A fluorophore attached to nicotinic acetylcholine receptor beta M2 detects productive binding of agonist to the alpha delta site

To study conformational transitions at the muscle nicotinic acetylcholine (ACh) receptor (nAChR), a rhodamine fluorophore was tethered to a Cys side chain introduced at the beta-19' position in the M2 region of the nAChR expressed in Xenopus oocytes. This procedure led to only minor changes in receptor function. During agonist application, fluorescence increased by (Delta-F/F) approximate to 10%, and the emission peak shifted to lower wavelengths, indicating a more hydrophobic environment for the fluorophore. The dose-response relations for Delta-F agreed well with those for epibatidine-induced currents, but were shifted approximate to 100-fold to the left of those for ACh-induced currents. Because (i) epibatidine binds more tightly to the alpha-gamma-binding site than to the alpha-delta site and (ii) ACh binds with reverse-site selectivity, these data suggest that Delta-F monitors an event linked to binding specifically at the alpha-delta-subunit interface. In experiments with flash-applied agonists, the earliest detectable Delta-F occurs within milliseconds, i.e., during activation. At low [ACh] (less than or equal to 10 muM), a phase of Delta-F occurs with the same time constant as desensitization, presumably monitoring an increased population of agonist-bound receptors. However, recovery from Delta-F is complete before the slowest phase of recovery from desensitization (time constant approximate to 250 s), showing that one or more desensitized states have fluorescence like that of the resting channel. That conformational transitions at the alpha-delta-binding site are not tightly coupled to channel activation suggests that sequential rather than fully concerted transitions occur during receptor gating. Thus, time-resolved fluorescence changes provide a powerful probe of nAChR conformational changes

Inferring hidden Markov models from noisy time sequences: a method to alleviate degeneracy in molecular dynamics

We present a new method for inferring hidden Markov models from noisy time sequences without the necessity of assuming a model architecture, thus allowing for the detection of degenerate states. This is based on the statistical prediction techniques developed by Crutchfield et al., and generates so called causal state models, equivalent to hidden Markov models. This method is applicable to any continuous data which clusters around discrete values and exhibits multiple transitions between these values such as tethered particle motion data or Fluorescence Resonance Energy Transfer (FRET) spectra. The algorithms developed have been shown to perform well on simulated data, demonstrating the ability to recover the model used to generate the data under high noise, sparse data conditions and the ability to infer the existence of degenerate states. They have also been applied to new experimental FRET data of Holliday Junction dynamics, extracting the expected two state model and providing values for the transition rates in good agreement with previous results and with results obtained using existing maximum likelihood based methods.Comment: 19 pages, 9 figure

Corporate tax: what do stakeholders expect?

Motivated by the ongoing controversy surrounding corporate tax, this article presents a study that explores stakeholder expectations of corporate tax in the context of UK business. We conduct a qualitative analysis of in-depth interviews with representatives of community groups (NGOs/think tanks and special interest groups), as well as interviews with those representing business groups (business leaders and industry representatives). We then identify eight themes that together describe “what” companies need to do, “how” they need to do it, and “why” they need to do it, if they wish to appeal to a wide group of interested parties. We discuss our findings based on the corporate social responsibility literature and propose novel ways for community groups and business groups to connect on the topic of corporate tax, suggesting opportunities and themes for dialogue and potential steps to co-create solutions in a stakeholder society

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844–848

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000–3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons—Leu844, Cys845, Ala846, Leu847, and Gly848—located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844–848 exists and will be valuable in the management and genetic counseling of a significant number of individuals

A Mechanism for the Polarity Formation of Chemoreceptors at the Growth Cone Membrane for Gradient Amplification during Directional Sensing

Accurate response to external directional signals is essential for many physiological functions such as chemotaxis or axonal guidance. It relies on the detection and amplification of gradients of chemical cues, which, in eukaryotic cells, involves the asymmetric relocalization of signaling molecules. How molecular events coordinate to induce a polarity at the cell level remains however poorly understood, particularly for nerve chemotaxis. Here, we propose a model, inspired by single-molecule experiments, for the membrane dynamics of GABA chemoreceptors in nerve growth cones (GCs) during directional sensing. In our model, transient interactions between the receptors and the microtubules, coupled to GABA-induced signaling, provide a positive-feedback loop that leads to redistribution of the receptors towards the gradient source. Using numerical simulations with parameters derived from experiments, we find that the kinetics of polarization and the steady-state polarized distribution of GABA receptors are in remarkable agreement with experimental observations. Furthermore, we make predictions on the properties of the GC seen as a sensing, amplification and filtering module. In particular, the growth cone acts as a low-pass filter with a time constant ∼10 minutes determined by the Brownian diffusion of chemoreceptors in the membrane. This filtering makes the gradient amplification resistent to rapid fluctuations of the external signals, a beneficial feature to enhance the accuracy of neuronal wiring. Since the model is based on minimal assumptions on the receptor/cytoskeleton interactions, its validity extends to polarity formation beyond the case of GABA gradient sensing. Altogether, it constitutes an original positive-feedback mechanism by which cells can dynamically adapt their internal organization to external signals

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins

Working with Commercially Available Quantum Dots for Immunofluorescence on Tissue Sections

Quantum dots are semiconductor fluorescent nanocrystals that exhibit excellent characteristics compared with more commonly used organic fluorescent dyes. For many years quantum dot conjugated products have been available in multiple forms for fluorescence imaging of tissue sections under the trademark name Qdot®. They have much increased brightness, narrow emission spectrum, large Stokes shift and photostability compared with conventional organic fluorescent dyes, which together make them the fluorophores of choice for demanding requirements. Vivid Qdots are recent replacements for original Qdots, modified to improve brightness, however this has affected the fluorescence stability in commonly used conditions for immunohistochemistry. We present here our investigation of the stability of original and Vivid Qdots in solution and in immunohistochemistry, highlight the potential pitfalls and propose a protocol for stable and reliable multiplex staining with current commercially available original and Vivid Qdots

The Human Phenotype Ontology in 2024: phenotypes around the world.

The Human Phenotype Ontology (HPO) is a widely used resource that comprehensively organizes and defines the phenotypic features of human disease, enabling computational inference and supporting genomic and phenotypic analyses through semantic similarity and machine learning algorithms. The HPO has widespread applications in clinical diagnostics and translational research, including genomic diagnostics, gene-disease discovery, and cohort analytics. In recent years, groups around the world have developed translations of the HPO from English to other languages, and the HPO browser has been internationalized, allowing users to view HPO term labels and in many cases synonyms and definitions in ten languages in addition to English. Since our last report, a total of 2239 new HPO terms and 49235 new HPO annotations were developed, many in collaboration with external groups in the fields of psychiatry, arthrogryposis, immunology and cardiology. The Medical Action Ontology (MAxO) is a new effort to model treatments and other measures taken for clinical management. Finally, the HPO consortium is contributing to efforts to integrate the HPO and the GA4GH Phenopacket Schema into electronic health records (EHRs) with the goal of more standardized and computable integration of rare disease data in EHRs