Identification of sequence changes in myosin II that adjust muscle contraction velocity.

The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (ß/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of ß-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human ß-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human ß-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of ß-myosin as species mass increased to enable a reduced contraction velocity and heart rate.

A Gold(I)-Acetylene Complex Synthesised using Single-Crystal Reactivity.

Using single-crystal to single-crystal solid/gas reactivity the gold(I) acetylene complex [Au(L1)(η2-HC≡CH)][BArF 4] is cleanly synthesized by addition of acetylene gas to single crystals of [Au(L1)(CO)][BArF 4] [L1=tris-2-(4,4'-di-tert-butylbiphenyl)phosphine, ArF=3,5-(CF3)2C6H3]. This simplest gold-alkyne complex has been characterized by single crystal X-ray diffraction, solution and solid-state NMR spectroscopy and periodic DFT. Bonding of HC≡CH with [Au(L1)]+ comprises both σ-donation and π-backdonation with additional dispersion interactions within the cavity-shaped phosphine.

Vasopressin receptor 1a, oxytocin receptor, and oxytocin knockout male and female mice display normal perceptual abilities towards non-social odorants.

Genetic knockouts of the vasopressin receptor 1a (Avpr1a), oxytocin receptor (Oxtr), or oxytocin (Oxt) gene in mice have helped cement the causal relationship between these neuropeptide systems and various social behaviors (e.g., social investigation, recognition, and communication, as well as territoriality and aggression). In mice, these social behaviors depend upon the olfactory system. Thus, it is critical to assess the olfactory capabilities of these knockout models to accurately interpret the observed differences in social behavior. Prior studies utilizing these transgenic mice have sought to test for baseline deficits in olfactory processing; predominantly through use of odor habituation/dishabituation tasks, buried food tests, or investigation assays using non-social odorants. While informative, these assays rely on the animal's intrinsic motivation and locomotor behavior to measure olfactory capabilities and thus, often yield mixed results. Instead, psychophysical analyses using operant conditioning procedures and flow-dilution olfactometry are ideally suited to precisely quantify olfactory perception. In the present study, we used these methods to assess the main olfactory capabilities of adult male and female Avpr1a, Oxtr, and Oxt transgenic mice to volatile non-social odorants. Our results indicate that homozygous and heterozygous knockout mice of all three strains have the same sensitivity and discrimination ability as their wild-type littermates. These data strongly support the hypothesis that the observed social deficits of these global knockout mice are not due to baseline deficits of their main olfactory system.

A qualitative study to examine meaningful change in physical function associated with weight-loss.

PURPOSE: This study explored perceptions of meaningful weight-loss and the level of change on two patient-reported outcome (PRO) measures, the 36-item Short Form Health Survey® [SF-36v2®] and Impact of Weight on Quality of Life Lite-Clinical Trials© [IWQOL-Lite-CT©], that individuals living with overweight or obesity consider to be meaningful and indicative of treatment success. METHODS: Thirty-three qualitative interviews were conducted in the US with adults living with overweight or obesity. Concept elicitation explored perceptions of minimally important/meaningful weight-loss using open-ended questions. Cognitive debriefing was used to understand thresholds for meaningful change on both measures. RESULTS: Most participants (n = 23/33) expected a 5% total body weight-loss to yield some benefit in physical functioning, while all participants expected a 10% weight-loss to provide a meaningful and noticeable improvement in their physical functioning. Participants indicated that an item-level 1-point score change on each measure would represent a noticeable improvement in physical functioning and indicate treatment success. CONCLUSIONS: Participants expected moderate weight-losses to be noticeable, with ≥ 10% weight-loss yielding the most consistent results. The findings suggested that both measures provide strong opportunity to demonstrate treatment benefit in relation to physical functioning as a small change on the response scale would represent a noticeable improvement in participants' daily lives.

Alpha and beta myosin isoforms and human atrial and ventricular contraction.

Human atrial and ventricular contractions have distinct mechanical characteristics including speed of contraction, volume of blood delivered and the range of pressure generated. Notably, the ventricle expresses predominantly ß-cardiac myosin while the atrium expresses mostly the α-isoform. In recent years exploration of the properties of pure α- & ß-myosin isoforms have been possible in solution, in isolated myocytes and myofibrils. This allows us to consider the extent to which the atrial vs ventricular mechanical characteristics are defined by the myosin isoform expressed, and how the isoform properties are matched to their physiological roles. To do this we Outline the essential feature of atrial and ventricular contraction; Explore the molecular structural and functional characteristics of the two myosin isoforms; Describe the contractile behaviour of myocytes and myofibrils expressing a single myosin isoform; Finally we outline the outstanding problems in defining the differences between the atria and ventricles. This allowed us consider what features of contraction can and cannot be ascribed to the myosin isoforms present in the atria and ventricles.

The ATPase cycle of human muscle myosin II isoforms: Adaptation of a single mechanochemical cycle for different physiological roles.

Striated muscle myosins are encoded by a large gene family in all mammals, including humans. These isoforms define several of the key characteristics of the different striated muscle fiber types, including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of seven different human myosin isoforms to define the actin·myosin cross-bridge cycle in solution. Here, we present data on an eighth isoform, the perinatal, which has not previously been characterized. The perinatal is distinct from the embryonic isoform, appearing to have features in common with the adult fast-muscle isoforms, including weak affinity of ADP for actin·myosin and fast ADP release. We go on to use a recently developed modeling approach, MUSICO, to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction, including duty ratio, shortening velocity, ATP economy, and load dependence of these parameters. The work shows that the parameters of the cross-bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics.

Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties.

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the ß-cardiac myosin heavy chain gene (ß-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any ß-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP-driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin·myosin·ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (the fraction of myosin in strongly attached force-holding states), and the measured ATPases all changed in parallel (in both sign and degree of change) compared with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

A Physician-Completed Digital Tool for Evaluating Disease Progression (Multiple Sclerosis Progression Discussion Tool): Validation Study.

BACKGROUND: Defining the transition from relapsing-remitting multiple sclerosis (RRMS) to secondary progressive multiple sclerosis (SPMS) can be challenging and delayed. A digital tool (MSProDiscuss) was developed to facilitate physician-patient discussion in evaluating early, subtle signs of multiple sclerosis (MS) disease progression representing this transition. OBJECTIVE: This study aimed to determine cut-off values and corresponding sensitivity and specificity for predefined scoring algorithms, with or without including Expanded Disability Status Scale (EDSS) scores, to differentiate between RRMS and SPMS patients and to evaluate psychometric properties. METHODS: Experienced neurologists completed the tool for patients with confirmed RRMS or SPMS and those suspected to be transitioning to SPMS. In addition to age and EDSS score, each patient's current disease status (disease activity, symptoms, and its impacts on daily life) was collected while completing the draft tool. Receiver operating characteristic (ROC) curves determined optimal cut-off values (sensitivity and specificity) for the classification of RRMS and SPMS. RESULTS: Twenty neurologists completed the draft tool for 198 patients. Mean scores for patients with RRMS (n=89), transitioning to SPMS (n=47), and SPMS (n=62) were 38.1 (SD 12.5), 55.2 (SD 11.1), and 69.6 (SD 12.0), respectively (P<.001, each between-groups comparison). Area under the ROC curve (AUC) including and excluding EDSS were for RRMS (including) AUC 0.91, 95% CI 0.87-0.95, RRMS (excluding) AUC 0.88, 95% CI 0.84-0.93, SPMS (including) AUC 0.91, 95% CI 0.86-0.95, and SPMS (excluding) AUC 0.86, 95% CI 0.81-0.91. In the algorithm with EDSS, the optimal cut-off values were ≤51.6 for RRMS patients (sensitivity=0.83; specificity=0.82) and ≥58.9 for SPMS patients (sensitivity=0.82; specificity=0.84). The optimal cut-offs without EDSS were ≤46.3 and ≥57.8 and resulted in similar high sensitivity and specificity (0.76-0.86). The draft tool showed excellent interrater reliability (intraclass correlation coefficient=.95). CONCLUSIONS: The MSProDiscuss tool differentiated RRMS patients from SPMS patients with high sensitivity and specificity. In clinical practice, it may be a useful tool to evaluate early, subtle signs of MS disease progression indicating the evolution of RRMS to SPMS. MSProDiscuss will help assess the current level of progression in an individual patient and facilitate a more informed physician-patient discussion.

Social Media Listening to Understand the Lived Experience of Presbyopia: Systematic Search and Content Analysis Study.

BACKGROUND: Presbyopia is defined as the age-related deterioration of near vision over time which is experienced in over 80% of people aged 40 years or older. Individuals with presbyopia have difficulty with tasks that rely on near vision. It is not currently possible to stop or reverse the aging process that causes presbyopia; generally, it is corrected with glasses, contact lenses, surgery, or the use of a magnifying glass. OBJECTIVE: This study aimed to explore how individuals used social media to describe their experience of presbyopia with regard to the symptoms experienced and the impacts of presbyopia on their quality of life. METHODS: Social media sources including Twitter, forums, blogs, and news outlets were searched using a predefined search string relating to symptoms and impacts of presbyopia. The data that were downloaded, based on the keywords, underwent manual review to identify relevant data points. Relevant posts were further manually analyzed through a process of data tagging, categorization, and clustering. Key themes relating to symptoms, impacts, treatment, and lived experiences were identified. RESULTS: A total of 4456 social media posts related to presbyopia were identified between May 2017 and August 2017. Using a random sampling methodology, we selected 2229 (50.0%) posts for manual review, with 1470 (65.9%) of these 2229 posts identified as relevant to the study objectives. Twitter was the most commonly used channel for discussions on presbyopia compared to forums and blogs. The majority of relevant posts originated in Spain (559/1470, 38.0%) and the United States (426/1470, 29.0%). Of the relevant posts, 270/1470 (18.4%) were categorized as posts written by individuals who have presbyopia, of which 37 of the 270 posts (13.7%) discussed symptoms. On social media, individuals with presbyopia most frequently reported experiencing difficulty reading small print (24/37, 64.9%), difficulty focusing on near objects (15/37, 40.5%), eye strain (12/37, 32.4%), headaches (9/37, 24.3%), and blurred vision (8/37, 21.6%). 81 of the 270 posts (30.0%) discussed impacts of presbyopia-emotional burden (57/81, 70.4%), functional or daily living impacts (46/81, 56.8%), such as difficulty reading (46/81, 56.8%) and using electronic devices (21/81, 25.9%), and impacts on work (3/81, 3.7%). CONCLUSIONS: Findings from this social media listening study provided insight into how people with presbyopia discuss their condition online and highlight the impact of presbyopia on individuals' quality of life. The social media listening methodology can be used to generate insights into the lived experience of a condition, but it is recommended that this research be combined with prospective qualitative research for added rigor and for confirmation of the relevance of the findings.

Synthesis of a Perfluorinated Phenoxyphosphorane and Conversion to Its Hexacoordinate Anions.

We report the synthesis and structural characterization of a neutral PV Lewis acid, P(OC6 F5 )5 , and salts containing the six-coordinate anions [P(OC6 F5 )5 F]- and [P(OC6 F5 )6 ]- . The latter anion exhibits a rare example of F-πarene interactions in both the solid and the solution phase, which has been quantitatively studied by variable-temperature (VT) NMR spectroscopy. The Lewis acid strength of P(OC6 F5 )5 has been assessed through experimental fluoride ion competition experiments and quantum-chemical calculations of its fluoride ion affinity (FIA) and global electrophilicity index (GEI). Our findings highlight the importance of considering solvent effects in electrophilicity calculations, even when neutral Lewis acids are involved, and show a rare divergence between FIA and GEI trends. The coordinating abilities of the [P(OC6 F5 )6 ]- and [P(OC6 F5 )5 F]- anions towards the trityl cation, as a prototypical electrophile, have been assessed.

Psychometric evaluation of a patient-reported outcome measure in pancreatic exocrine insufficiency (PEI).

BACKGROUND/OBJECTIVES: Pancreatic exocrine insufficiency (PEI) is commonly caused by chronic pancreatitis (CP) or cystic fibrosis (CF). There are no PEI-specific patient-reported assessments of symptoms and impacts. The PEI Questionnaire (PEI-Q) was developed through qualitative research with PEI patients and expert clinical input. This study evaluated the psychometric properties of the PEI-Q. METHODS: 162 PEI patients (CF = 71 and CP = 91), 62 diarrhoea-specific irritable bowel syndrome (IBS-D) patients and 60 healthy controls completed the 26-item PEI-Q and the Gastrointestinal Quality of Life Index (GIQLI) at baseline. PEI patients completed the measures again two weeks later to assess the test-retest reliability of the PEI-Q. Analyses supported item reduction and scoring algorithm development, followed by psychometric evaluation. RESULTS: Over 90% of PEI patients completed at least 23 of the 26 items at baseline. Item responses and clinical relevance supported retention of 18 items. Factor analysis supported a three-factor solution (abdominal symptoms, bowel movements, impacts) with adequate model fit. PEI-Q scores had good internal consistency (Cronbach's alpha: 0.77-0.82) and test-retest reliability (ICC: 0.73-0.87). Correlations between PEI-Q and GIQLI supported convergent validity. Known-groups and receiver operating characteristic analyses demonstrated that PEI-Q scores discriminated (p < 0.001) between differing PEI severities, and PEI patients and controls. CONCLUSIONS: The PEI-Q has good validity and reliability. Results indicate that the PEI-Q could be used to aid identification and diagnosis of PEI, assist in the management of patients already diagnosed with PEI, ensuring correct and optimum treatment as well as enhance patient-clinician communication.

Temperature sensitive point mutations in fission yeast tropomyosin have long range effects on the stability and function of the actin-tropomyosin copolymer.

The actin cytoskeleton is modulated by regulatory actin-binding proteins which fine-tune the dynamic properties of the actin polymer to regulate function. One such actin-binding protein is tropomyosin (Tpm), a highly-conserved alpha-helical dimer which stabilises actin and regulates interactions with other proteins. Temperature sensitive mutants of Tpm are invaluable tools in the study of actin filament dependent processes, critical to the viability of a cell. Here we investigated the molecular basis of the temperature sensitivity of fission yeast Tpm mutants which fail to undergo cytokinesis at the restrictive temperatures. Comparison of Contractile Actomyosin Ring (CAR) constriction as well as cell shape and size revealed the cdc8.110 or cdc8.27 mutant alleles displayed significant differences in their temperature sensitivity and impact upon actin dependent functions during the cell cycle. In vitro analysis revealed the mutant proteins displayed a different reduction in thermostability, and unexpectedly yield two discrete unfolding domains when acetylated on their amino-termini. Our findings demonstrate how subtle changes in structure (point mutations or acetylation) alter the stability not simply of discrete regions of this conserved cytoskeletal protein but of the whole molecule. This differentially impacts the stability and cellular organisation of this essential cytoskeletal protein.

Exploring the Effect of Ligand Structural Isomerism in Langmuir-Blodgett Films of Chiral Luminescent Eu(III) Self-Assemblies.

Here we have investigated the influence of the antenna group position on both the formation of chiral amphiphilic Eu(III) -based self-assemblies in CH3 CN solution and, on the ability to form monolayers on the surface of quartz substrates using the Langmuir-Blodgett technique, by changing from the 1-naphthyl (2(R), 2(S)) to the 2-naphthyl (1(R), 1(S)) position. The evaluation of binding constants of the self- assemblies in CH3 CN solution was achieved using conventional techniques such as UV/Visible and luminescence spectroscopies along with more specific circular dichroism (CD) spectroscopy. The binding constants obtained for EuL, EuL2 and EuL3 species in the case of 2-naphthyl derivatives were comparable to those obtained for 1-naphthyl derivatives. The analysis of the changes in the CD spectra of 1(R) and 1(S) upon addition of Eu(III) not only allowed us to evaluate the values of the binding constants but the resulting recalculated spectra may also be used as fingerprints for assignment of the chiral self-assembly species formed in solution. The obtained monolayers were predominantly formed from EuL3 (≈85 %) with the minor species present in ≈15 % EuL2 .

"Tail" tuning of iron(II) spin crossover temperature by 100 K.

Two new Rdpt ligands featuring long "tails", padpt (N-4H-1,2,4-triazole-3,5-di(2-pyridyl)palmitamide) and hpdpt (4-(4-heptadecafluoroctylphenyl)-3,5-bis(2-pyridyl)-4H-1,2,4-triazole), were made and reacted with [Fe(II)(py)4(NCS)2] to give pinkish-red [Fe(II)(padpt)2(SCN)2] (1) and purple-red [Fe(II)(hpdpt)2(SCN)2] (2) as solvent-free crystals. Magnetic measurements reveal that both 1 and 2 exhibit complete and reproducible spin crossovers, with a far lower T1/2 for the amide-alkyl tailed 1 (182 K) than for the fluorocarbon tailed 2 (248 K), which in turn is far lower than the T1/2 of 290 K previously reported for the nonamide-alkyl tailed analogue [Fe(II)(C16dpt)2(SCN)2]·(2)/3H2O (3). Structure determinations for 1 and 2 in both the high spin (HS) and low spin (LS) states confirm the expected trans-NCS conformation and reveal that (a) the "tails" interdigitate and (b) the LS forms are less distorted than the HS forms (Σ = 58-70° vs 47-54°). DSC and Raman spectroscopy confirmed the high tail-dependence of the SCO events in 1 and 2, as well as in 3, with the Raman data giving T1/2 values of 190, 243, and 285 K, respectively. Bright orange single crystals of the solvatomorph [Fe(II)(hpdpt)2(SCN)2]·MeOH·H2O (2solv) were also structurally and magnetically characterized and, in contrast to 2, found to remain HS down to 4 K, providing further evidence of the huge impact of crystal packing on SCO. Both 1 and 2 form stable Langmuir films at an air-water interface, a single layer of which can be transferred to a solid support.

The two C. elegans class VI myosins, SPE-15/HUM-3 and HUM-8, share similar motor properties, but have distinct developmental and tissue expression patterns.

Myosins of class VI move toward the minus-end of actin filaments and play vital roles in cellular processes such as endocytosis, autophagy, protein secretion, and the regulation of actin filament dynamics. In contrast to the majority of metazoan organisms examined to date which contain a single MYO6 gene, C. elegans, possesses two MYO6 homologues, SPE-15/HUM-3 and HUM-8. Through a combination of in vitro biochemical/biophysical analysis and cellular assays, we confirmed that both SPE-15/HUM-3 and HUM-8 exhibit reverse directionality, velocities, and ATPase activity similar to human MYO6. Our characterization also revealed that unlike SPE-15/HUM-3, HUM-8 is expressed as two distinct splice isoforms, one with an additional unique 14 amino acid insert in the cargo-binding domain. While lipid and adaptor binding sites are conserved in SPE-15/HUM-3 and HUM-8, this conservation does not enable recruitment to endosomes in mammalian cells. Finally, we performed super-resolution confocal imaging on transgenic worms expressing either mNeonGreen SPE-15/HUM-3 or wrmScarlet HUM-8. Our results show a clear distinction in tissue distribution between SPE-15/HUM-3 and HUM-8. While SPE-15/HUM-3 exhibited specific expression in the gonads and neuronal tissue in the head, HUM-8 was exclusively localized in the intestinal epithelium. Overall, these findings align with the established tissue distributions and localizations of human MYO6.

A drug cocktail of rapamycin, acarbose, and phenylbutyrate enhances resilience to features of early-stage Alzheimer's disease in aging mice.

The process of aging is defined by the breakdown of critical maintenance pathways leading to an accumulation of damage and its associated phenotypes. Aging affects many systems and is considered the greatest risk factor for a number of diseases. Therefore, interventions aimed at establishing resilience to aging should delay or prevent the onset of age-related diseases. Recent studies have shown a three-drug cocktail consisting of rapamycin, acarbose, and phenylbutyrate delayed the onset of physical, cognitive, and biological aging phenotypes in old mice. To test the ability of this drug cocktail to impact Alzheimer's disease (AD), an adeno-associated-viral vector model of AD was created. Mice were fed the drug cocktail 2 months prior to injection and allowed 3 months for phenotypic development. Cognitive phenotypes were evaluated through a spatial navigation learning task. To quantify neuropathology, immunohistochemistry was performed for AD proteins and pathways of aging. Results suggested the drug cocktail was able to increase resilience to cognitive impairment, inflammation, and AD protein aggregation while enhancing autophagy and synaptic integrity, preferentially in female cohorts. In conclusion, female mice were more susceptible to the development of early stage AD neuropathology and learning impairment, and more responsive to treatment with the drug cocktail in comparison to male mice. Translationally, a model of AD where females are more susceptible would have greater value as women have a greater burden and incidence of disease compared to men. These findings validate past results and provide the rationale for further investigations into enhancing resilience to early-stage AD by enhancing resilience to aging.

Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor.

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.