants qu'aux États-Unis. Après avoir tenu compte des départs à la retraite et des apports de l'extérieur, le nombre de neurologues pédiatriques ajoutés chaque année a été en moyenne de 4,9. Pendant une période de 10 ans, on anticipe donc qu'on devrait passer de 151 à 200 neurologues pédiatriques canadiens.Conclusions:En dépit d'une hausse du nombre de neurologues pédiatriques canadiens au cours des deux dernières décennies, nous ne prédisons pas de problèmes de sous-emploi au cours de la prochaine décennie.
Assuntos
Mão de Obra em Saúde/estatística & dados numéricos , Neurologistas/provisão & distribuição , Pediatras/provisão & distribuição , Canadá , Humanos , Neurologia/estatística & dados numéricos , Pediatria/estatística & dados numéricosRESUMO
Deep-sequencing reveals extensive variation in the sequence of endogenously expressed microRNAs (termed 'isomiRs') in human cell lines and tissues, especially in relation to the 3' end. From the immunoprecipitation of the microRNA-binding protein Argonaute and the sequencing of associated small RNAs, we observe extensive 3'-isomiR variation, including for miR-222 where the majority of endogenously expressed miR-222 is extended by 1-5 nt compared to the canonical sequence. We demonstrate this 3' heterogeneity has dramatic implications for the phenotype of miR-222 transfected cells, with longer isoforms promoting apoptosis in a size (but not 3' sequence)-dependent manner. The transfection of longer miR-222 isomiRs did not induce an interferon response, but did downregulate the expression of many components of the pro-survival PI3K-AKT pathway including PIK3R3, a regulatory subunit whose knockdown phenocopied the expression of longer 222 isoforms in terms of apoptosis and the inhibition of other PI3K-AKT genes. As this work demonstrates the capacity for 3' isomiRs to mediate differential functions, we contend more attention needs to be given to 3' variance given the prevalence of this class of isomiR.
Assuntos
Apoptose/genética , Proliferação de Células/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Isoformas de RNA/genética , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Immunoblotting , Células MCF-7 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genéticaRESUMO
PURPOSE: The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data. METHODS: We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry. RESULTS: There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent. CONCLUSIONS: This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.
Assuntos
Genes da Neurofibromatose 1 , Neurofibromatose 1/diagnóstico , Neurofibromatose 1/genética , Adulto , Idoso , Biomarcadores Tumorais , Análise Mutacional de DNA , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Estadiamento de Neoplasias , Neurofibromatose 1/epidemiologia , Neurofibromatose 1/mortalidadeRESUMO
A series of azobenzene-containing peptidic boronate esters was prepared and the activity of the thermally adapted states (TAS), enriched in trans isomer, and the photostationary states (PSS), enriched in cis isomer, for each compound were evaluated against ß5 and ß1 proteasome subunits. Compounds with a sterically demanding phenyl-substituted azobenzene at P2 (4c), and a less sterically demanding unsubstituted azobenzene at the N-terminus (5a), showed the greatest difference in activity between the two states. In both cases, the more active trans-enriched TAS had activity comparable to bortezomib and delanzomib. Furthermore, cis-enriched 4c inhibited tumor growth in both breast and colorectal carcinoma cell lines. Significantly, the initial trans-enriched TAS of 4c was not cytotoxic against the non-malignant MCF-10A cells.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos Azo/química , Compostos Azo/farmacologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Isomerismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Processos Fotoquímicos , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
BACKGROUND: Little knowledge exists on the availability of academic and community paediatric neurology positions. This knowledge is crucial for making workforce decisions. Our study aimed to: 1) obtain information regarding the availability of positions for paediatric neurologists in academic centres; 2) survey paediatric neurology trainees regarding their perceptions of employment issues and career plans; 3) survey practicing community paediatric neurologists 4) convene a group of paediatric neurologists to develop consensus regarding how to address these workforce issues. METHODS: Surveys addressing workforce issues regarding paediatric neurology in Canada were sent to: 1) all paediatric neurology program directors in Canada (n=9) who then solicited information from division heads and from paediatric neurologists in surrounding areas; 2) paediatric neurology trainees in Canada (n=57) and; 3) community paediatric neurologists (n=27). A meeting was held with relevant stakeholders to develop a consensus on how to approach employment issues. RESULTS: The response rate was 100% from program directors, 57.9% from residents and 44% from community paediatric neurologists. We found that the number of projected positions in academic paediatric neurology is fewer than the number of paediatric neurologists that are being trained over the next five to ten years, despite a clinical need for paediatric neurologists. Paediatric neurology residents are concerned about job availability and desire more career counselling. CONCLUSIONS: There is a current and projected clinical demand for paediatric neurologists despite a lack of academic positions. Training programs should focus on community neurology as a viable career option.
Assuntos
Consenso , Neurologia , Pediatras/provisão & distribuição , Canadá , Feminino , Humanos , Masculino , Inquéritos e Questionários , Recursos HumanosRESUMO
FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Proteínas F-Box/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Fase G2 , Humanos , Ligação Proteica , Proteólise , Proteínas Supressoras de Tumor/genética , UbiquitinaçãoRESUMO
Mutations in ANKRD11 have recently been reported to cause KBG syndrome, an autosomal dominant condition characterized by intellectual disability (ID), behavioral problems, and macrodontia. To understand the pathogenic mechanism that relates ANKRD11 mutations with the phenotype of KBG syndrome, we studied the cellular characteristics of wild-type ANKRD11 and the effects of mutations in humans and mice. We show that the abundance of wild-type ANKRD11 is tightly regulated during the cell cycle, and that the ANKRD11 C-terminus is required for the degradation of the protein. Analysis of 11 pathogenic ANKRD11 variants in humans, including six reported in this study, and one reported in the Ankrd11 (Yod/+) mouse, shows that all mutations affect the C-terminal regions and that the mutant proteins accumulate aberrantly. In silico analysis shows the presence of D-box sequences that are signals for proteasome degradation. We suggest that ANKRD11 C-terminus plays an important role in regulating the abundance of the protein, and a disturbance of the protein abundance due to the mutations leads to KBG syndrome.
Assuntos
Anormalidades Múltiplas , Doenças do Desenvolvimento Ósseo , Ciclo Celular/genética , Proteínas de Ligação a DNA , Fácies , Deficiência Intelectual , Mutação , Proteólise , Proteínas Repressoras , Anormalidades Dentárias , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Animais , Doenças do Desenvolvimento Ósseo/genética , Doenças do Desenvolvimento Ósseo/metabolismo , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Masculino , Camundongos , Camundongos Mutantes , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Anormalidades Dentárias/genética , Anormalidades Dentárias/metabolismoRESUMO
OBJECTIVE: Epilepsy is a common neurologic disorder of childhood. To determine the genetic diagnostic yield in epileptic encephalopathy, we performed a retrospective cohort study in a single epilepsy genetics clinic. METHODS: We included all patients with intractable epilepsy, global developmental delay, and cognitive dysfunction seen between January 2012 and June 2014 in the Epilepsy Genetics Clinic. Electronic patient charts were reviewed for clinical features, neuroimaging, biochemical investigations, and molecular genetic investigations including targeted next-generation sequencing of epileptic encephalopathy genes. RESULTS: Genetic causes were identified in 28% of the 110 patients: 7% had inherited metabolic disorders including pyridoxine dependent epilepsy caused by ALDH7A1 mutation, Menkes disease, pyridox(am)ine-5-phosphate oxidase deficiency, cobalamin G deficiency, methylenetetrahydrofolate reductase deficiency, glucose transporter 1 deficiency, glycine encephalopathy, and pyruvate dehydrogenase complex deficiency; 21% had other genetic causes including genetic syndromes, pathogenic copy number variants on array comparative genomic hybridization, and epileptic encephalopathy related to mutations in the SCN1A, SCN2A, SCN8A, KCNQ2, STXBP1, PCDH19, and SLC9A6 genes. Forty-five percent of patients obtained a genetic diagnosis by targeted next-generation sequencing epileptic encephalopathy panels. It is notable that 4.5% of patients had a treatable inherited metabolic disease. SIGNIFICANCE: To the best of our knowledge, this is the first study to combine inherited metabolic disorders and other genetic causes of epileptic encephalopathy. Targeted next-generation sequencing panels increased the genetic diagnostic yield from <10% to >25% in patients with epileptic encephalopathy.
Assuntos
Predisposição Genética para Doença , Mutação/genética , Espasmos Infantis/diagnóstico , Espasmos Infantis/genética , Adolescente , Caderinas , Criança , Pré-Escolar , Transtornos Cognitivos/complicações , Transtornos Cognitivos/genética , Estudos de Coortes , Deficiências do Desenvolvimento/complicações , Deficiências do Desenvolvimento/genética , Feminino , Humanos , Lactente , Canal de Potássio KCNQ2 , Masculino , Proteínas Munc18 , Canal de Sódio Disparado por Voltagem NAV1.2 , Protocaderinas , Trocadores de Sódio-Hidrogênio , Espasmos Infantis/complicaçõesRESUMO
Autosomal dominant mutations in the sodium-gated potassium channel subunit gene KCNT1 have been associated with two distinct seizure syndromes, nocturnal frontal lobe epilepsy (NFLE) and malignant migrating focal seizures of infancy (MMFSI). To further explore the phenotypic spectrum associated with KCNT1, we examined individuals affected with focal epilepsy or an epileptic encephalopathy for mutations in the gene. We identified KCNT1 mutations in 12 previously unreported patients with focal epilepsy, multifocal epilepsy, cardiac arrhythmia, and in a family with sudden unexpected death in epilepsy (SUDEP), in addition to patients with NFLE and MMFSI. In contrast to the 100% penetrance so far reported for KCNT1 mutations, we observed incomplete penetrance. It is notable that we report that the one KCNT1 mutation, p.Arg398Gln, can lead to either of the two distinct phenotypes, ADNFLE or MMFSI, even within the same family. This indicates that genotype-phenotype relationships for KCNT1 mutations are not straightforward. We demonstrate that KCNT1 mutations are highly pleiotropic and are associated with phenotypes other than ADNFLE and MMFSI. KCNT1 mutations are now associated with Ohtahara syndrome, MMFSI, and nocturnal focal epilepsy. They may also be associated with multifocal epilepsy and cardiac disturbances.
Assuntos
Epilepsias Parciais/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Canais de Potássio/genética , Adolescente , Idade de Início , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Canais de Potássio Ativados por Sódio , Morte Súbita do Lactente/genéticaRESUMO
Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole-containing macrocyclic protease inhibitors pre-organized into a ß-strand conformation and an evaluation of their activity against a panel of proteases. Acyclic azido-alkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpainâ II, cathepsinâ L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent macrocyclic inhibitors of cathepsinâ S were identified. These adopt a well-defined ß-strand geometry as shown by NMR spectroscopy, X-ray analysis, and molecular docking studies.
Assuntos
Compostos Macrocíclicos/síntese química , Peptídeos/química , Inibidores de Proteases/síntese química , Triazóis/síntese química , Calpaína/antagonistas & inibidores , Catepsina L/antagonistas & inibidores , Química Click , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Conformação Molecular , Ressonância Magnética Nuclear Biomolecular , Peptidomiméticos , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Triazóis/química , Triazóis/farmacologiaRESUMO
BACKGROUND: Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. METHODS: A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. RESULTS: Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. CONCLUSIONS: The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/análogos & derivados , Mama/citologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas Luminescentes/metabolismo , Nitrorredutases/metabolismo , Antineoplásicos/farmacologia , Azacitidina/farmacologia , Proteínas de Transporte/genética , Células Cultivadas , Citomegalovirus/genética , Metilação de DNA , Decitabina , Epigênese Genética , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos , Humanos , Ácidos Hidroxâmicos/farmacologia , Proteínas Luminescentes/genética , Nitrorredutases/genética , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Lipossomas Unilamelares , Vorinostat , Proteína Vermelha FluorescenteRESUMO
A significant proportion of transcription factors encoded by the human genome are classical C(2) H(2) zinc finger proteins that regulate gene expression by directly interacting with their cognate DNA binding motifs. We previously showed that one such C(2) H(2) zinc finger DNA binding protein, ZNF652 (zinc finger protein 652), specifically and functionally interacts with CBFA2T3 to repress transcription of genes involved in breast oncogenesis. To identify potential targets by which ZNF652 exerts its putative tumour suppressive function, its promoter-specific cistrome was mapped by ChIP-chip. De novo motif scanning of the ZNF652 binding sites identified a novel ZNF652 recognition motif that closely resembles the previously characterised in vitro binding site, being a 10 nucleotide core of that 13 nucleotide sequence. Genes with ZNF652 binding sites function in diverse cellular pathways, and many are involved in cancer development and progression. Characterisation of the in vivo ZNF652 DNA binding motif and identification of potential ZNF652 target genes are key steps towards elucidating the function(s) of this transcription factor in the normal and malignant breast cell.
Assuntos
Neoplasias da Mama/genética , Mapeamento Cromossômico/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Humanos , Hidroliases/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The p53 tumour suppressor plays a pivotal role in the prevention of oncogenic transformation. Cancers frequently evade the potent antitumour surveillance mechanisms of p53 through mutation of the TP53 gene, with approximately 50% of all human malignancies expressing dysfunctional, mutated p53 proteins. Interestingly, genetic lesions in the TP53 gene are only observed in 10% of Ewing Sarcomas, with the majority of these sarcomas expressing a functional wild-type p53. In addition, the p53 downstream signaling pathways and DNA-damage cell cycle checkpoints remain functionally intact in these sarcomas. This paper summarizes recent insights into the functional capabilities and regulation of p53 in Ewing Sarcoma, with a particular focus on the cross-talk between p53 and the EWS-FLI1 gene rearrangement frequently associated with this disease. The development of several activators of p53 is discussed, with recent evidence demonstrating the potential of small molecule p53 activators as a promising systemic therapeutic approach for the treatment of Ewing Sarcomas with wild-type p53.
RESUMO
We report two rare genetic aberrations in a schizophrenia patient that may act together to confer disease susceptibility. A previously unreported balanced t(9;17)(q33.2;q25.3) translocation was observed in two schizophrenia-affected members of a small family with diverse psychiatric disorders. The proband also carried a 1.5 Mbp microduplication at 16p13.1 that could not be investigated in other family members. The duplication has been reported to predispose to schizophrenia, autism and mental retardation, with incomplete penetrance and variable expressivity. The t(9;17) (q33.2;q25.3) translocation breakpoint occurs within the open reading frames of KIAA1618 on 17q25.3, and TTLL11 (tyrosine tubulin ligase like 11) on 9q33.2, causing no change in the expression level of KIAA1618 but leading to loss of expression of one TTLL11 allele. TTLL11 belongs to a family of enzymes catalyzing polyglutamylation, an unusual neuron-specific post-translational modification of microtubule proteins, which modulates microtubule development and dynamics. The 16p13.1 duplication resulted in increased expression of NDE1, encoding a DISC1 protein partner mediating DISC1 functions in microtubule dynamics. We hypothesize that concomitant TTLL11-NDE1 deregulation may increase mutation load, among others, also on the DISC1 pathway, which could contribute to disease pathogenesis through multiple effects on neuronal development, synaptic plasticity, and neurotransmission. Our data illustrate the difficulties in interpreting the contribution of multiple potentially pathogenic changes likely to emerge in future next-generation sequencing studies, where access to extended families will be increasingly important.
Assuntos
Cromossomos Humanos Par 16 , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 9/genética , Esquizofrenia/genética , Duplicações Segmentares Genômicas , Translocação Genética , Adulto , Alelos , Família , Humanos , Masculino , Mutação/genética , Proteínas do Tecido Nervoso/genética , Linhagem , Processamento de Proteína Pós-Traducional , Esquizofrenia/metabolismo , Esquizofrenia/patologiaRESUMO
We report on the role of PAMAM dendrimer concentration and generation (G2, G4, G6) on cell growth and cytotoxicity in HEK293T and HeLa cell lines and make comparisons with dendrimer-induced leakage from liposomes to probe the mechanisms in action. Specifically, we observed a striking transition from cell growth enhancement to a reduction in cell viability at a critical PAMAM dendrimer concentration, that is, approximately 500 nM. Confocal microscopy studies show evidence of a transition from cell membrane adhesion to cell internalization and cell nucleus interaction at equivalent dendrimer concentrations. A dendrimer concentration window of 500-700 nM was identified for effective cell internalization without significant cytotoxicity. Though liposome leakage correlated with cytotoxicity, no quantitative agreement was observed, that is, cells are 100 times (based on surface coverage) more resistant to dendrimers than liposomes. These findings have significant implications in the design of effective drug/gene delivery vehicles based on dendrimers.
Assuntos
Materiais Biocompatíveis/toxicidade , Proliferação de Células/efeitos dos fármacos , Citotoxinas/toxicidade , Dendrímeros/toxicidade , Materiais Biocompatíveis/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Citotoxinas/metabolismo , Dendrímeros/metabolismo , Células HeLa , Humanos , Lipossomos/metabolismoRESUMO
Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.