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During the write-up of the meeting summary reports from the 2019 dissolution similarity workshop held at the University of Maryland's Center of Excellence in Regulatory Science and Innovation (M-CERSI), several coauthors continued their discussions to develop a "best-practice" document defining the steps required to assess dissolution profiles in support of certain biowaivers and postapproval changes. In previous reports, current challenges related to dissolution profile studies were discussed such that the steps outlined in the two flow charts ("decision trees") presented here can be applied. These decision trees include both recommendations for the use of equivalence procedures between reference and test products as well as application of the dissolution safe space concept. Common approaches towards establishing dissolution safe spaces are described. This paper encourages the preparation of protocols clearly describing why and how testing is performed along with the expected pass/fail criteria prior to generating data on the materials to be evaluated. The target audience of this manuscript includes CMC regulatory scientists, laboratory analysts, as well as statisticians from industry and regulatory health agencies involved in the assessment of product quality via in vitro dissolution testing. Building upon previous publications, this manuscript provides a solution to the current ambiguity related to dissolution profile comparison. The principles outlined in this and previous manuscripts provide a basis for global regulatory alignment in the application of dissolution profile assessment to support manufacturing changes and biowaiver requests.
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SolubilidadRESUMEN
BACKGROUND: Despite widespread use of learning collaboratives, few randomized trials have evaluated their effectiveness as a strategy for implementing evidence based practices. This randomized trial evaluated the effectiveness of a virtual learning collaborative (VLC) in the implementation of a health promotion program for persons with serious mental illness (SMI) aimed at reducing cardiovascular risk reduction in routine mental health settings, compared to routine technical assistance (TA). METHODS: Fifty-five mental health provider organizations were recruited to participate in a Hybrid Type 3 cluster randomized implementation-effectiveness trial of the InSHAPE health promotion program for persons with SMI. Sites were stratified by size and randomized prior to implementation to an 18-month group-based VLC with monthly learning sessions or individual site TA with four scheduled conference calls over 18 months. Primary implementation and service outcomes were InSHAPE program fidelity, participation, and reach. Primary clinical outcomes were weight loss, cardiorespiratory fitness, and cardiovascular risk reduction (≥ 5% weight loss or > 50 m increase on the 6-Minute Walk Test). Program fidelity was assessed at 6, 12, and 24 months; program participation and participant-level outcomes were assessed at 3, 6, 9, and 12 months. RESULTS: VLC (N = 27) and TA (N = 28) sites were similar in organizational characteristics (all p > 0.05). At 12-month follow-up mean program fidelity score was higher in VLC compared to TA (90.5 vs. 79.1; p = 0.002), with over double the proportion with good fidelity (VLC = 73.9% vs. TA = 34.8%; p = 0.009). Over half of individuals in both VLC and TA achieved cardiovascular risk-reduction at 6-month follow-up (VLC: 51.0%; TA: 53.5%; p = 0.517) and at 12-month follow-up (62% VLC and TA; p = 0.912). At 12-month follow-up VLC compared to TA was associated with greater participation (VLC 69.5% vs. TA 56.4% attending at least 50% of sessions, p = 0.002); larger caseloads (VLC = 16 vs. TA = 11; p = 0.024); greater reach consisting of 45% greater number of participants receiving InSHAPE (VLC = 368 vs. TA = 253), and 58% greater number of participants achieving cardiovascular risk reduction (VLC = 150 vs. TA = 95). CONCLUSION: Virtual learning collaboratives compared to routine technical assistance as an implementation strategy for evidence-based health promotion promote greater intervention fidelity, greater levels of intervention participation, greater reach, and a greater number of participants achieving clinically significant risk reduction outcomes, while achieving similarly high levels of intervention effectiveness for participants who completed at least 6 months of the program.
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Educación a Distancia , Salud Mental , Humanos , Promoción de la Salud , Pérdida de Peso , Práctica Clínica Basada en la EvidenciaRESUMEN
This report summarizes podium presentations and breakout sessions from the second day of the 2019 M-CERSI workshop on In Vitro Dissolution Similarity Assessment in Support of Drug Product Quality: What, How, and When? Presenters from the U.S. Food and Drug Administration (FDA), Health Canada (HC), European Medicines Agency (EMA), Brazilian Health Surveillance Agency (ANVISA), and the pharmaceutical industry shared experiences/concerns with dissolution profile similarity assessment supporting minor/moderate Chemistry, Manufacturing and Control (CMC) changes. Members from regulatory agencies explained that dissolution profile similarity testing is only part of the overall assessment of the acceptability of the proposed changes; decisions are usually made based on aggregate weight of evidence. Scientific shortcomings of f2 were highlighted but no proposal on how to replace it was made. Controlling dissolution timepoint variability and application of pairwise batch-to-batch comparisons (PBC) of dissolution profiles caused considerable debate. Several industry participants suggested increased sample sizes to raise confidence in decision-making and to avoid PBC. They proposed identification of a single mathematical method with predefined acceptance criteria and suggested that dissolution timepoint selection should follow EMA and HC guidance. A majority of meeting attendees favored applying clinically relevant dissolution specifications (CRDS) and dissolution safe space to determine the impact of minor/moderate CMC changes as opposed to dissolution profile similarity assessment via statistical methods. Day 2 of the workshop highlighted the need and opportunities for global harmonization including variability, timepoint selection, role of CRDS, and statistical methods to address the ambiguity globally operating pharmaceutical companies are currently facing.
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Industria Farmacéutica , Motivación , Humanos , Preparaciones Farmacéuticas , Solubilidad , Estados Unidos , United States Food and Drug AdministrationRESUMEN
In yeast, many proteins are found in both the cytoplasmic and extracellular compartments, and consequently it can be difficult to distinguish nonconventional secretion from cellular leakage. Therefore, we monitored the extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of intact cells as a specific marker for nonconventional secretion. Extracellular GAPDH activity was proportional to the number of cells assayed, increased with incubation time, and was dependent on added substrates. Preincubation of intact cells with 100 µM dithiothreitol increased the reaction rate, consistent with increased access of the enzyme after reduction of cell wall disulfide cross-links. Such treatment did not increase cell permeability to propidium iodide, in contrast to effects of higher concentrations of reducing agents. An amine-specific membrane-impermeant biotinylation reagent specifically inactivated extracellular GAPDH. The enzyme was secreted again after a 30- to 60-min lag following the inactivation, and there was no concomitant increase in propidium iodide staining. There were about 4 × 104 active GAPDH molecules per cell at steady state, and secretion studies showed replenishment to that level 1 h after inactivation. These results establish conditions for specific quantitative assays of cell wall proteins in the absence of cytoplasmic leakage and for subsequent quantification of secretion rates in intact cells.IMPORTANCE Eukaryotic cells secrete many proteins, including many proteins that do not follow the classical secretion pathway. Among these, the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is unexpectedly found in the walls of yeasts and other fungi and in extracellular space in mammalian cell cultures. It is difficult to quantify extracellular GAPDH, because leakage of just a little of the very large amount of cytoplasmic enzyme can invalidate the determinations. We used enzymatic assays of intact cells while also maintaining membrane integrity. The results lead to estimates of the amount of extracellular enzyme and its rate of secretion to the wall in intact cells. Therefore, enzyme assays under controlled conditions can be used to investigate nonconventional secretion more generally.
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Pared Celular/enzimología , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Secreciones Corporales/metabolismo , Permeabilidad de la Membrana Celular , Citoplasma/enzimología , Citometría de Flujo , Gliceraldehído-3-Fosfato Deshidrogenasas/química , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Proteins are secreted from eukaryotic cells by several mechanisms besides the well-characterized classical secretory system. Proteins destined to enter the classical secretory system contain a signal peptide for translocation into the endoplasmic reticulum. However, many proteins lacking a signal peptide are secreted nonetheless. Contrary to conventional belief, these proteins are not just released as a result of membrane damage leading to cell leakage, but are actively packaged for secretion in alternative pathways. They are called unconventionally secreted proteins, and the best-characterized are from fungi and mammals. These proteins have extracellular functions including cell signaling, immune modulation, as well as moonlighting activities different from their well-described intracellular functions. Among the pathways for unconventional secretion are direct transfer across the plasma membrane, release within plasma membrane-derived microvesicles, use of elements of autophagy, or secretion from endosomal/multivesicular body-related components. We review the fungal and metazoan unconventional secretory pathways and their regulation, and propose experimental criteria to identify their mode of secretion.
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Dental enamel is a principal component of teeth1, and has evolved to bear large chewing forces, resist mechanical fatigue and withstand wear over decades2. Functional impairment and loss of dental enamel, caused by developmental defects or tooth decay (caries), affect health and quality of life, with associated costs to society3. Although the past decade has seen progress in our understanding of enamel formation (amelogenesis) and the functional properties of mature enamel, attempts to repair lesions in this material or to synthesize it in vitro have had limited success4-6. This is partly due to the highly hierarchical structure of enamel and additional complexities arising from chemical gradients7-9. Here we show, using atomic-scale quantitative imaging and correlative spectroscopies, that the nanoscale crystallites of hydroxylapatite (Ca5(PO4)3(OH)), which are the fundamental building blocks of enamel, comprise two nanometric layers enriched in magnesium flanking a core rich in sodium, fluoride and carbonate ions; this sandwich core is surrounded by a shell with lower concentration of substitutional defects. A mechanical model based on density functional theory calculations and X-ray diffraction data predicts that residual stresses arise because of the chemical gradients, in agreement with preferential dissolution of the crystallite core in acidic media. Furthermore, stresses may affect the mechanical resilience of enamel. The two additional layers of hierarchy suggest a possible new model for biological control over crystal growth during amelogenesis, and hint at implications for the preservation of biomarkers during tooth development.
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Amelogénesis , Esmalte Dental/química , Ácidos/química , Calcio/química , Carbonatos/química , Cristalización , Teoría Funcional de la Densidad , Esmalte Dental/ultraestructura , Durapatita/química , Fluoruros/química , Humanos , Magnesio/química , Microscopía Electrónica de Transmisión de Rastreo , Sodio/química , Tomografía , Difracción de Rayos XRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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BACKGROUND: Despite widespread use of learning collaboratives in health care, few randomized trials have evaluated their effectiveness. The primary aim of this cluster randomized implementation trial is to evaluate the effectiveness of a virtual learning collaborative (VLC) in the implementation of a lifestyle intervention for persons with serious mental illness (SMI) in routine mental health settings, compared to standard individual technical assistance. METHODS: Forty-eight mental health provider organizations from across the United States will be recruited to participate in the trial. The evidence-based practice to be implemented is the InSHAPE health promotion intervention for persons with SMI. Sites will be stratified by size and randomized to receive an 18-month intensive group-based VLC with monthly learning sessions or individual technical assistance with four scheduled conference calls over 18â¯months. Sites will be enrolled in three blocks of 16 sites each. The primary outcomes are InSHAPE program participation and fidelity, and participant weight loss; secondary outcomes are program operation, program uptake, participant health behaviors of physical activity and nutrition, organizational change, and program sustainment. Implementation outcomes are measured at 3, 6, 12, 18, and 24â¯months after the program start-up. Participant-level outcomes are measured at fixed intervals every 3â¯months after each participant enrolls in the study. DISCUSSION: This study will determine whether VLCs are an effective implementation strategy among resource-limited providers when the new practice necessitates a shift in mission, scope of practice, type of services delivered, and new financing. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03891368 Registered 25 March 2019, retrospectively registered. https://clinicaltrials.gov/ct2/show/NCT03891368?term=NCT03891368&rank=1.
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Instrucción por Computador/métodos , Conductas Relacionadas con la Salud , Educación en Salud/métodos , Trastornos Mentales/epidemiología , Servicios de Salud Mental/organización & administración , Conducta Cooperativa , Dieta , Ejercicio Físico , Humanos , Capacitación en Servicio , Cooperación del Paciente , Estados Unidos/epidemiología , Pérdida de PesoRESUMEN
This manuscript represents the perspective of the Dissolution Working Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) and of two focus groups of the American Association of Pharmaceutical Scientists (AAPS): Process Analytical Technology (PAT) and In Vitro Release and Dissolution Testing (IVRDT). The intent of this manuscript is to show recent progress in the field of in vitro predictive dissolution modeling and to provide recommended general approaches to developing in vitro predictive dissolution models for both early- and late-stage formulation/process development and batch release. Different modeling approaches should be used at different stages of drug development based on product and process understanding available at those stages. Two industry case studies of current approaches used for modeling tablet dissolution are presented. These include examples of predictive model use for product development within the space explored during formulation and process optimization, as well as of dissolution models as surrogate tests in a regulatory filing. A review of an industry example of developing a dissolution model for real-time release testing (RTRt) and of academic case studies of enabling dissolution RTRt by near-infrared spectroscopy (NIRS) is also provided. These demonstrate multiple approaches for developing data-rich empirical models in the context of science- and risk-based process development to predict in vitro dissolution. Recommendations of modeling best practices are made, focused primarily on immediate-release (IR) oral delivery products for new drug applications. A general roadmap is presented for implementation of dissolution modeling for enhanced product understanding, robust control strategy, batch release testing, and flexibility toward post-approval changes.
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Química Farmacéutica/métodos , Desarrollo de Medicamentos/métodos , Liberación de Fármacos , Modelos Biológicos , Administración Oral , Cápsulas , ComprimidosRESUMEN
This publication summarizes the proceedings and key outcomes of the first day ("Day 1") of the 3-day workshop on "Dissolution and Translational Modeling Strategies Enabling Patient-Centric Product Development." The overall aims of the workshop were to foster a productive dialog between industry and regulatory agencies and to discuss current strategies toward the development and implementation of clinically relevant dissolution specifications as an integral part of enhanced drug product understanding and effective drug product life-cycle management. The Day 1 podium presentations covered existing challenges and concerns for implementing highly valuable, yet often unique and novel experimental dissolution setups as quality control tools. In addition, several podium presentations highlighted opportunities to replace conventional dissolution testing with surrogate test methods to enable robust drug product and process understanding within the context of quality by design (QbD), new manufacturing technologies, and real-time release testing (RTRT). The topics covered on Day 1 laid the foundation for subsequent discussions which focused on the challenges related to establishing an in vitro-in vivo link and approaches for establishing clinically relevant drug product specifications which are becoming an expectation in regulatory submissions. Clarification of dissolution-related terminology used inconsistently among the scientific community, and the purpose of various testing approaches were key discussion topics of the Day 1 breakout sessions. The outcome of these discussions along with creative ways to overcome challenges related to bridging "exploratory dissolution approaches" with methods suitable for end-product control testing are captured within this report.
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Desarrollo de Medicamentos/métodos , Control de Calidad , Animales , Congresos como Asunto , Desarrollo de Medicamentos/normas , Humanos , SolubilidadRESUMEN
OBJECTIVE: The purpose of this study was to evaluate health outcomes of a state-supported implementation in community mental health settings of an evidence-based lifestyle intervention for overweight and obese adults with serious mental illness. METHODS: Weight and fitness outcomes were evaluated for 122 overweight or obese adults with serious mental illness in four community mental health centers (CMHCs) that were participating in a phased statewide implementation of the In SHAPE lifestyle intervention. Six- and 12-month outcomes were compared between two CMHCs that implemented In SHAPE in the first 12 months and two CMHCs with similar characteristics that implemented In SHAPE in a subsequent phase in the statewide implementation 12 months later. RESULTS: Participants in the two In SHAPE sites (N=63 participants) lost significantly more weight (p=.003) and showed greater improvement in fitness (p=.011) compared with participants at the two usual care control sites (N=59 participants). At six months, nearly half (49%) of In SHAPE participants and at 12 months more than half (60%) of In SHAPE participants showed clinically significant cardiovascular risk reduction defined as ≥5% weight loss or improved fitness (>50 m [164 feet] increase on the six-minute walk test). The difference between the In SHAPE and control groups was not statistically significant. CONCLUSIONS: This natural experiment demonstrated promising public health benefits of a practical implementation of health promotion programming for overweight and obese adults with serious mental illness and offers a potential model for reducing risk of early mortality among individuals served by state-funded mental health centers nationwide.
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Servicios Comunitarios de Salud Mental/métodos , Promoción de la Salud/métodos , Trastornos Mentales/rehabilitación , Evaluación de Resultado en la Atención de Salud , Sobrepeso/terapia , Educación del Paciente como Asunto/métodos , Conducta de Reducción del Riesgo , Adulto , Centros Comunitarios de Salud Mental , Comorbilidad , Femenino , Estudios de Seguimiento , Humanos , Masculino , Trastornos Mentales/epidemiología , Persona de Mediana Edad , Sobrepeso/epidemiologíaRESUMEN
This article intends to summarize the current views of the IQ Consortium Dissolution Working Group, which comprises various industry companies, on the roles of dissolution testing throughout pharmaceutical product development, registration, commercialization, and beyond. Over the past 3 decades, dissolution testing has evolved from a routine and straightforward test as a component of end-product release into a comprehensive set of tools that the developer can deploy at various stages of the product life cycle. The definitions of commonly used dissolution approaches, how they relate to one another and how they may be applied in modern drug development, and life cycle management is described in this article. Specifically, this article discusses the purpose, advantages, and limitations of quality control, biorelevant, and clinically relevant dissolution methods.
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Preparaciones Farmacéuticas/química , Animales , Química Farmacéutica/métodos , Humanos , Control de Calidad , SolubilidadRESUMEN
This manuscript represents the perspective of the Dissolution Analytical Working Group of the IQ Consortium. The intent of this manuscript is to highlight the challenges of, and to provide a recommendation on, the development of clinically relevant dissolution specifications (CRS) for immediate release (IR) solid oral dosage forms. A roadmap toward the development of CRS for IR products containing active ingredients with a non-narrow therapeutic window is discussed, within the context of mechanistic dissolution understanding, supported by in-human pharmacokinetic (PK) data. Two case studies present potential outcomes of following the CRS roadmap and setting dissolution specifications. These cases reveal some benefits and challenges of pursuing CRS with additional PK data, in light of current regulatory positions, including that of the US Food and Drug Administration (FDA), who generally favor this approach, but with the understanding that both industry and regulatory agency perspectives are still evolving in this relatively new field. The CRS roadmap discussed in this manuscript also describes a way to develop clinically relevant dissolution specifications based primarily on dissolution data for batches used in pivotal clinical studies, acknowledging that not all IR product development efforts need to be supported by additional PK studies, albeit with the associated risk of potentially unnecessarily tight manufacturing controls. Recommendations are provided on what stages during the life cycle investment into in vivo studies may be valuable. Finally, the opportunities for CRS within the context of post-approval changes, Modeling and Simulation (M&S), and the application of biowaivers, are briefly discussed.
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Solubilidad , Administración Oral , Humanos , Modelos Biológicos , ComprimidosRESUMEN
DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation.
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Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas E1A de Adenovirus/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas E1A de Adenovirus/química , Sitios de Unión , Proteínas Portadoras/química , Dictyostelium/metabolismo , Células HeLa , Humanos , Fosforilación , Unión Proteica , Conformación Proteica , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química , Fracciones Subcelulares/metabolismo , Quinasas DyrKRESUMEN
The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A's role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs.
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Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Imitación Molecular , Proteínas de Anclaje a la Quinasa A/química , Adenoviridae/química , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/química , Secuencia de Aminoácidos , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas Quinasas Dependientes de AMP Cíclico/química , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunoprecipitación , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Secundaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Dental enamel, a hierarchical material composed primarily of hydroxylapatite nanowires, is susceptible to degradation by plaque biofilm-derived acids. The solubility of enamel strongly depends on the presence of Mg(2+), F(-), and CO3(2-). However, determining the distribution of these minor ions is challenging. We showusing atom probe tomography, x-ray absorption spectroscopy, and correlative techniquesthat in unpigmented rodent enamel, Mg(2+) is predominantly present at grain boundaries as an intergranular phase of Mg-substituted amorphous calcium phosphate (Mg-ACP). In the pigmented enamel, a mixture of ferrihydrite and amorphous iron-calcium phosphate replaces the more soluble Mg-ACP, rendering it both harder and more resistant to acid attack. These results demonstrate the presence of enduring amorphous phases with a dramatic influence on the physical and chemical properties of the mature mineralized tissue.
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Fosfatos de Calcio/química , Esmalte Dental/química , Esmalte Dental/ultraestructura , Animales , Incisivo/química , Incisivo/ultraestructura , Ratones , Microscopía Electrónica de Rastreo , Espectroscopía de Absorción de Rayos XRESUMEN
Metastable precursors are thought to play a major role in the ability of organisms to create mineralized tissues. Of particular interest are the hard and abrasion-resistant teeth formed by chitons, a class of rock-grazing mollusks. The formation of chiton teeth relies on the precipitation of metastable ferrihydrite (Fh) in an organic scaffold as a precursor to magnetite. Inâ vitro synthesis of Fh under physiological conditions has been challenging. Using a combination of X-ray absorption and electron paramagnetic resonance spectroscopy, we show that, prior to Fh formation in the chiton tooth, iron ions are complexed by the organic matrix. Inâ vitro experiments demonstrate that such complexes facilitate the formation of Fh under physiological conditions. These results indicate that acidic molecules may be integral to controlling Fh formation in the chiton tooth. This biological approach to polymorph selection is not limited to specialized proteins and can be expropriated using simple chemistry.
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Compuestos Férricos/síntesis química , Poliplacóforos/química , Animales , Espectroscopía de Resonancia por Spin del Electrón , Espectroscopía de Absorción de Rayos X , Difracción de Rayos XRESUMEN
The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285-289, as well as a second basic patch situated between residues 258 and 263 ((258)RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP(289)). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs.
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Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Señales de Localización Nuclear/fisiología , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/genética , Secuencia de Aminoácidos , Línea Celular , Regulación Viral de la Expresión Génica/fisiología , Humanos , Datos de Secuencia MolecularRESUMEN
The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell.
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Proteínas E1A de Adenovirus/genética , Adenovirus Humanos/genética , Señales de Localización Nuclear , Humanos , Saccharomyces cerevisiae/genéticaRESUMEN
TOPIC: A majority of people with serious mental illnesses want to return to work as part of their recovery, but access to effective employment services is limited. This report highlights the role that families can take to advocate for access to high-quality services for their relatives with mental illnesses. PURPOSE: This report summarizes major accomplishments of the family advocacy for the Individual Placement and Support (IPS) supported employment project, which is part of the Johnson & Johnson-Dartmouth Community Mental Health Program. SOURCES USED: This description draws on published literature related to the role of families on behalf of individuals living with serious mental health conditions and supported employment services. The experiences of family members as advocates for change are also included. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: Family teams can play a key role in changing public policy to increase access to evidence-based employment services and in promoting high-quality services at the local level in partnership with state mental health and vocational rehabilitation agency leaders responsible for employment services. In addition, family teams can educate their peers to better understand the role of work in recovery.