RESUMO
During central nervous system development, neural progenitors are patterned to form discrete neurogenic and non-neurogenic zones. In the zebrafish hindbrain, neurogenesis is organised by Fgf20a emanating from neurons located at each segment centre that inhibits neuronal differentiation in adjacent progenitors. Here, we have identified a molecular mechanism that clusters fgf20a-expressing neurons in segment centres and uncovered a requirement for this positioning in the regulation of neurogenesis. Disruption of hindbrain boundary cell formation alters the organisation of fgf20a-expressing neurons, consistent with a role of chemorepulsion from boundaries. The semaphorins Sema3fb and Sema3gb, which are expressed by boundary cells, and their receptor Nrp2a are required for clustering of fgf20a-expressing neurons at segment centres. The dispersal of fgf20a-expressing neurons that occurs following the disruption of boundaries or of Sema3fb/Sema3gb signalling leads to reduced FGF target gene expression in progenitors and an increased number of differentiating neurons. Sema3 signalling from boundaries thus links hindbrain segmentation to the positioning of fgf20a-expressing neurons that regulates neurogenesis.
Assuntos
Neurogênese/fisiologia , Rombencéfalo/embriologia , Peixe-Zebra/embriologia , Animais , Sequência de Bases , Padronização Corporal/genética , Padronização Corporal/fisiologia , Primers do DNA/genética , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Modelos Neurológicos , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Neuropilina-2/antagonistas & inibidores , Neuropilina-2/genética , Neuropilina-2/metabolismo , Rombencéfalo/metabolismo , Semaforinas/antagonistas & inibidores , Semaforinas/genética , Semaforinas/metabolismo , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Patient experience data (PED), provided by patients/their carers without interpretation by clinicians, directly capture what matters more to patients on their medical condition, treatment and impact of healthcare. PED can be collected through different methodologies and these need to be robust and validated for its intended use. Medicine regulators are increasingly encouraging stakeholders to generate, collect and submit PED to support both scientific advice in development programs and regulatory decisions on the approval and use of these medicines. This article reviews the existing definitions and types of PED and demonstrate the potential for use in different settings of medicines' life cycle, focusing on Patient-Reported Outcomes (PRO) and Patient Preferences (PP). Furthermore, it addresses some challenges and opportunities, alluding to important regulatory guidance that has been published, methodological aspects and digitalization, highlighting the lack of guidance as a key hurdle to achieve more systematic inclusion of PED in regulatory submissions. In addition, the article discusses opportunities at European and global level that could be implemented to leverage PED use. New digital tools that allow patients to collect PED in real time could also contribute to these advances, but it is equally important not to overlook the challenges they entail. The numerous and relevant initiatives being developed by various stakeholders in this field, including regulators, show their confidence in PED's value and create an ideal moment to address challenges and consolidate PED use across medicines' life cycle.
RESUMO
The COVID-19 pandemic posed challenges to communicating accurate information about vaccines because of the spread of misinformation. The European Medicines Agency (EMA) tried to reassure the public by communicating early on about the development and approval of COVID-19 vaccines. The EMA surveyed patients/consumers, healthcare professional organizations, and individual stakeholders, both at the EU level and in an Italian regional context. The objectives of the study were to see if the EMA's core information materials were informative and well-understood and which communication channels were preferred by the public. The main findings showed that individual patients/consumers generally prefer to obtain information about COVID-19 vaccines from the internet or mass media, while organizations and individual healthcare professionals prefer to obtain information from national and international health authorities. Both at EU and local levels, participants had a good understanding of the key messages from regulators and found the materials useful and relevant. However, some improvements were recommended to the visual, text, and dissemination formats, including publishing more information on safety and using a more public-friendly language. Also, it was recommended to maintain the EMA's approach of using media, stakeholder engagement, and web-based formats to communicate about COVID-19 vaccines. In conclusion, user-testing of proactive communication materials aimed to prebunk misinformation during a public health crisis helps to ensure that users understand the development and safety of novel vaccine technologies. This information can then be used as a basis for further evidence-based communication activities by regulators and public health bodies in an emergency context.
RESUMO
The EU Medicines Regulatory Network (EMRN), comprised of the European Medicines Agency (EMA), the medicines regulatory authorities of the Member States and the European Commission (EC), is operating amid a complex crisis that has positioned regulators centre stage due to their key role in the development, approval and safety monitoring of vaccines and treatments for COVID-19. Here we consider the EMA's and EMRN's response to the pandemic and some of the early learnings that will help reshape medicines regulation in the post COVID-19 era. We also reflect on how some of these learnings will be formally followed up under revised EU legislation to extend EMA's mandate, reinforcing its role in crisis preparedness and response.
RESUMO
During development, dynamic remodeling of the actin cytoskeleton allows the precise placement and morphology of tissues. Morphogens such as Sonic hedgehog (Shh) and local cues such as receptor protein tyrosine phosphatases (RPTPs) mediate this process, but how they regulate the cytoskeleton is poorly understood. We previously identified Basal cell carcinoma-enriched gene 4 (BEG4)/Missing in Metastasis (MIM), a Shh-inducible, Wiskott-Aldrich homology 2 domain-containing protein that potentiates Gli transcription (Callahan, C.A., T. Ofstad, L. Horng, J.K. Wang, H.H. Zhen, P.A. Coulombe, and A.E. Oro. 2004. Genes Dev. 18:2724-2729). Here, we show that endogenous MIM is induced in a patched1-dependent manner and regulates the actin cytoskeleton. MIM functions by bundling F-actin, a process that requires self-association but is independent of G-actin binding. Cytoskeletal remodeling requires an activation domain distinct from sequences required for bundling in vitro. This domain associates with RPTPdelta and, in turn, enhances RPTPdelta membrane localization. MIM-dependent cytoskeletal changes can be inhibited using a soluble RPTPdelta-D2 domain. Our data suggest that the hedgehog-responsive gene MIM cooperates with RPTP to induce cytoskeletal changes.
Assuntos
Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Actinas/análise , Actinas/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular , Linhagem Celular Tumoral , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/fisiologia , Citoesqueleto/efeitos dos fármacos , Células Epiteliais/química , Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Adesões Focais/química , Proteína GAP-43/genética , Expressão Gênica , Proteínas de Homeodomínio/análise , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas com Homeodomínio LIM , Proteínas de Membrana , Camundongos , Proteínas dos Microfilamentos/genética , Microscopia de Fluorescência , Mutação , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/análise , Células PC12 , Receptores Patched , Receptor Patched-1 , Fosfatidilinositol 4,5-Difosfato/farmacologia , Ligação Proteica , Transporte Proteico/fisiologia , Proteínas Tirosina Fosfatases/genética , Proteínas/genética , Proteínas/metabolismo , Ratos , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores , Receptores de Superfície Celular , Medula Espinal/citologia , Medula Espinal/embriologia , Medula Espinal/metabolismo , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo , Fibras de Estresse/ultraestrutura , Fatores de Transcrição , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Vanadatos/farmacologiaRESUMO
The pace of innovation is accelerating, and so medicines regulators need to actively innovate regulatory science to protect human and animal health. This requires consideration and consultation across all stakeholder groups. To this end, the European Medicines Agency worked with stakeholders to draft its Regulatory Science Strategy to 2025 and launched it for public consultation. The responses to this consultation were analyzed qualitatively, using framework analysis and quantitatively, to derive stakeholders' aggregate scores for the proposed recommendations. This paper provides a comprehensive resource of stakeholder positions on key regulatory science topics of the coming 5 years. These stakeholder positions have implications for the development and regulatory approval of both human and veterinary medicines.
RESUMO
With the imminent arrival of oncology biosimilars in the therapeutic paradigm, stakeholders including a clinician, specialist nurse, patient advocate, regulator and economist provide their perspective on optimising the uptake of these new agents in the treatment of cancer. A number of key messages emerge, based on the discussion that took place during a session of the European Society for Medical Oncology's Annual Congress, ESMO Madrid 2017. First, for successful integration of biosimilars into the global healthcare paradigm, informing and educating the full scope of stakeholders, including clinicians, nurses, pharmacists and patients, is primordial. Success is dependent on providing solid evidence and ensuring all voices are heard. Second, for oncology medicines, much can be learnt from the growing experience of approved biosimilars in other disease indications, with success stories for patients, their healthcare providers and healthcare budgets alike. Finally, effective sustainability of the impact on healthcare budgets and the redirection of these savings require education and transparency.
RESUMO
Colorectal cancers from the mutator phenotype pathway display distinctive pathological features and confer a lesser aggressiveness than colorectal adenocarcinomas originated by the suppressor pathway. The goal of this work was to test whether tumors developed through the mutator pathway could show a decrease in matrix metalloproteinase (MMP) activity. We evaluated levels and activity of gelatinase A (MMP-2) and gelatinase B (MMP-9), as well as stromelysin-1 (MMP-3) expression in 101 sporadic colorectal tumors in consideration of the microsatellite instability (MSI) status of the groups. Gelatinases were analyzed by ELISA and zymography. The MMP-3 study was performed by real-time quantitative PCR. MMP-9 total levels were significantly higher in MSI-H tumors. However, levels of the active MMP-9 form were significantly much lower in this group of tumors. Data from real-time quantitative PCR indicated that levels of MMP-3 for MSI-L/MSS tumors were much higher as compared with those observed in MSI-H cancers (P = 0.033). Moreover, all MSI-H tumors showed nucleotide insertions and/or deletions in MMP-3 promoter. These mutations were not observed in the group of MSI-L/MSS tumors. Our data indicate that the MMP-3 promoter constitutes a novel target of the defective mismatch repair machinery in sporadic colorectal tumors, resulting in a dramatic decrease in the levels of the active MMP-9 form, which may result in a lessened capacity for invasion.
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Repetições de Microssatélites/genética , Mutação , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Sequência de Bases , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , DNA de Neoplasias/genética , Ativação Enzimática/genética , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/biossíntese , Dados de Sequência Molecular , Estadiamento de Neoplasias , Regiões Promotoras GenéticasAssuntos
Desenvolvimento de Medicamentos/organização & administração , Organizações sem Fins Lucrativos/organização & administração , Produção de Droga sem Interesse Comercial/métodos , Desenvolvimento de Medicamentos/legislação & jurisprudência , União Europeia , Órgãos Governamentais , Humanos , Produção de Droga sem Interesse Comercial/legislação & jurisprudênciaRESUMO
We report the genomic organization of a novel human gene mapped to chromosome 6p21, encoding a putative glycosylphosphatidylinositol (GPI) anchored protein containing a MAM (meprin, A5 antigen, protein tyrosine phosphatase mu) domain, that we have termed as GPIM (GPI and MAM) protein. GPIM gene consists of an 8.9 kb transcript composed of 17 coding exons spanning about 65.5 kb of genomic DNA. The deduced polypeptide consists of 955 amino acids and exhibits structural features found in different types of cell adhesion molecules (CAMs), such as the presence of immunoglobulin domains, the presence of a MAM domain or the capacity to anchor to the cell membrane by a GPI motif. Expression analysis in normal human tissues revealed that this gene is expressed as a 5 kb and 9.5 kb mRNA. Furthermore, the smaller transcript is highly expressed in some human cancer cell lines, as well as in different primary tumors (lung, colon, uterus, stomach and breast). Interestingly, the gene was higher expressed in several tumor tissues analysed as compared to their corresponding normal tissues. Thus, GPIM is a novel gene codifying a protein with structural features characteristics of some CAMs, which might be involved in the tumor progression.
Assuntos
Moléculas de Adesão Celular/genética , Cromossomos Humanos Par 6/genética , Genes , Proteínas de Neoplasias/genética , Neoplasias/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/química , DNA de Neoplasias/genética , Progressão da Doença , Etiquetas de Sequências Expressas , Feminino , Proteínas Ligadas por GPI , Expressão Gênica , Glicosilfosfatidilinositóis/metabolismo , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/química , Neoplasias/metabolismo , Neoplasias/patologia , Moléculas de Adesão de Célula Nervosa , Especificidade de Órgãos , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico , Células Tumorais Cultivadas/metabolismoRESUMO
PURPOSE: Telomerase activity and p16 expression can be considered two of the most important molecular markers implicated in tumorigenesis. Our main aim was to study the cooperative role of both molecular alterations in the prognosis of patients surgically resected for non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We have determined telomerase activity and p16 expression in a series of 98 prospectively collected NSCLC specimens obtained from patients who had undergone surgery without other treatment. Telomerase activity was investigated by a telomeric repeat amplification protocol enzyme-linked immunosorbent assay-based procedure, and p16 expression was examined by Western blot. Associations with survival were evaluated. RESULTS: Positive results for telomerase activity were found in 82% of the cases, and this variable correlated with poor differentiation and recurrence of tumors. Lack of p16 expression was observed in 61% of tumors, and a significant association with tumor recurrence was also observed. By univariate analysis, both negative telomerase activity and p16-positive expression were significantly correlated with a better prognosis. Moreover, statistics for equality of survival distributions for telomerase, adjusted for p16, indicated a positive interaction between both parameters. For telomerase-positive tumors, p16 expression emerged as a significant independent protective variable, as indicated by Cox multivariate analysis (relative risk [RR], 0.214; P =.014). This protective effect was maintained only for stage I and II tumors (RR, 0.108; P =.046). CONCLUSION: These results suggest that the combined telomerase activity and p16 expression analyses may be of prognostic importance in NSCLC, especially for patients affected by stage I and II tumors.
Assuntos
Biomarcadores Tumorais/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Neoplasias Pulmonares/genética , Telomerase/biossíntese , Idoso , Análise de Variância , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos , Risco , Espanha , Taxa de SobrevidaRESUMO
BACKGROUND: Although the painful shoulder is one of the most common dysfunctions of the locomotor apparatus, and is frequently treated both at primary healthcare centres and by specialists, little evidence has been reported to support or refute the effectiveness of the treatments most commonly applied. According to the bibliography reviewed, physiotherapy, which is the most common action taken to alleviate this problem, has not yet been proven to be effective, because of the small size of sample groups and the lack of methodological rigor in the papers published on the subject. No reviews have been made to assess the effectiveness of acupuncture in treating this complaint, but in recent years controlled randomised studies have been made and these demonstrate an increasing use of acupuncture to treat pathologies of the soft tissues of the shoulder. In this study, we seek to evaluate the effectiveness of physiotherapy applied jointly with acupuncture, compared with physiotherapy applied with a TENS-placebo, in the treatment of painful shoulder caused by subacromial syndrome (rotator cuff tendinitis and subacromial bursitis). METHODS/DESIGN: Randomised controlled multicentre study with blind evaluation by an independent observer and blind, independent analysis. A study will be made of 465 patients referred to the rehabilitation services at participating healthcare centres, belonging to the regional public health systems of Andalusia and Murcia, these patients presenting symptoms of painful shoulder and a diagnosis of subacromial syndrome (rotator cuff tendinitis and subacromial bursitis). The patients will be randomised into two groups: 1) experimental (acupuncture + physiotherapy); 2) control (TENS-placebo + physiotherapy); the administration of rescue medication will also be allowed. The treatment period will have a duration of three weeks. The main result variable will be the change produced on Constant's Shoulder Function Assessment (SFA) Scale; as secondary variables, we will record the changes in diurnal pain intensity on a visual analogue scale (VAS), nocturnal pain intensity on the VAS, doses of non-steroid anti-inflammatory drugs (NSAIDs) taken during the study period, credibility scale for the treatment, degree of improvement perceived by the patient and degree of improvement perceived by the evaluator. A follow up examination will be made at 3, 6 and 12 months after the study period has ended. Two types of population will be considered for analysis: per protocol and per intention to treat. DISCUSSION: The discussion will take into account the limitations of the study, together with considerations such as the choice of a simple, safe method to treat this shoulder complaint, the choice of the control group, and the blinding of the patients, evaluators and those responsible for carrying out the final analysis.
Assuntos
Analgesia por Acupuntura/métodos , Artralgia/terapia , Estudos Multicêntricos como Assunto/métodos , Ensaios Clínicos Controlados Aleatórios como Assunto/métodos , Projetos de Pesquisa , Síndrome de Colisão do Ombro/terapia , Artralgia/etiologia , Artralgia/reabilitação , Doença Crônica , Crioterapia , Seguimentos , Humanos , Medição da Dor , Seleção de Pacientes , Modalidades de Fisioterapia , Síndrome de Colisão do Ombro/complicações , Síndrome de Colisão do Ombro/reabilitação , Estimulação Elétrica Nervosa TranscutâneaAssuntos
Ensaios Clínicos como Assunto/normas , Aprovação de Drogas/legislação & jurisprudência , Indústria Farmacêutica/legislação & jurisprudência , União Europeia/organização & administração , Preparações Farmacêuticas/normas , Aprovação de Drogas/organização & administração , Desenvolvimento de Medicamentos , HumanosRESUMO
Inactivation of p16 has been reported as one of the more frequent events in human carcinogenesis. In order to contribute to the knowledge of the impact of p16 silencing by promoter methylation, we have investigated p16 expression and inactivation of p16 by methylation in two of the major types of human cancer, non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). p16 expression was evaluated by Western blot, and p16 promoter methylation by a methylation-specific PCR procedure (MSP). Clinical correlations were established using the chi-square test, and distributions of disease-free survival (DFS) were estimated with the Kaplan-Meier method. Analyses for p16 revealed that 61.22% (60 of 98) of NSCLCs, and 32.9% (26 of 79) of CRCs here considered, lacked p16 expression. Moreover, 36.7% (22/60) of the non-small cell lung tumours without p16 expression showed p16 promoter methylation, detecting a significant correlation between p16 methylation and the histological subtype of squamous cell carcinomas (SCC) (P=0.04). With respect to CRCs, p16 promoter methylation was observed in 26.9% of tumours that lacked p16 expression (7/26), all tumours studied showing partial methylation. Survival studies demonstrated a clear correlation between p16 negative expression and poor prognosis in NSCLC patients. Moreover, we found a trend toward poor clinical evolution in the group of patients with tumours showing total p16 methylation, in NSCLC, without statistically significant differences in CRC. In conclusion, our results indicate that p16 alterations constitute a major molecular abnormality in NSCLC with a considerable prognosis impact, promoter methylation being an important mechanism involved in p16 silencing. In CRC, however, p16 promoter methylation could be considered as a less definitive molecular factor without prognostic implication, since partial methylation constitutes a prevalent mechanism.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Colorretais/genética , Inativação Gênica , Genes p16 , Neoplasias Pulmonares/genética , Regiões Promotoras Genéticas , Idoso , Western Blotting , Metilação de DNA , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Prognóstico , Fatores de TempoRESUMO
Cell diversity and organization in the neural tube depend on the integration of extrinsic signals acting along orthogonal axes. These are believed to specify distinct cellular identities by triggering all-or-none changes in expression of combinations of transcription factors. Under the influence of a common dorsoventral signal, sonic hedgehog, and distinct anterior-posterior (A-P) inductive signals, two topographically related progenitor pools that share a common transcriptional code produce serotonergic and V3 neurons in the hindbrain and spinal cord, respectively. These neurons have different physiological properties, functions, and connectivity. Serotonergic involvement in neuropsychiatric diseases has prompted greater characterization of their postmitotic repertoire of fate determinants, which include Gata2, Lmx1b, and Pet1, whereas V3 neurons express Sim1. How distinct serotonergic and V3 neuronal identities emerge from progenitors that share a common transcriptional code is not understood. Here, we show that changes in retinoid activity in these two progenitor pools determine their fates. Retinoids, via Notch signaling, control the expression level in progenitors of the transcription factor Ascl1, which selects serotonergic and V3 neuronal identities in a dose-dependent manner. Therefore, quantitative differences in the expression of a single component of a transcriptional code can select distinct cell fates.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Tubo Neural/metabolismo , Neurogênese , Retinoides/metabolismo , Animais , Embrião de Galinha , Camundongos , Camundongos Transgênicos , Tubo Neural/citologia , Tubo Neural/embriologia , Receptores Notch/metabolismo , Neurônios Serotoninérgicos/citologia , Transdução de SinaisRESUMO
Precise regulation of neurogenesis is achieved in specific regions of the vertebrate nervous system by formation of distinct neurogenic and nonneurogenic zones. We have investigated how neurogenesis becomes confined to zones adjacent to rhombomere boundaries in the zebrafish hindbrain. The nonneurogenic zone at segment centers comprises a distinct progenitor population that expresses fibroblast growth factor (fgfr) 2, erm, sox9b, and the retinoic acid degrading enzyme, cyp26b1. FGF receptor activation upregulates expression of these genes and inhibits neurogenesis in segment centers. Cyp26 activity is a key effector inhibiting neuronal differentiation, suggesting antagonistic interactions with retinoid signaling. We identify the critical FGF ligand, fgf20a, which is expressed by specific neurons located in the mantle region at the center of segments, adjacent to the nonneurogenic zone. Fgf20a mutants have ectopic neurogenesis and lack the segment center progenitor population. Our findings reveal how signaling from neurons induces formation of a nonneurogenic zone of neural progenitors.