An artificial metalloenzyme for a bimolecular Diels–Alder reaction

The Diels–Alder reaction, one of the most important in organic chemistry, forms functionalized six-membered cycloadducts in a single step. While widely used to construct complex biologically active molecules in the laboratory, [4+2] cycloadditions are rarely employed for natural product biosynthesis in cells owing to the lack of appropriate enzymes. Creating artificial metalloenzymes able to exploit Lewis acid catalysis for substrate activation could change this situation. Embedding a metal ion in a chiral protein binding pocket potentially combines the best aspects of two worlds – transition metal and enzymatic catalysis – to achieve both high activity and selectivity. Here we report the transformation of a zinc-binding helical bundle into an artificial metalloenzyme that efficiently catalyzes a hetero-Diels–Alder reaction between 3-vinyl indole and an azachalcone derivative by a process of design and laboratory evolution. The best enzyme, DA7, performed \u3e15,000 turnovers per active site and produced only a single product stereoisomer (\u3e99% ee). Detailed kinetic analysis showed that this catalyst is more than two orders of magnitude more proficient than other known Diels–Alderases, including many designed catalysts and natural enzymes involved in polyketide natural products biosynthesis. The remarkable activity of DA7 can be ascribed to the Zn(II) ion, which activates the heterodiene for reaction, and a shape complementary binding pocket that preorganized the reactants for efficient reaction and exacting control over chemo-, diastereo-, and enantioselectivity. These results establish the feasibility of combining design and evolution to harness the structural and functional properties of metal ions to produce remarkably active enzymes for an important abiological reaction. Extending this approach to metal ions other than zinc, and to scaffolds beyond helical bundles, can be expected to produce proficient custom-metalloenzymes for a wide spectrum of unnatural chemical transformations

Ebola Virion Attachment and Entry into Human Macrophages Profoundly Effects Early Cellular Gene Expression

Zaire ebolavirus (ZEBOV) infections are associated with high lethality in primates. ZEBOV primarily targets mononuclear phagocytes, which are activated upon infection and secrete mediators believed to trigger initial stages of pathogenesis. The characterization of the responses of target cells to ZEBOV infection may therefore not only further understanding of pathogenesis but also suggest possible points of therapeutic intervention. Gene expression profiles of primary human macrophages exposed to ZEBOV were determined using DNA microarrays and quantitative PCR to gain insight into the cellular response immediately after cell entry. Significant changes in mRNA concentrations encoding for 88 cellular proteins were observed. Most of these proteins have not yet been implicated in ZEBOV infection. Some, however, are inflammatory mediators known to be elevated during the acute phase of disease in the blood of ZEBOV-infected humans. Interestingly, the cellular response occurred within the first hour of Ebola virion exposure, i.e. prior to virus gene expression. This observation supports the hypothesis that virion binding or entry mediated by the spike glycoprotein (GP1,2) is the primary stimulus for an initial response. Indeed, ZEBOV virions, LPS, and virus-like particles consisting of only the ZEBOV matrix protein VP40 and GP1,2 (VLPVP40-GP) triggered comparable responses in macrophages, including pro-inflammatory and pro-apoptotic signals. In contrast, VLPVP40 (particles lacking GP1,2) caused an aberrant response. This suggests that GP1,2 binding to macrophages plays an important role in the immediate cellular response

Cell-Sorting at the A/P Boundary in the Drosophila Wing Primordium: A Computational Model to Consolidate Observed Non-Local Effects of Hh Signaling

Non-intermingling, adjacent populations of cells define compartment boundaries; such boundaries are often essential for the positioning and the maintenance of tissue-organizers during growth. In the developing wing primordium of Drosophila melanogaster, signaling by the secreted protein Hedgehog (Hh) is required for compartment boundary maintenance. However, the precise mechanism of Hh input remains poorly understood. Here, we combine experimental observations of perturbed Hh signaling with computer simulations of cellular behavior, and connect physical properties of cells to their Hh signaling status. We find that experimental disruption of Hh signaling has observable effects on cell sorting surprisingly far from the compartment boundary, which is in contrast to a previous model that confines Hh influence to the compartment boundary itself. We have recapitulated our experimental observations by simulations of Hh diffusion and transduction coupled to mechanical tension along cell-to-cell contact surfaces. Intriguingly, the best results were obtained under the assumption that Hh signaling cannot alter the overall tension force of the cell, but will merely re-distribute it locally inside the cell, relative to the signaling status of neighboring cells. Our results suggest a scenario in which homotypic interactions of a putative Hh target molecule at the cell surface are converted into a mechanical force. Such a scenario could explain why the mechanical output of Hh signaling appears to be confined to the compartment boundary, despite the longer range of the Hh molecule itself. Our study is the first to couple a cellular vertex model describing mechanical properties of cells in a growing tissue, to an explicit model of an entire signaling pathway, including a freely diffusible component. We discuss potential applications and challenges of such an approach

A regulatory receptor network directs the range and output of the Wingless signal

The potent activity of Wnt/Wingless (Wg) signals necessitates sophisticated mechanisms that spatially and temporally regulate their distribution and range of action. The two main receptor components for Wg - Arrow (Arr) and Frizzled 2 (Fz2) - are transcriptionally downregulated by Wg signaling, thus forming gradients that oppose that of Wg. Here, we analyze the relevance of this transcriptional regulation for the formation of the Wg gradient in the Drosophila wing disc by combining in vivo receptor overexpression with an in silico model of Wg receptor interactions. Our experiments show that ubiquitous upregulation of Arr and Fz2 has no significant effects on Wg output, whereas clonal overexpression of these receptors leads to signaling discontinuities that have detrimental phenotypic consequences. These findings are supported by our in silico model for Wg diffusion and signal transduction, which suggests that abrupt changes in receptor levels causes discontinuities in Wg signaling. Furthermore, we identify a 200 bp regulatory element in the arr locus that can account for the Arr gradient, and we show that this is indirectly negatively controlled by Wg activity. Finally, we analyze the role of Frizzled 3 (Fz3) in this system and find that its expression, which is induced by Wg, contributes to the establishment of the Arr and Fz2 gradients through counteracting canonical signaling. Taken together, our results provide a model in which the regulatory network of Wg and the three receptor components account for the range and shape of this prototypical morphogen system

Graded Exposure for Chronic Low Back Pain in Older Adults: A Pilot Study

Background and Purpose: Fear-avoidance beliefs in older adults with chronic low back pain (CLBP) can lead to disability. Graded exposure-based active physical therapy could be an option to enhance physical ability in older patients with CLBP. The purpose of this study was to develop a standardized graded exposure treatment according to the fear-avoidance model of musculoskeletal pain for older patients with CLBP and to examine its effectiveness and feasibility in the German health care system. Methods: The study represents a phase I/phase II trial of a complex intervention. Taking a first step into the hierarchy of growing empirical evidence, a prospective 1-factor observational study was conducted with repeated measurements 1 week before and within 2 weeks after the intervention. Three physical therapists, who completed an introductory workshop, provided the treatment in the form of individual therapies. Sixteen participants 65 years or older with CLBP and perceived physical limitations were recruited. Four patient-reported outcome measures and semistructured interviews were conducted. The primary outcome was physical ability measured with the Hanover Functional Ability Questionnaire. Secondary outcomes were the numerical pain rating scale, and an age-specific and adapted 11-item short-form of the Patient Anxiety Symptom Scale, the KVS-D 65+, which quantified catastrophizing and avoidance beliefs. Fear of falling was measured with the Falls Efficacy Scale-International. For the analysis, Wilcoxon signed-rank test for paired samples and an a level of .05 were chosen. For the qualitative evaluation, semistructured interviews were conducted with the patients and physical therapists explored indicators of feasibility such as demands, acceptability, satisfaction, adaptation needs, and implementation. For content analysis, codes were primarily derived deductively and complemented by inductively derived new themes. Results: A significant increase in physical ability after the treatment was observed with an effect size (ES) of 0.95 (P = .008). With regard to secondary outcomes, there was a statistically signifi cant decrease in pain intensity (P = .029) and a reduction in catastrophizing (ES = 0.91; P = .021) and avoidance beliefs (ES = 1.37; P = .001). The interviews revealed good acceptance and satisfaction of the treatment by the patients and physical therapists. Conclusion: On the whole, the treatment appears effective and feasible. Apart from the benefits achieved by the participants, the study provides a basis for designing future studies at a higher level of evidence

Graded exposure for chronic low back pain in older adults: A pilot study

Fear-avoidance beliefs in older adults with chronic low back pain (CLBP) can lead to disability. Graded exposure-based active physical therapy could be an option to enhance physical ability in older patients with CLBP. The purpose of this study was to develop a standardized graded exposure treatment according to the fear-avoidance model of musculoskeletal pain for older patients with CLBP and to examine its effectiveness and feasibility in the German health care system.status: publishe