Laboratory Evolution of Fast-Folding Green Fluorescent Protein Using Secretory Pathway Quality Control

Green fluorescent protein (GFP) has undergone a long history of optimization to become one of the most popular proteins in all of cell biology. It is thermally and chemically robust and produces a pronounced fluorescent phenotype when expressed in cells of all types. Recently, a superfolder GFP was engineered with increased resistance to denaturation and improved folding kinetics. Here we report that unlike other well-folded variants of GFP (e.g., GFPmut2), superfolder GFP was spared from elimination when targeted for secretion via the SecYEG translocase. This prompted us to hypothesize that the folding quality control inherent to this secretory pathway could be used as a platform for engineering similar ‘superfolded’ proteins. To test this, we targeted a combinatorial library of GFPmut2 variants to the SecYEG translocase and isolated several superfolded variants that accumulated in the cytoplasm due to their enhanced folding properties. Each of these GFP variants exhibited much faster folding kinetics than the parental GFPmut2 protein and one of these, designated superfast GFP, folded at a rate that even exceeded superfolder GFP. Remarkably, these GFP variants exhibited little to no loss in specific fluorescence activity relative to GFPmut2, suggesting that the process of superfolding can be accomplished without altering the proteins' normal function. Overall, we demonstrate that laboratory evolution combined with secretory pathway quality control enables sampling of largely unexplored amino-acid sequences for the discovery of artificial, high-performance proteins with properties that are unparalleled in their naturally occurring analogues

Convergence among Non-Sister Dendritic Branches: An Activity-Controlled Mean to Strengthen Network Connectivity

The manner by which axons distribute synaptic connections along dendrites remains a fundamental unresolved issue in neuronal development and physiology. We found in vitro and in vivo indications that dendrites determine the density, location and strength of their synaptic inputs by controlling the distance of their branches from those of their neighbors. Such control occurs through collective branch convergence, a behavior promoted by AMPA and NMDA glutamate receptor activity. At hubs of convergence sites, the incidence of axo-dendritic contacts as well as clustering levels, pre- and post-synaptic protein content and secretion capacity of synaptic connections are higher than found elsewhere. This coupling between synaptic distribution and the pattern of dendritic overlapping results in ‘Economical Small World Network’, a network configuration that enables single axons to innervate multiple and remote dendrites using short wiring lengths. Thus, activity-mediated regulation of the proximity among dendritic branches serves to pattern and strengthen neuronal connectivity

Similar Genetic Mechanisms Underlie the Parallel Evolution of Floral Phenotypes

The repeated origin of similar phenotypes is invaluable for studying the underlying genetics of adaptive traits; molecular evidence, however, is lacking for most examples of such similarity. The floral morphology of neotropical Malpighiaceae is distinctive and highly conserved, especially with regard to symmetry, and is thought to result from specialization on oil-bee pollinators. We recently demonstrated that CYCLOIDEA2–like genes (CYC2A and CYC2B) are associated with the development of the stereotypical floral zygomorphy that is critical to this plant–pollinator mutualism. Here, we build on this developmental framework to characterize floral symmetry in three clades of Malpighiaceae that have independently lost their oil bee association and experienced parallel shifts in their floral morphology, especially in regard to symmetry. We show that in each case these species exhibit a loss of CYC2B function, and a strikingly similar shift in the expression of CYC2A that is coincident with their shift in floral symmetry. These results indicate that similar floral phenotypes in this large angiosperm clade have evolved via parallel genetic changes from an otherwise highly conserved developmental program

Imaging biomarker roadmap for cancer studies.

Imaging biomarkers (IBs) are integral to the routine management of patients with cancer. IBs used daily in oncology include clinical TNM stage, objective response and left ventricular ejection fraction. Other CT, MRI, PET and ultrasonography biomarkers are used extensively in cancer research and drug development. New IBs need to be established either as useful tools for testing research hypotheses in clinical trials and research studies, or as clinical decision-making tools for use in healthcare, by crossing 'translational gaps' through validation and qualification. Important differences exist between IBs and biospecimen-derived biomarkers and, therefore, the development of IBs requires a tailored 'roadmap'. Recognizing this need, Cancer Research UK (CRUK) and the European Organisation for Research and Treatment of Cancer (EORTC) assembled experts to review, debate and summarize the challenges of IB validation and qualification. This consensus group has produced 14 key recommendations for accelerating the clinical translation of IBs, which highlight the role of parallel (rather than sequential) tracks of technical (assay) validation, biological/clinical validation and assessment of cost-effectiveness; the need for IB standardization and accreditation systems; the need to continually revisit IB precision; an alternative framework for biological/clinical validation of IBs; and the essential requirements for multicentre studies to qualify IBs for clinical use.Development of this roadmap received support from Cancer Research UK and the Engineering and Physical Sciences Research Council (grant references A/15267, A/16463, A/16464, A/16465, A/16466 and A/18097), the EORTC Cancer Research Fund, and the Innovative Medicines Initiative Joint Undertaking (grant agreement number 115151), resources of which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and European Federation of Pharmaceutical Industries and Associations (EFPIA) companies' in kind contribution

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Metopic synostosis

Premature closure of the metopic suture results in a growth restriction of the frontal bones, which leads to a skull malformation known as trigonocephaly. Over the course of recent decades, its incidence has been rising, currently making it the second most common type of craniosynostosis. Treatment consists of a cranioplasty, usually preformed before the age of 1 year. Metopic synostosis is linked with an increased level of neurodevelopmental delays. Theories on the etiology of these delays range from a reduced volume of the anterior cranial fossa to intrinsic malformations of the brain. This paper aims to provide an overview of this entity by giving an update on the epidemiology, etiology, evolution of treatment, follow-up, and neurodevelopment of metopic synostosis

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment