RESUMO
Characterising post-translational regulation of key transcriptional activators is crucial for understanding how cell division and differentiation are coordinated in developing organisms and cycling cells. One important mode of protein post-translational control is by regulation of half-life via ubiquitin-mediated proteolysis. Two key basic Helix-Loop-Helix transcription factors, Neurogenin 2 (Ngn2) and NeuroD, play central roles in development of the central nervous system but despite their homology, Ngn2 is a highly unstable protein whilst NeuroD is, by comparison, very stable. The basis for and the consequences of the difference in stability of these two structurally and functionally related proteins has not been explored. Here we see that ubiquitylation alone does not determine Ngn2 or NeuroD stability. By making chimeric proteins, we see that the N-terminus of NeuroD in particular has a stabilising effect, whilst despite their high levels of homology, the most conserved bHLH domains of these proneural proteins alone can confer significant changes in protein stability. Despite widely differing stabilities of Ngn2, NeuroD and the chimeric proteins composed of domains of both, there is little correlation between protein half-life and ability to drive neuronal differentiation. Therefore, we conclude that despite significant homology between Ngn2 and NeuroD, the regulation of their stability differs markedly and moreover, stability/instability of the proteins is not a direct correlate of their activity.
Assuntos
Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Meia-Vida , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Desdobramento de Proteína , Proteólise , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Neuronal differentiation is largely under the control of basic Helix-Loop-Helix (bHLH) proneural transcription factors that play key roles during development of the embryonic nervous system. In addition to well-characterised regulation of their expression, increasing evidence is emerging for additional post-translational regulation of proneural protein activity. Of particular interest is the bHLH proneural factor Neurogenin2 (Ngn2), which orchestrates progression from neural progenitor to differentiated neuron in several regions of the central nervous system. Previous studies have demonstrated a key role for cell cycle-dependent multi-site phosphorylation of Ngn2 protein at Serine-Proline (SP) sites for regulation of its neuronal differentiation activity, although the potential structural and functional consequences of phosphorylation at different regions of the protein are unclear. RESULTS: Here we characterise the role of phosphorylation of specific regions of Ngn2 on the stability of Ngn2 protein and on its neuronal differentiation activity in vivo in the developing embryo, demonstrating clearly that the location of SP sites is less important than the number of SP sites available for control of Ngn2 activity in vivo. We also provide structural evidence that Ngn2 contains large, intrinsically disordered regions that undergo phosphorylation by cyclin-dependent kinases (cdks). CONCLUSIONS: Phosphorylation of Ngn2 occurs in both the N- and C-terminal regions, either side of the conserved basic Helix-Loop-Helix domain. While these phosphorylation events do not change the intrinsic stability of Ngn2, phosphorylation on multiple sites acts to limit its ability to drive neuronal differentiation in vivo. Phosphorylated regions of Ngn2 are predicted to be intrinsically disordered and cdk-dependent phosphorylation of these intrinsically disordered regions contributes to Ngn2 regulation.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Xenopus/metabolismo , Animais , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas do Tecido Nervoso/química , Neurogênese , Fosforilação , Estrutura Terciária de Proteína , Proteínas de Xenopus/química , Xenopus laevisRESUMO
Careful, clean and controlled preparation of samples for mass spectrometry proteomics is crucial to obtain reproducible and reliable data. This is especially important when carrying out quantitative proteomics by chemical isobaric labeling (aka tandem mass tagging), since the differentially labeled samples are combined quite late during the sample processing. Addressing this need for robust and reliable sample processing for quantitative proteomics, we describe here iFASP, a simple protocol for combining isobaric mass tagging with the recently introduced filter-aided sample preparation (FASP) method. iFASP provides a quick, simple and effective method for obtaining clean samples, ensuring efficient digestion and providing excellent labeling yields for quantitative proteomics experiments. We have carried out our iFASP protocol using several highly complex Xenopus laevis egg and embryo lysates and compared the labeling yields and number of high-confidence peptide identifications to a standard in-solution digestion and labeling protocol. Although the labeling efficiency with both techniques is in the 99+% range, the number of peptides identified with a 1% false discovery rate and the corresponding number of quantified peptide spectral matches are as much as doubled with iFASP compared to the corresponding non-FASP-based method.
Assuntos
Proteínas de Anfíbios/química , Embrião não Mamífero/química , Proteômica/métodos , Coloração e Rotulagem/métodos , Zigoto/química , Proteínas de Anfíbios/isolamento & purificação , Animais , Cromatografia Líquida , Feminino , Filtração , Peso Molecular , Espectrometria de Massas em Tandem , Xenopus laevis/metabolismoRESUMO
Education about scientific publishing and manuscript peer review is not universally provided in undergraduate science courses. Since peer review is integral to the scientific process and central to the identity of a scientist, we envision a paradigm shift where teaching peer review becomes integral to undergraduate science education. We hypothesize that teaching undergraduates how to peer review scientific manuscripts may facilitate their development of scientific literacy and identity formation. To this end, we developed a constructivist, service-learning curriculum for biology undergraduates to learn about the mechanisms of peer review using preprints and then to write and publish their own peer reviews of preprints as a way to authentically join the scientific community of practice. The curriculum was implemented as a semester-long intervention in one class and, in another class, as an embedded module intervention. Students' scientific literacy and peer review ability were assessed using quantitative methods. Student's perceptions of their scientific literacy and identity were assessed using thematic analysis of students' reflective writing. Here, we present data on the improvement in the peer review ability of undergraduates in both classes and data on the curriculum's interrelated impact on students' development of scientific literacy, identity, and belonging in peer and professional discourse spaces. These data suggest that undergraduates can and should be trained in peer review to foster the interrelated development of their scientific literacy, scientific identity, and sense of belonging in science.
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Science, technology, engineering, mathematics, and medicine (STEMM) fields change rapidly and are increasingly interdisciplinary. Commonly, STEMM practitioners use short-format training (SFT) such as workshops and short courses for upskilling and reskilling, but unaddressed challenges limit SFT's effectiveness and inclusiveness. Education researchers, students in SFT courses, and organizations have called for research and strategies that can strengthen SFT in terms of effectiveness, inclusiveness, and accessibility across multiple dimensions. This paper describes the project that resulted in a consensus set of 14 actionable recommendations to systematically strengthen SFT. A diverse international group of 30 experts in education, accessibility, and life sciences came together from 10 countries to develop recommendations that can help strengthen SFT globally. Participants, including representation from some of the largest life science training programs globally, assembled findings in the educational sciences and encompassed the experiences of several of the largest life science SFT programs. The 14 recommendations were derived through a Delphi method, where consensus was achieved in real time as the group completed a series of meetings and tasks designed to elicit specific recommendations. Recommendations cover the breadth of SFT contexts and stakeholder groups and include actions for instructors (e.g., make equity and inclusion an ethical obligation), programs (e.g., centralize infrastructure for assessment and evaluation), as well as organizations and funders (e.g., professionalize training SFT instructors; deploy SFT to counter inequity). Recommendations are aligned with a purpose-built framework-"The Bicycle Principles"-that prioritizes evidenced-based teaching, inclusiveness, and equity, as well as the ability to scale, share, and sustain SFT. We also describe how the Bicycle Principles and recommendations are consistent with educational change theories and can overcome systemic barriers to delivering consistently effective, inclusive, and career-spanning SFT.
Assuntos
Estudantes , Tecnologia , Humanos , Consenso , EngenhariaRESUMO
Early career researchers are frequent and valuable contributors to peer review. Systemic changes that acknowledge this fact would result in ethical co-reviewing, peer reviews of greater quality, and a reduction in peer reviewer burden.
Assuntos
Autoria , Revisão da Pesquisa por Pares/tendências , Má Conduta Científica/ética , Humanos , Revisão da Pesquisa por Pares/normas , Pesquisadores/psicologia , Má Conduta Científica/tendênciasRESUMO
Poly-ubiquitin chains targeting proteins for 26S proteasomal degradation are classically anchored on internal lysines of substrates via iso-peptide linkages. However recently, linkage of ubiquitin moieties to non-canonical nucleophilic residues, such as cysteines, serines and threonines, has been demonstrated in a small number of cases. Non-canonical ubiquitylation of the proneural protein Ngn2 has previously been seen in Xenopus egg extract, but it was not clear whether such highly unusual modes of ubiquitylation were restricted to the environment of egg cytoplasm. Here we show that Ngn2 is, indeed, ubiquitylated on non-canonical sites in extracts from neurula stage Xenopus embryos, when Ngn2 is usually active. Moreover, in the P19 mammalian embryonal carcinoma cell line capable of differentiating into neurons, xNgn2 is ubiquitylated on both canonical and non-canonical sites. We see that mutation of cysteines alone results stabilisation of the protein in P19 cells, indicating that non-canonical ubiquitylation on these residues normally contributes to the fast turnover of xNgn2 in mammalian cells.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Ubiquitinação , Proteínas de Xenopus/metabolismo , Animais , Embrião não Mamífero , Feminino , Camundongos , Proteínas do Tecido Nervoso/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
In contrast to efforts focusing on improving inclusion in STEM classrooms from kindergarten through undergraduate (K-16), efforts to improve inclusion in scientific meetings and conferences, important hubs of STEM culture, are more recent. Markers of inclusion that are sometimes overlooked at these events can include the composition of panels, how workshops are run, the affordability of conferences, and various other mechanisms that maintain pre-existing hierarchies and norms that limit the participation of early-career researchers and individuals of minoritized cultural, linguistic, and economic backgrounds. The Inclusive Environments and Metrics in Biology Education and Research (iEMBER) network coordinates efforts of researchers from many fields interested in diversity and inclusion in biology education. Given the concerns regarding inclusion at professional meetings, iEMBER has developed and implemented several practices in planning and executing our meetings to make them more inclusive. In this report, we share our experiences developing inclusive meetings on biology education research and discuss the outcomes of such efforts. Specifically, we present our approach to planning and executing the iEMBER 2019 conference and the National Association of Biology Teachers iEMBER 2019 workshop. This report adds to the growing body of resources on inclusive meetings, provides readers with an account of how such an attempt at implementation might unfold, and complements existing theories and work relating to the importance and functioning of such meetings in terms of representation in STEM.
RESUMO
Xenopus extract systems have been used to study ubiquitylation of proteins, and to uncover some of the fundamental processes of the ubiquitylation pathway itself. They provide a simple, quick, and robust method for studying ubiquitylation. In this protocol, methods are provided for studying protein ubiquitylation using Xenopus egg or embryo extracts and in vitro radiolabeled proteins. These methods also enable examination of whether proteins undergo noncanonical ubiquitylation, through modification of the protein by covalent linkage to ubiquitin through residues other than lysine, such as cysteine, serine, and threonine.
Assuntos
Bioquímica/métodos , Extratos Celulares/química , Ubiquitinação , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Bioensaio , Biossíntese de Proteínas , Compostos Radiofarmacêuticos/química , Transcrição GênicaRESUMO
The Xenopus extract system has been used extensively as a simple, quick, and robust method for assessing the stability of proteins against proteasomal degradation. In this protocol, methods are provided for assessing the half-life of in vitro translated radiolabeled proteins using Xenopus egg or embryo extracts.
Assuntos
Bioquímica/métodos , Extratos Celulares/química , Proteólise , Proteínas de Xenopus/metabolismo , Animais , Bioensaio , Biossíntese de Proteínas , Compostos Radiofarmacêuticos/química , Transcrição Gênica , Xenopus laevisRESUMO
Many early-career researchers are involved in the peer review of manuscripts for scientific journals, typically under the guidance of or jointly with their advisor, but most of the evidence about this activity is anecdotal. Here we report the results of a literature review and a survey of researchers, with an emphasis on co-reviewing and 'ghostwriting'. The literature review identified 36 articles that addressed the involvement of early-career researchers in peer review, most of them about early-career researchers and their advisors co-reviewing manuscripts for the purposes of training: none of them addressed the topic of ghostwriting in detail. About three quarters of the respondents to the survey had co-reviewed a manuscript. Most respondents believe co-reviewing to be a beneficial (95%) and ethical (73%) form of training in peer review. About half of the respondents have ghostwritten a peer review report, despite 81% responding that ghostwriting is unethical and 82% agreeing that identifying co-reviewers to the journal is valuable. Peer review would benefit from changes in both journal policies and lab practices that encourage mentored co-review and discourage ghostwriting.
Assuntos
Autoria , Pesquisa Biomédica/ética , Revisão por Pares , Pesquisadores , HumanosRESUMO
The Inclusive Environments and Metrics in Biology Education and Research (iEMBER) network is a newly forming national community of practice that engages diversity, equity, and inclusion stakeholders in interdisciplinary collaborative projects. iEMBER was initiated with incubator funding from the National Science Foundation program for Research Coordination Networks in Undergraduate Biology Education. In June 2017, biology education researchers, social scientists, biologists, and program and policy administrators, all with interests in diversity, equity, and inclusion, met to lay the foundation for the iEMBER network. iEMBER provides a distinct forum to coordinate efforts through networking, professional development, and the initiation of collaborative research. iEMBER advances science, technology, engineering, and mathematics reform focused on diversity, equity, and inclusion through the initiation of research teams at the iEMBER biennial conference and outreach efforts at discipline-specific meetings and conferences. The focus of iEMBER is on understanding how to create inclusive, supportive, and engaging environments to foster the success of all biology students and trainees. This report focuses on the structure of the iEMBER network, two takeaways that emerged from the 2017 conference (interdisciplinary networking/collaboration and intradisciplinary broadening participation strategies), and ways for prospective members to engage in ongoing dialogue and future events. Learn more at http://iember.org .
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Biologia/educação , Diversidade Cultural , Pesquisa/educação , Congressos como Assunto , Comportamento Cooperativo , Humanos , Estudos Interdisciplinares , Estudos ProspectivosRESUMO
Being successful in an academic environment places many demands on junior scientists. Science communication currently may not be adequately valued and rewarded, and yet communication to multiple audiences is critical for ensuring that it remains a priority in today's society. Due to the potential for science communication to produce better scientists, facilitate scientific progress, and influence decision-making at multiple levels, training junior scientists in both effective and ethical science communication practices is imperative, and can benefit scientists regardless of their chosen career path. However, many challenges exist in addressing specific aspects of this training. Principally, science communication training and resources should be made readily available to junior scientists at institutions, and there is a need to scale up existing science communication training programs and standardize core aspects of these programs across universities, while also allowing for experimentation with training. We propose a comprehensive core training program be adopted by universities, utilizing a centralized online resource with science communication information from multiple stakeholders. In addition, the culture of science must shift toward greater acceptance of science communication as an essential part of training. For this purpose, the science communication field itself needs to be developed, researched and better understood at multiple levels. Ultimately, this may result in a larger cultural change toward acceptance of professional development activities as valuable for training scientists.
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Background: On December 1, 2016, the Fair Labor Standards Act (FLSA) was due to be updated by the U.S. Department of Labor. Key changes included an increase in the salary threshold for exemption from overtime for working more than 40 hours per week, and indexing the salary level so that it is updated automatically every 3 years. This was predicted to have a profound effect on academe as postdoctoral researchers were mostly paid at a salary below the new threshold. On November 22, 2016, an injunction was granted nationwide, delaying implementation of the updates, which were finally struck down entirely on August 31, 2017. Here we review the key changes to the FLSA, how they came about, and how the postdoctoral population was affected. Methods: We describe recent data collection efforts to uncover what institutions with postdocs were doing to comply with the FLSA. Results: Our data showed that 57% of institutions checked (containing 41% of the estimated postdoctoral workforce in science, engineering and health) had not decided or had no public decision available one month prior to implementation, and only 35.5% of institutions were planning to raise salaries to the new minimum. After the injunction, a number of institutions and the NIH continued with their plans to raise salaries. Overall, despite the removal of a federal mandate, approximately 60% of postdocs are at institutions whose policy is to raise salaries. Conclusions: Our data show uncertainty in postdoctoral salaries in the U.S. prior to implementation of the FLSA ruling. In addition, while some institutions did suspend plans to raise postdoctoral salaries after the injunction, many continued with the raise. The implementation of postdoctoral salary raises may be inconsistent, however, as the legal minimum is still $23,660.
RESUMO
The small protein modifier, ubiquitin, can be covalently attached to proteins in the process of ubiquitylation, resulting in a variety of functional outcomes. In particular, the most commonly-associated and well-studied fate for proteins modified with ubiquitin is their ultimate destruction: degradation by the 26S proteasome via the ubiquitin-proteasome system, or digestion in lysosomes by proteolytic enzymes. From the earliest days of ubiquitylation research, a reliable and versatile "cell-in-a-test-tube" system has been employed in the form of cytoplasmic extracts from the eggs and embryos of the frog Xenopus laevis. Biochemical studies of ubiquitin and protein degradation using this system have led to significant advances particularly in the study of ubiquitin-mediated proteolysis, while the versatility of Xenopus as a developmental model has allowed investigation of the in vivo consequences of ubiquitylation. Here we describe the use and history of Xenopus extract in the study of ubiquitin-mediated protein degradation, and highlight the versatility of this system that has been exploited to uncover mechanisms and consequences of ubiquitylation and proteolysis.
Assuntos
Sistema Livre de Células/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/metabolismo , Xenopus laevis/metabolismo , Animais , UbiquitinaçãoRESUMO
Consistently-biased left-right (LR) patterning is required for the proper placement of organs including the heart and viscera. The LR axis is especially fascinating as an example of multi-scale pattern formation, since here chiral events at the subcellular level are integrated and amplified into asymmetric transcriptional cascades and ultimately into the anatomical patterning of the entire body. In contrast to the other two body axes, there is considerable controversy about the earliest mechanisms of embryonic laterality. Many molecular components of asymmetry have not been widely tested among phyla with diverse bodyplans, and it is unknown whether parallel (redundant) pathways may exist that could reverse abnormal asymmetry states at specific checkpoints in development. To address conservation of the early steps of LR patterning, we used the Xenopus laevis (frog) embryo to functionally test a number of protein targets known to direct asymmetry in plants, fruit fly, and rodent. Using the same reagents that randomize asymmetry in Arabidopsis, Drosophila, and mouse embryos, we show that manipulation of the microtubule and actin cytoskeleton immediately post-fertilization, but not later, results in laterality defects in Xenopus embryos. Moreover, we observed organ-specific randomization effects and a striking dissociation of organ situs from effects on the expression of left side control genes, which parallel data from Drosophila and mouse. Remarkably, some early manipulations that disrupt laterality of transcriptional asymmetry determinants can be subsequently "rescued" by the embryo, resulting in normal organ situs. These data reveal the existence of novel corrective mechanisms, demonstrate that asymmetric expression of Nodal is not a definitive marker of laterality, and suggest the existence of amplification pathways that connect early cytoskeletal processes to control of organ situs bypassing Nodal. Counter to alternative models of symmetry breaking during neurulation (via ciliary structures absent in many phyla), our data suggest a widely-conserved role for the cytoskeleton in regulating left-right axis formation immediately after fertilization of the egg. The novel mechanisms that rescue organ situs, even after incorrect expression of genes previously considered to be left-side master regulators, suggest LR patterning as a new context in which to explore multi-scale redundancy and integration of patterning from the subcellular structure to the entire bodyplan.
Assuntos
Padronização Corporal/fisiologia , Citoesqueleto/fisiologia , Animais , Arabidopsis , Padronização Corporal/genética , Drosophila , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Microtúbulos/fisiologia , Miosinas/genética , Miosinas/metabolismo , Processamento de Proteína Pós-Traducional , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genéticaRESUMO
While components of the pathway that establishes left-right asymmetry have been identified in diverse animals, from vertebrates to flies, it is striking that the genes involved in the first symmetry-breaking step remain wholly unknown in the most obviously chiral animals, the gastropod snails. Previously, research on snails was used to show that left-right signaling of Nodal, downstream of symmetry breaking, may be an ancestral feature of the Bilateria [1 and 2]. Here, we report that a disabling mutation in one copy of a tandemly duplicated, diaphanous-related formin is perfectly associated with symmetry breaking in the pond snail. This is supported by the observation that an anti-formin drug treatment converts dextral snail embryos to a sinistral phenocopy, and in frogs, drug inhibition or overexpression by microinjection of formin has a chirality-randomizing effect in early (pre-cilia) embryos. Contrary to expectations based on existing models [3, 4 and 5], we discovered asymmetric gene expression in 2- and 4-cell snail embryos, preceding morphological asymmetry. As the formin-actin filament has been shown to be part of an asymmetry-breaking switch in vitro [6 and 7], together these results are consistent with the view that animals with diverse body plans may derive their asymmetries from the same intracellular chiral elements [8].
Assuntos
Padronização Corporal , Proteínas Fetais/genética , Lymnaea/genética , Proteínas dos Microfilamentos/genética , Proteínas Nucleares/genética , Transdução de Sinais , Xenopus laevis/genética , Animais , Proteínas Fetais/metabolismo , Forminas , Lymnaea/embriologia , Lymnaea/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Fenótipo , Xenopus laevis/embriologia , Xenopus laevis/metabolismoRESUMO
There is a common misconception that the United States is suffering from a "STEM shortage," a dearth of graduates with scientific, technological, engineering, and mathematical backgrounds. In biomedical science, however, we are likely suffering from the opposite problem and could certainly better tailor training to actual career outcomes. At the Future of Research Symposium, various workshops identified this as a key issue in a pipeline traditionally geared toward academia. Proposals for reform all ultimately come up against the same problem: there is a shocking lack of data at institutional and national levels on the size, shape, and successful careers of participants in the research workforce. In this paper, we call for improved institutional reporting of the number of graduate students and postdocs and their training and career outcomes.