Control of meiotic entry by dual inhibition of a key mitotic transcription factor.

The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a functional homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.

Coaching with audiovisual technology in acute-care hospital settings: systematic review.

BACKGROUND: Surgical coaching programmes are a means of improving surgeon performance. Embedded audiovisual technology has the potential to further enhance participant benefit and scalability of coaching. The objective of this systematic review was to evaluate how audiovisual technology has augmented coaching in the acute-care hospital setting and to characterize its impact on outcomes. METHODS: A systematic review was conducted, searching PubMed, Ovid MEDLINE, Embase, PsycInfo, and CINAHL databases using PRISMA. Eligible studies described a coaching programme that utilized audiovisual technology, involved at least one coach-coachee interaction, and included healthcare professionals from the acute-care hospital environment. The risk of bias 2 tool and grading of recommendations, assessment, development, and evaluations (GRADE) framework were used to evaluate studies. Synthesis without meta-analysis was performed, creating harvest plots of three coaching outcomes: technical skills, self-assessment/feedback, and non-technical skills. RESULTS: Of 10 458 abstracts screened, 135 full texts were reviewed, and 21 studies identified for inclusion. Seventeen studies were conducted within surgical specialties and six classes of audiovisual technology were utilized. An overall positive direction of effect was demonstrated for studies measuring improvement of either technical skills or non-technical skills. Direction of effect for self-assessment/feedback was weakly positive. CONCLUSION: Audiovisual technology has been used successfully in coaching programmes within acute-care hospital settings to facilitate or assess coaching, with a positive impact on outcome measures. Future studies may address the additive benefits of video over in-person observation and enhance the certainty of evidence that coaching impacts on surgeon performance, surgeon well-being, and patient outcomes.

Evaluating eating behavior traits of virtual targets: Attitudes and empathy.

There are many common stereotypes related to food consumption and eating behaviors that are considered indicative of one's personal character. Negative evaluations of an individual based on their eating behaviors may lead to stigmatization and other harmful psychosocial outcomes. Using vignette scenarios, we examined 582 participants' attitudes towards two target characters who exhibited bitter food dislike and high reward-based eating drive respectively. In open-ended text responses, participants were more likely to respond negatively and use stigmatizing language when describing the character with high reward-based eating drive versus the character with bitter food dislike. In addition, empathic responses depended on whether participants believed they, themselves, exhibited reward-based eating drive. Participants tended to be more empathetic and more positive towards the target who shared their own reward-based eating behaviors. Interestingly, the same was not true for those who shared bitter food distaste. These results suggest that eating behaviors that are perceived as more controllable and unusual may be more negatively perceived. Targeted education or support to reduce negative attitudes about such traits may be beneficial.

A positively tuned voltage indicator for extended electrical recordings in the brain.

Genetically encoded voltage indicators (GEVIs) enable optical recording of electrical signals in the brain, providing subthreshold sensitivity and temporal resolution not possible with calcium indicators. However, one- and two-photon voltage imaging over prolonged periods with the same GEVI has not yet been demonstrated. Here, we report engineering of ASAP family GEVIs to enhance photostability by inversion of the fluorescence-voltage relationship. Two of the resulting GEVIs, ASAP4b and ASAP4e, respond to 100-mV depolarizations with ≥180% fluorescence increases, compared with the 50% fluorescence decrease of the parental ASAP3. With standard microscopy equipment, ASAP4e enables single-trial detection of spikes in mice over the course of minutes. Unlike GEVIs previously used for one-photon voltage recordings, ASAP4b and ASAP4e also perform well under two-photon illumination. By imaging voltage and calcium simultaneously, we show that ASAP4b and ASAP4e can identify place cells and detect voltage spikes with better temporal resolution than commonly used calcium indicators. Thus, ASAP4b and ASAP4e extend the capabilities of voltage imaging to standard one- and two-photon microscopes while improving the duration of voltage recordings.

Improving the quality of co-evolution intermolecular contact prediction with DisVis.

The steep rise in protein sequences and structures has paved the way for bioinformatics approaches to predict residue-residue interactions in protein complexes. Multiple sequence alignments are commonly used in contact predictions to identify co-evolving residues. These contacts, however, often include false positives (FPs), which may impair their use to predict three dimensional structures of biomolecular complexes and affect the accuracy of the generated models. Previously, we have developed DisVis to identify FP in mass spectrometry cross-linking data. DisVis allows to assess the accessible interaction space between two proteins consistent with a set of distance restraints. Here, we investigate if a similar approach could be applied to co-evolution predicted contacts in order to improve their precision prior to using them for modeling. We analyze co-evolution contact predictions with DisVis for a set of 26 protein-protein complexes. The DisVis-reranked and the original co-evolution contacts are then used to model the complexes with our integrative docking software HADDOCK using different filtering scenarios. Our results show that HADDOCK is robust with respect to the precision of the predicted contacts due to the 50% random contact removal during docking and can enhance the quality of docking predictions when combined with DisVis filtering for low precision contact data. DisVis can thus have a beneficial effect on low quality data, but overall HADDOCK can accommodate FP restraints without negatively impacting the quality of the resulting models. Other more precision-sensitive docking protocols might, however, benefit from the increased precision of the predicted contacts after DisVis filtering.

Control of meiotic entry by dual inhibition of a key mitotic transcription factor.

The mitosis to meiosis transition requires dynamic changes in gene expression, but whether and how the mitotic transcriptional machinery is regulated during this transition is unknown. In budding yeast, SBF and MBF transcription factors initiate the mitotic gene expression program. Here, we report two mechanisms that work together to restrict SBF activity during meiotic entry: repression of the SBF-specific Swi4 subunit through LUTI-based regulation and inhibition of SBF by Whi5, a homolog of the Rb tumor suppressor. We find that untimely SBF activation causes downregulation of early meiotic genes and delays meiotic entry. These defects are largely driven by the SBF-target G1 cyclins, which block the interaction between the central meiotic regulator Ime1 and its cofactor Ume6. Our study provides insight into the role of SWI4LUTI in establishing the meiotic transcriptional program and demonstrates how the LUTI-based regulation is integrated into a larger regulatory network to ensure timely SBF activity.

Cyclization and Docking Protocol for Cyclic Peptide-Protein Modeling Using HADDOCK2.4.

An emerging class of therapeutic molecules are cyclic peptides with over 40 cyclic peptide drugs currently in clinical use. Their mode of action is, however, not fully understood, impeding rational drug design. Computational techniques could positively impact their design, but modeling them and their interactions remains challenging due to their cyclic nature and their flexibility. This study presents a step-by-step protocol for generating cyclic peptide conformations and docking them to their protein target using HADDOCK2.4. A dataset of 30 cyclic peptide-protein complexes was used to optimize both cyclization and docking protocols. It supports peptides cyclized via an N- and C-terminus peptide bond and/or a disulfide bond. An ensemble of cyclic peptide conformations is then used in HADDOCK to dock them onto their target protein using knowledge of the binding site on the protein side to drive the modeling. The presented protocol predicts at least one acceptable model according to the critical assessment of prediction of interaction criteria for each complex of the dataset when the top 10 HADDOCK-ranked single structures are considered (100% success rate top 10) both in the bound and unbound docking scenarios. Moreover, its performance in both bound and fully unbound docking is similar to the state-of-the-art software in the field, Autodock CrankPep. The presented cyclization and docking protocol should make HADDOCK a valuable tool for rational cyclic peptide-based drug design and high-throughput screening.

Interface refinement of low- to medium-resolution Cryo-EM complexes using HADDOCK2.4.

A wide range of cellular processes requires the formation of multimeric protein complexes. The rise of cryo-electron microscopy (cryo-EM) has enabled the structural characterization of these protein assemblies. The density maps produced can, however, still suffer from limited resolution, impeding the process of resolving structures at atomic resolution. In order to solve this issue, monomers can be fitted into low- to medium-resolution maps. Unfortunately, the models produced frequently contain atomic clashes at the protein-protein interfaces (PPIs), as intermolecular interactions are typically not considered during monomer fitting. Here, we present a refinement approach based on HADDOCK2.4 to remove intermolecular clashes and optimize PPIs. A dataset of 14 cryo-EM complexes was used to test eight protocols. The best-performing protocol, consisting of a semi-flexible simulated annealing refinement with centroid restraints on the monomers, was able to decrease intermolecular atomic clashes by 98% without significantly deteriorating the quality of the cryo-EM density fit.

Gαi1 inhibition mechanism of ATP-bound adenylyl cyclase type 5.

Conversion of adenosine triphosphate (ATP) to the second messenger cyclic adenosine monophosphate (cAMP) is an essential reaction mechanism that takes place in eukaryotes, triggering a variety of signal transduction pathways. ATP conversion is catalyzed by the enzyme adenylyl cyclase (AC), which can be regulated by binding inhibitory, Gαi, and stimulatory, Gαs subunits. In the past twenty years, several crystal structures of AC in isolated form and complexed to Gαs subunits have been resolved. Nevertheless, the molecular basis of the inhibition mechanism of AC, induced by Gαi, is still far from being fully understood. Here, classical molecular dynamics simulations of the isolated holo AC protein type 5 and the holo binary complex AC5:Gαi have been analyzed to investigate the conformational impact of Gαi association on ATP-bound AC5. The results show that Gαi appears to inhibit the activity of AC5 by preventing the formation of a reactive ATP conformation.

Structure of a GRK5-Calmodulin Complex Reveals Molecular Mechanism of GRK Activation and Substrate Targeting.

The phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) facilitates arrestin binding and receptor desensitization. Although this process can be regulated by Ca2+-binding proteins such as calmodulin (CaM) and recoverin, the molecular mechanisms are poorly understood. Here, we report structural, computational, and biochemical analysis of a CaM complex with GRK5, revealing how CaM shapes GRK5 response to calcium. The CaM N and C domains bind independently to two helical regions at the GRK5 N and C termini to inhibit GPCR phosphorylation, though only the C domain interaction disrupts GRK5 membrane association, thereby facilitating cytoplasmic translocation. The CaM N domain strongly activates GRK5 via ordering of the amphipathic αN-helix of GRK5 and allosteric disruption of kinase-RH domain interaction for phosphorylation of cytoplasmic GRK5 substrates. These results provide a framework for understanding how two functional effects, GRK5 activation and localization, can cooperate under control of CaM for selective substrate targeting by GRK5.

Regulation of adenylyl cyclase 5 in striatal neurons confers the ability to detect coincident neuromodulatory signals.

Long-term potentiation and depression of synaptic activity in response to stimuli is a key factor in reinforcement learning. Strengthening of the corticostriatal synapses depends on the second messenger cAMP, whose synthesis is catalysed by the enzyme adenylyl cyclase 5 (AC5), which is itself regulated by the stimulatory Gαolf and inhibitory Gαi proteins. AC isoforms have been suggested to act as coincidence detectors, promoting cellular responses only when convergent regulatory signals occur close in time. However, the mechanism for this is currently unclear, and seems to lie in their diverse regulation patterns. Despite attempts to isolate the ternary complex, it is not known if Gαolf and Gαi can bind to AC5 simultaneously, nor what activity the complex would have. Using protein structure-based molecular dynamics simulations, we show that this complex is stable and inactive. These simulations, along with Brownian dynamics simulations to estimate protein association rates constants, constrain a kinetic model that shows that the presence of this ternary inactive complex is crucial for AC5's ability to detect coincident signals, producing a synergistic increase in cAMP. These results reveal some of the prerequisites for corticostriatal synaptic plasticity, and explain recent experimental data on cAMP concentrations following receptor activation. Moreover, they provide insights into the regulatory mechanisms that control signal processing by different AC isoforms.

Association of Both Inhibitory and Stimulatory Gα Subunits Implies Adenylyl Cyclase 5 Deactivation.

Adenylyl cyclase (AC) generates cyclic AMP required for a variety of cellular functions, and its regulation plays a major role in cellular signal transduction in eukaryotes and prokaryotes. All membrane-bound AC isoforms in eukaryotes can be activated by stimulatory G-proteins, but only AC1, AC5, and AC6 can be both stimulated and inhibited by active Gα subunits, Gαs and Gαi, respectively. In principle, these Gαi-sensitive AC isoforms could form both binary and ternary complexes with Gα subunits due to the noncompetitive association of inhibitory and stimulatory Gα. However, the formation and possible catalytic activity of a putative ternary complex have not yet been experimentally confirmed due to its proposed short-lived nature. Here, the catalytic activity of such a ternary complex consisting of apo AC5, stimulatory Gαolf, and inhibitory Gαi1 is investigated via classical molecular dynamics simulations. Trajectories of inhibited and stimulated binary complexes, AC5:Gαi1 and AC5:Gαolf, respectively, as well as Gα-free AC5 were also obtained to compare the sampled AC5 conformation in the ternary complex to those sampled under different Gα conditions. This comparison suggests that association of both Gα subunits results in an AC5 conformation similar to that sampled by the AC5:Gαi1 complex, indicating that the ternary complex mainly samples an inactive conformation.

Erratum: Publisher's Note: "Implications of short time scale dynamics on long time processes" (Struct. Dyn. 4, 061507 (2017)].

[This corrects the article DOI: 10.1063/1.4996448.].

How Rhodopsin Tunes the Equilibrium between Protonated and Deprotonated Forms of the Retinal Chromophore.

Rhodopsin is a photoactive G-protein-coupled receptor (GPCR) that converts dim light into a signal for the brain, leading to eyesight. Full activation of this GPCR is achieved after passing through several steps of the protein's photoactivation pathway. Key events of rhodopsin activation are the initial cis-trans photoisomerization of the covalently bound retinal moiety followed by conformational rearrangements and deprotonation of the chromophore's protonated Schiff base (PSB), which ultimately lead to full activation in the meta II state. PSB deprotonation is crucial for achieving full activation of rhodopsin; however, the specific structural rearrangements that have to take place to induce this pKa shift are not well understood. Classical molecular dynamics (MD) simulations were employed to identify intermediate states after the cis-trans isomerization of rhodopsin's retinal moiety. In order to select the intermediate state in which PSB deprotonation is experimentally known to occur, the validity of the intermediate configurations was checked through an evaluation of the optical properties in comparison with experiment. Subsequently, the selected state was used to investigate the molecular factors that enable PSB deprotonation at body temperature to obtain a better understanding of the difference between the protonated and the deprotonated state of the chromophore. To this end, the deprotonation reaction has been investigated by applying QM/MM MD simulations in combination with thermodynamic integration. The study shows that, compared to the inactive 11-cis-retinal case, trans-retinal rhodopsin is able to undergo PSB deprotonation due to a change in the conformation of the retinal and a consequent alteration in the hydrogen-bond (HB) network in which PSB and the counterion Glu113 are embedded. Besides the retinal moiety and Glu113, also two water molecules as well as Thr94 and Gly90 that are related to congenital night blindness are part of this essential HB network.

Does Proton Conduction in the Voltage-Gated H+ Channel hHv1 Involve Grotthuss-Like Hopping via Acidic Residues?

Hv1s are ubiquitous highly selective voltage-gated proton channels involved in male fertility, immunology, and the invasiveness of certain forms of breast cancer. The mechanism of proton extrusion in Hv1 is not yet understood, while it constitutes the first step toward the design of high-affinity drugs aimed at this important pharmacological target. In this contribution, we explore the details of the mechanism via an integrative approach, using classical and QM/MM molecular dynamics simulations of a monomeric hHv1 model. We propose that protons localize in three binding sites along the channel lumen, formed by three pairs of conserved negatively charged residues lining the pore: D174/E153, D112/D185, and E119/D123. Local rearrangements, involving notably a dihedral transition of F150, a conserved phenylalanine lining the permeation pathway, appear to allow protons to hop from one acidic residue to the next through a bridging water molecule. These results constitute a first attempt at rationalizing hHv1 selectivity for H+ and the role played by D112 in this process. They pave the way for further quantitative characterization of H+ transport in hHv1.

Effect of N-Terminal Myristoylation on the Active Conformation of Gαi1-GTP.

G proteins are part of the G-protein-coupled receptor (GPCR) signal transduction cascade in which they transfer a signal from the membrane-embedded GPCR to other proteins in the cell. In the case of the inhibitory G-protein heterotrimer, permanent N-terminal myristoylation can transiently localize the Gαi subunit at the membrane as well as crucially influence Gαi's function in the GTP-bound conformation. The attachment of lipids to proteins is known to be essential for membrane trafficking; however, our results suggest that lipidation is also important for protein-protein interactions during signal transduction. Here we investigate the effect of myristoylation on the structure and dynamics of soluble Gαi1 and its possible implication for signal transduction. A 2 µs classical molecular dynamics simulation of a myristoylated Gαi1-GTP complex suggests that the myristoyl-induced conformational changes of the switch II and alpha helical domains create new possibilities for protein-protein interactions and emphasize the importance of permanent lipid attachment for the conformation and functional tunability of signaling proteins.

Implications of short time scale dynamics on long time processes.

This review provides a comprehensive overview of the structural dynamics in topical gas- and condensed-phase systems on multiple length and time scales. Starting from vibrationally induced dissociation of small molecules in the gas phase, the question of vibrational and internal energy redistribution through conformational dynamics is further developed by considering coupled electron/proton transfer in a model peptide over many orders of magnitude. The influence of the surrounding solvent is probed for electron transfer to the solvent in hydrated I-. Next, the dynamics of a modified PDZ domain over many time scales is analyzed following activation of a photoswitch. The hydration dynamics around halogenated amino acid side chains and their structural dynamics in proteins are relevant for iodinated TyrB26 insulin. Binding of nitric oxide to myoglobin is a process for which experimental and computational analyses have converged to a common view which connects rebinding time scales and the underlying dynamics. Finally, rhodopsin is a paradigmatic system for multiple length- and time-scale processes for which experimental and computational methods provide valuable insights into the functional dynamics. The systems discussed here highlight that for a comprehensive understanding of how structure, flexibility, energetics, and dynamics contribute to functional dynamics, experimental studies in multiple wavelength regions and computational studies including quantum, classical, and more coarse grained levels are required.

Representation of pheromones, interspecific signals, and plant odors in higher olfactory centers; mapping physiologically identified antennal-lobe projection neurons in the male heliothine moth.

The arrangement of anatomically separated systems for information about general and pheromone odorants is well documented at the initial levels of the olfactory pathway both in vertebrates and insects. In the primary olfactory center of the moth brain, for example, a few enlarged glomeruli situated dorsally, at the entrance of the antennal nerve, are devoted to information about female-produced substances whereas a set of more numerous ordinary glomeruli (OG) receives input about general odorants. Heliothine moths are particularly suitable for studying central chemosensory mechanisms not only because of their anatomically separated systems for plant odors and pheromones but also due to their use of female-produced substances in communication across the species. Thus, the male-specific system of heliothine moths includes two sub-arrangements, one ensuring attraction and mating behavior by carrying information about pheromones released by conspecifics, and the other inhibition of attraction via signal information emitted from heterospecifics. Based on previous tracing experiments, a general chemotopic organization of the male-specific glomeruli has been demonstrated in a number of heliothine species. As compared to the well explored organization of the moth antennal lobe (AL), demonstrating a non-overlapping representation of the biologically relevant stimuli, less is known about the neural arrangement residing at the following synaptic level, i.e., the mushroom body calyces and the lateral horn. In the study presented here, we have labeled physiologically characterized antennal-lobe projection neurons in males of the two heliothine species, Heliothis virescens and Helicoverpa assulta, for the purpose of mapping their target regions in the protocerebrum. In order to compare the representation of plant odors, pheromones, and interspecific signals in the higher brain regions of each species, we have created standard brain atlases and registered three-dimensional models of distinct uniglomerular projection neuron types into the relevant atlas.

Validation and attempts of revision of the MDS-recommended tests for the screening of Parkinson's disease dementia.

The Movement Disorders Society (MDS) formulated diagnostic criteria and assessment guidelines for the screening of dementia in Parkinson's disease (PD). We carried out a validation of the cognitive measures suggested in the screening algorithm (i.e. the Mini Mental State Examination - MMSE - total score, serial 7s subtraction, 3-word recall, pentagons copy, and one minute letter fluency) in 86 patients with PD. Thirty-six percent of participants were diagnosed with dementia using the MDS algorithm, but with the Dementia Rating Scale instead of the MMSE. The original MDS procedure misclassified 11 patients (12.8%) as false negatives and 3 (3.5%) as false positives, leading to 65% sensitivity and 95% specificity. The main reason for misdiagnoses was insensitivity of the MMSE total score. Three attempts were made to reach a better screening performance, which warrants high sensitivity more than high specificity: 1. exclusion of the MMSE total score as a diagnostic requirement; 2. determination of a better cut off through Receiver Operating Characteristic curve analysis; 3. replacement of the MMSE with the equally undemanding, but more PD-specific, Mini Mental Parkinson. The first two strategies generally yielded high sensitivity, but poor specificity. The best outcome was achieved using a Mini Mental Parkinson total score <27 as cognitive criterion: sensitivity was 87% and negative predictive value was 90%; however, specificity was only 67%. Our findings seem to suggest that MDS practical guidelines are specific, but might benefit from the use of more PD-oriented tools than the MMSE in terms of sensitivity.