Science Communication Training Imparts Confidence and Influences Public Engagement Activity.

The impacts of science are felt across all socio-ecological levels, ranging from the individual to societal. In order to adapt or respond to scientific discoveries, novel technologies, or biomedical or environmental challenges, a fundamental understanding of science is necessary. However, antiscientific rhetoric, mistrust in science, and the dissemination of misinformation hinder the promotion of science as a necessary and beneficial component of our world. Scientists can promote scientific literacy by establishing dialogues with nonexperts, but they may find a lack of formal training as a barrier to public engagement. To address this, the American Society for Biochemistry and Molecular Biology (ASBMB) launched the Art of Science Communication course in 2015 in order to provide scientists at all career stages with introductory science communication training. In 2020, we conducted a retrospective survey of former participants to evaluate how the course had impacted participants' science communication behaviors and their confidence engaging with nonexperts, as well as other benefits to their professional development. We found that scientists were significantly more likely to communicate with nonexpert audiences following the course compared to before (77% versus 51%; P < 0.0001). In addition, quantitative and qualitative data suggested that scientists were more confident in their ability to communicate science after completing the course (median of 8, standard deviation [SD] of 0.98 versus median of 5, SD of 1.57; P < 0.0001). Qualitative responses from participants supported quantitative findings. This suggested that the Art of Science Communication course is highly effective at improving the confidence of scientists to engage with the public and other nonexpert audiences regardless of career status. These data-driven perspectives provide a rationale for the implementation of broadly accessible science communication training programs that promote public engagement with science.

An AMPK biosensor for Caenorhabditis elegans.

Adenosine monophosphate-activated kinase (AMPK) functions in a broad spectrum of cellular stress response pathways. Investigation of AMPK activity has been limited to whole-organism analyses in Caenorhabditis elegans which does not allow for observations of cellular heterogeneity, temporal dynamics, or correlation with physiological states in real time. We codon adapted the genetically-coded AMPK biosensor, called AMPKAR-EV, for use in C. elegans . We report heterogeneity of activation in different tissues (intestine, neurons, muscle) and test the biosensor in the context of two missense mutations affecting residues T243 and S244 on the AMPK α subunit, AAK-2, which are predicted regulatory sites.

Interneurons Regulate Locomotion Quiescence via Cyclic Adenosine Monophosphate Signaling During Stress-Induced Sleep in Caenorhabditis elegans.

Sleep is evolutionarily conserved, thus studying simple invertebrates such as Caenorhabditis elegans can provide mechanistic insight into sleep with single cell resolution. A conserved pathway regulating sleep across phylogeny involves cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger that functions in neurons by activating protein kinase A. C. elegans sleep in response to cellular stress caused by environmental insults [stress-induced sleep (SIS)], a model for studying sleep during sickness. SIS is controlled by simple neural circuitry, thus allowing for cellular dissection of cAMP signaling during sleep. We employed a red-light activated adenylyl cyclase, IlaC22, to identify cells involved in SIS regulation. We found that pan-neuronal activation of IlaC22 disrupts SIS through mechanisms independent of the cAMP response element binding protein. Activating IlaC22 in the single DVA interneuron, the paired RIF interneurons, and in the CEPsh glia identified these cells as wake-promoting. Using a cAMP biosensor, epac1-camps, we found that cAMP is decreased in the RIF and DVA interneurons by neuropeptidergic signaling from the ALA neuron. Ectopic overexpression of sleep-promoting neuropeptides coded by flp-13 and flp-24, released from the ALA, reduced cAMP in the DVA and RIFs, respectively. Overexpression of the wake-promoting neuropeptides coded by pdf-1 increased cAMP levels in the RIFs. Using a combination of optogenetic manipulation and in vivo imaging of cAMP we have identified wake-promoting neurons downstream of the neuropeptidergic output of the ALA. Our data suggest that sleep- and wake-promoting neuropeptides signal to reduce and heighten cAMP levels during sleep, respectively.

Phosphodiesterase expression in the normal and failing heart.

The number of patients with heart failure with reduced ejection fraction (HFrEF) and preserved ejection fraction (HFpEF) is increasing, and for HFpEF, no therapies have clinical benefit. It has been hypothesized that PKG attenuates pathological remodelling, and increasing cGMP would be beneficial for patients with HF. However, neither the RELAX nor NEAT-HFpEF trial showed benefit. But there is still enthusiasm for increasing cGMP in patients with HF, which highlight the need to determine the expression of PDEs in cardiac muscle. This study used immunoblotting to examine the expression of the PDEs that have been suggested to be targets for therapy of HF in both canines (normal and HFpEF) and humans (normal and HFrEF). Our results demonstrate PDE1C and PDE3A are expressed in cardiac muscle, but we could not detect the expression of PDE2A, PDE5A, PDE7A and PDE9A in cardiac tissue lysates from either normal or failing hearts. Thus, one should not expect a clinical benefit for a therapy targeting these PDEs in heart failure, which highlights the importance of rigorous demonstration of the target of therapy prior to undertaking a clinical trial.

Sofosbuvir and simeprevir is effective for recurrent hepatitis C in liver transplant recipients.

BACKGROUND & AIMS: Hepatitis C is the most common indication for liver transplantation (LT). Recurrent infection is universal and can lead to progressive liver disease. Widespread use of interferon-based therapy has been limited by intolerability and adverse effects. METHODS: We retrospectively evaluated the safety, tolerability, and efficacy of sofosbuvir and simeprevir in the treatment of recurrent hepatitis C in adult (age >18) LT recipients. RESULTS: Seventy-six percent of the recipients were male and the mean age [±standard deviation (SD)] was 61 (±6.0) years. The mean time (±SD) from LT to treatment initiation was 71.8 (±77.1) months. Of the 26 patients with viral levels measured 4 weeks after starting antiviral therapy, 58% were undetectable. At the end of therapy, viral load was undetectable in all transplant recipients. The 12 week sustained viral response (SVR) was 93%. All recipients were able to complete therapy and no patients required growth factors of blood product transfusion during treatment. No patient required drug interruption of their immunosuppressant therapy. CONCLUSION: The use of sofosbuvir and simeprevir is efficacious, safe, and tolerable and should be considered in LT recipients with recurrent HCV who are candidates for antiviral therapy.

The pathogenic A391E mutation in FGFR3 induces a structural change in the transmembrane domain dimer.

Fibroblast growth factor receptor 3 (FGFR3) is a single-pass membrane protein and a member of the receptor tyrosine kinase family of proteins that is involved in the regulation of skeletal growth and development. FGFR3 has three distinct domains: the ligand binding extracellular domain, the cytosolic kinase domain and the transmembrane domain (TMD). Previous work with the isolated FGFR3 TMD has shown that it has the ability to dimerize. Clinical and genetic studies have also correlated mutations in the TMD with a variety of skeletal and cranial dysplasias and cancer. Although the structures of the extracellular and cytosolic domains of FGFR3 have been solved, the structure of the TMD dimer is still unknown. Furthermore, very little is known regarding the effects of pathogenic mutations on the TMD dimer structure. We, therefore, carried out ToxR activity assays to determine the role of the SmXXXSm motif in the dimerization of the FGFR3 TMD. This motif has been shown to drive the association of many transmembrane proteins. Our results indicate that the interaction between wild-type FGFR3 TMDs is not mediated by two adjacent SmXXXSm motifs. In contrast, studies using the TMD carrying the pathogenic A391E mutation suggest that the motifs play a role in the dimerization of the mutant TMD. Based on these observations, here we report a new mechanistic model in which the pathogenic A391E mutation induces a structural change that leads to the formation of a more stable dimer.

Increased expression of the integral membrane proteins EGFR and FGFR3 in anti-apoptotic Chinese hamster ovary cell lines.

Membrane proteins such as receptor tyrosine kinases (RTKs) have a vital role in many cellular functions, making them potential targets for therapeutic research. In this study, we investigated the coexpression of the anti-apoptosis gene Bcl-x(L) with model membrane proteins as a means of increasing membrane protein expression in mammalian cells. Chinese hamster ovary (CHO) cells expressing heterologous Bcl-x(L) and wild-type CHO cells were transfected with either epidermal growth factor receptor or fibroblast growth factor receptor 3. The CHO-Bcl-x(L) cell lines showed increased expression of both RTK proteins as compared with the wild-type CHO cell lines in transient expression analysis, as detected by Western blot and flow cytometry after 15 days of antibiotic selection in stable expression pools. Increased expression was also seen in clonal isolates from the CHO-Bcl-x(L) cell lines, whereas the clonal cell line expression was minimal in wild-type CHO cell lines. Our results demonstrate that application of the anti-apoptosis gene Bcl-x(L) can increase expression of RTK proteins in CHO cells. This approach may be applied to improve stable expression of other membrane proteins in the future using mammalian cell lines with Bcl-x(L) or perhaps other anti-apoptotic genes.

Transmembrane helix dimerization: beyond the search for sequence motifs.

Studies of the dimerization of transmembrane (TM) helices have been ongoing for many years now, and have provided clues to the fundamental principles behind membrane protein (MP) folding. Our understanding of TM helix dimerization has been dominated by the idea that sequence motifs, simple recognizable amino acid sequences that drive lateral interaction, can be used to explain and predict the lateral interactions between TM helices in membrane proteins. But as more and more unique interacting helices are characterized, it is becoming clear that the sequence motif paradigm is incomplete. Experimental evidence suggests that the search for sequence motifs, as mediators of TM helix dimerization, cannot solve the membrane protein folding problem alone. Here we review the current understanding in the field, as it has evolved from the paradigm of sequence motifs into a view in which the interactions between TM helices are much more complex. This article is part of a Special Issue entitled: Membrane protein structure and function.

Assembly of the m2 tetramer is strongly modulated by lipid chain length.

The influenza virus matrix protein 2 (M2) assembles into a tetramer in the host membrane during viral uncoating and maturation. It has been used as a model system to understand the relative contributions of protein-lipid and protein-protein interactions to membrane protein structure and association. Here we investigate the effect of lipid chain length on the association of the M2 transmembrane domain into tetramers using Förster resonance energy transfer. We observe that the interactions between the M2 helices are much stronger in 1,2-dilauroyl-sn-glycero-3-phosphocholine than in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Thus, lipid chain length and bilayer thickness not only modulate peptide interactions, but could also be a major determinant of the association of transmembrane helices into functional membrane protein oligomers.

Receptor tyrosine kinase transmembrane domains: Function, dimer structure and dimerization energetics.

The transmembrane (TM) domains of receptor tyrosine kinases (RTKs) play an active role in signaling. They contribute to the stability of full-length receptor dimers and to maintaining a signaling-competent dimeric receptor conformation. In an exciting new development, two structures of RTK TM domains have been solved, a break-through achievement in the field. Here we review these structures, and we discuss recent studies of RTK TM domain dimerization energetics, possible synergies between domains, and the effects of pathogenic RTK TM mutations on structure and dimerization.

Increased expression of the integral membrane protein ErbB2 in Chinese hamster ovary cells expressing the anti-apoptotic gene Bcl-xL.

Receptor tyrosine kinases (RTKs) are the second largest family of membrane receptors and play a key role in the regulation of vital cellular processes, such as control of cell growth, differentiation, metabolism, and migration. The production of whole-length RTKs in large quantities for biophysical or structural characterization, however, is a challenge. In this study, a cell engineering strategy using the anti-apoptotic Bcl-2 family protein, Bcl-x(L), was tested as a potential method for increasing stable expression levels of a recombinant RTK membrane protein in Chinese hamster ovary (CHO) cells. Wild-type and CHO cells stably overexpressing heterologous Bcl-x(L) were transformed with the gene for a model RTK membrane protein, ErbB2, on a plasmid also containing the Zeocin resistance gene. While CHO cells exhibited a gradual decrease in expression with passaging, CHO-bcl-x(L) cells offered an increased and sustained level of ErbB2 expression following continuous passaging over more than 33 days in culture. The increased ErbB2 expression in CHO-bcl-x(L) cells was evident both in stable transfected pools and in clonal isolates, and demonstrated both in Western blot analysis and flow cytometry. Furthermore, the sustained high-level protein expression in CHO-bcl-x(L) cells does not alter the correct membrane localization of the ErbB2 protein. Our results demonstrate that cellular engineering, specifically anti-apoptosis engineering, can provide increased and stable ErbB2 membrane protein expression in mammalian cells. This approach may also be useful for other membrane proteins in which large quantities are needed for biophysical and structural studies.

Utility of surface-supported bilayers in studies of transmembrane helix dimerization.

This review focuses on the methods that are available to study transmembrane (TM) helix dimerization in membrane-like environments (either bacterial membranes or lipid bilayers, as mimics of the eukaryotic cellular membrane), with an emphasis on the utility of surface-supported bilayers in such studies.

Quantitative measurements of protein interactions in a crowded cellular environment.

Quantitative measurements of protein interaction strengths, crucial for describing signaling networks and predicting cellular responses to environmental stimuli, are typically performed in dilute buffer solutions. However, protein-protein interactions in cells occur within the context of a crowded system, which is characterized by a high macromolecular concentration. In this paper, we explore the utility of cell-derived vesicles as a model crowded environment for quantitative FRET measurements of protein-protein interactions. We show that the FRET efficiency, and the donor and acceptor concentrations, can be calculated in each vesicle. We also introduce the "quantitative imaging Foster resonance energy transfer" method as a tool that can yield protein interaction strengths within these vesicles.

Surface supported bilayer platform for studies of lateral association of proteins in membranes (Mini Review).

Here, the authors review how surface supported bilayers can be engineered and how Forster resonance energy transfer (FRET) can be used to quantify interactions between transmembrane peptides in these bilayers. The requirements for the surface supported platform are (1) lateral mobility of the peptides, (2) transmembrane orientation of the peptides, and (3) capabilities for FRET measurements. To satisfy these requirements, a new assembly method, termed "directed assembly" was developed. This assembly method could have broad utility in basic studies of proteins in membranes and in biotechnological applications.

Effect of pathogenic cysteine mutations on FGFR3 transmembrane domain dimerization in detergents and lipid bilayers.

Mutations in fibroblast growth factor receptors are known as the genetic basis of skeletal growth disorders. The mechanism of pathogenesis, as determined by mutation-induced changes in receptor structure, interactions, and function, is elusive. Here we study three pathogenic Cys mutations, associated with either thanatophoric dysplasia or achondroplasia, in the TM domain of fibroblast growth factor receptors 3 (FGFR3). We characterize the dimerization propensities of the mutant TM domains in detergents and in lipid bilayers, in the presence and absence of reducing agents, and compare them to previous measurements of wild-type. We find that the Cys mutations increase the propensity for dimerization in detergent, with the Cys370 mutant exhibiting the highest propensity for disulfide bond formation, the Cys371 mutant having an intermediate propensity, and Cys375 the lowest. Thus, disulfide bonds readily form in detergents, with efficiency that correlates with the severity of the phenotype. In lipid bilayers, however, the Cys370 mutant, which dimerizes strongly in detergent, behaves as the wild-type, suggesting that Cys370-mediated disulfide bonds do not form between the isolated TM domains in bilayers. Thus, the nature of the hydrophobic environment plays an important role in defining the structure and flexibility of transmembrane dimers. These results and previous findings from cellular studies lead us to propose a conformational flexibility mechanism of receptor stabilization as a basis for disregulated FGFR3 signaling in thanatophoric dysplasia and achondroplasia.

Surface-supported bilayers with transmembrane proteins: role of the polymer cushion revisited.

Protein lateral mobility in surface-supported bilayers is often much lower than the mobility of the lipids. In the present study we explore whether the incorporation of a PEG cushion between the bilayer and the substrate increases the lateral mobility of transmembrane proteins in bilayers produced via directed assembly, a method based on Langmuir-Blodgett deposition techniques. In our experiments, the PEG cushions were incorporated by adding PEG lipids to the protein/lipid monolayer at the air/water interface, at the first step of bilayer assembly. The protein and lipid mobilities in 160 different bilayers, with various PEG molecular weights and PEG lipid concentrations, were measured and compared. We found that the measured diffusion coefficients do not depend on the PEG molecular weight or the PEG lipid concentration and are very similar to the values measured in the absence of PEG. Therefore, contrary to our expectations, we found that a PEG cushion does not necessarily increase protein mobility, suggesting that the low protein mobility is not a consequence of protein-substrate interactions. Furthermore, we showed that the low protein mobility is not due to protein aggregation. The major determinant of protein mobility in surface-supported bilayer systems appears to be the method of bilayer assembly. While proteins were always mobile if the bilayers were prepared using the directed assembly method, in the presence and absence of a PEG cushion, other bilayer assembly protocols resulted in complete lack of protein mobility.

Spectral Förster resonance energy transfer detection of protein interactions in surface-supported bilayers.

Förster resonance energy transfer (FRET), a fluorescence detection technique, is often used for sensing molecular interactions in solution and in membranes. Here we show that (1) FRET spectra can be recorded in single bilayers, supported on a surface, and (2) the fluorescein/rhodamine dye pair is an adequate reporter of FRET when spectral detection is used. Thus, measurements pertaining to molecular interactions in membranes can be carried out in supported bilayers. Spectral FRET has advantages over imaging FRET, which monitors only signal amplitudes at certain wavelength. There are also advantages to performing spectral FRET measurements in supported bilayers as compared to free liposomes in suspension. However, the spectral properties of dyes can be altered in an unexpected manner in an ordered bilayer structure on a surface, such that fluorescence detection in surface-supported bilayers is not always trivial.

Role of receptor tyrosine kinase transmembrane domains in cell signaling and human pathologies.

Receptor tyrosine kinases (RTKs) conduct biochemical signals via lateral dimerization in the plasma membrane, and their transmembrane (TM) domains play an important role in the dimerization process. Here we present two models of RTK-mediated signaling, and we discuss the role of the TM domains within the framework of these two models. We summarize findings of single-amino acid mutations in RTK TM domains that induce unregulated signaling and, as a consequence, pathological phenotypes. We review the current knowledge of pathology induction mechanisms due to these mutations, focusing on the structural and thermodynamic basis of pathogenic dimer stabilization.

The achondroplasia mutation does not alter the dimerization energetics of the fibroblast growth factor receptor 3 transmembrane domain.

The Gly380 --> Arg mutation in the TM domain of fibroblast growth factor receptor 3 (FGFR3) of the RTK family is linked to achondroplasia, the most common form of human dwarfism. The molecular mechanism of pathology induction is under debate, and two different mechanisms have been proposed to contribute to pathogenesis: (1) Arg380-mediated FGFR3 dimer stabilization and (2) slow downregulation of the activated mutant receptors. Here we show that the Gly380 --> Arg mutation does not alter the dimerization energetics of the FGFR3 transmembrane domain in detergent micelles or in lipid bilayers. This result indicates that pathogenesis in achondroplasia cannot be explained simply by a higher dimerization propensity of the mutant FGFR3 TM domain, thus highlighting the importance of the observed slow downregulation in phenotype induction.

Transmembrane helix heterodimerization in lipid bilayers: probing the energetics behind autosomal dominant growth disorders.

Here, we show that the energetics of transmembrane helix heterodimer formation can be characterized in liposomes using Förster resonance energy transfer (FRET). We present the theory and the protocol for measuring the free energy of heterodimerization, and the total (hetero and homo-dimeric) dimer fraction. We use the presented methodology to determine the propensity for heterodimer formation between wild-type fibroblast growth factor receptor 3 (FGFR3) transmembrane domain and the Ala391Glu mutant, linked to Crouzon syndrome with acanthosis nigricans.