The flexibility of myosin 7a

Myosin 7a is a molecular motor found in hair cells of the ear and the photoreceptor cells of the eye. Myosin 7a is comprised of an actin-binding motor domain, a lever; which is composed of 5 IQ motifs that can potentially bind 5 light chains followed by a single alpha helical (SAH) domain, and a tail composed of 2 MyTH4-FERM domains. The lever is an essential mechanical element in myosin 7a function, but an understanding of its mechanical properties and how these derive from its substructure is lacking. It has been observed in vitro that myosin 7a is able to regulate its activity through a head-tail interaction. How the flexibility of the sub-domains of the lever allows the molecule to fold up is not completely understood. To address this, the first aim of this study was to look for evidence of novel light chain binding partners in myosin 7a, which revealed calmodulin to be the preferred light chain. My second aim was to study the structure and flexibility of the lever of full-length myosin 7a using single-particle image processing of images from negative stain electron microscopy (EM). Image averaging revealed the lever to be much shorter than expected. Additionally, there was evidence of thermally-driven flexing at the motor-lever junction. A stiffness of 78 pN.nm.rad-2 for the flexing was inferred, which represents a significant compliance in the head. An investigation into lever bending analysis, by monitoring the decay of tangent-tangent correlations of the lever shapes, yielded a persistence length of 38 ± 3 nm. Finally, long time molecular dynamics (MD) simulations were compared with a novel coarse-grained (CG) simulation technique called Fluctuating Finite Element Analysis (FFEA), which treats proteins as visco-elastic continua subject to thermal noise to probe the flexibility of myosin 7a. FFEA allows sufficiently long time simulations that are computationally less expensive than corresponding all-atom MD simulations to allow myosin 7a to explore its full range of configurations. Extraction of flexibility data from all-atom MD simulations calculated the bending stiffness of the SAH domain to be 60.5 pN.nm2, with reasonable overlap of the major modes of motion between the all-atom and CG simulation types

Early Vocabulary Development of Australian Indigenous Children: Identifying Strengths

The current study sought to increase our understanding of the factors involved in the early vocabulary development of Australian Indigenous children. Data from the Longitudinal Study of Indigenous Children were available for 573 Indigenous children (291 boys) who spoke English ( = 37.0 months, = 5.4 months, at wave 3). Data were also available for 86 children (51 boys) who spoke an Indigenous language ( = 37.1 months, = 6.0 months, at wave 3). As hypothesised, higher levels of parent-child book reading and having more children’s books in the home were associated with better English vocabulary development. Oral storytelling in Indigenous language was a significant predictor of the size of children’s Indigenous vocabular

The management of sexually transmitted infections: a scoping survey in primary care

Background National guidelines for sexually transmitted infections (STIs) in primary care exists but their management is uncertain. Aim To assess the management of STIs against national standards in primary care. Design & setting A questionnaire based study in London and Brighton. The survey was conducted in 2015 following reorganisation of sexual health services in England. Method Questionnaires were sent to GPs in London and Brighton about testing for STIs, treatment for gonorrhoea, specialist advice, and referral services. Results Of 119 GPs who responded, most expressed confidence in treating chlamydia (n = 105/119, 88%), trichomonas (n = 81/119, 68%), and herpes (n = 82/119, 69%) but not gonorrhoea (n = 32/119, 27%). Most referred cases of syphilis (n = 92/119, 77%) and genital warts (83/119, 70%) to genito-urinary medicine (GUM) as per guidance. Most GPs tested for gonorrhoea on patient request (n = 95/119, 80%), in tandem with chlamydia screening (n = 89/119, 75%), because of high risk status (n = 85/119, 71%) and genital symptoms (n = 108/119, 91%). Some GPs (n = 22/119, 18%) sampled urine for culture, 53/119 (45%) provided high vaginal swabs (HVS), and 28/119 (24%) provided self-taken vulvovaginal swabs (STVVS) for culture. These samples are not appropriate for gonococcal culture and not processed in the laboratory. Urethral swabs for men and endocervical swabs (ECS) are recommended for gonococcus culture. Over half (n = 60/102, 59%) of GPs did not treat gonorrhoea but some prescribed cefixime, ciprofloxacin, or azithromycin. Eighty-seven per cent (n = 104/119) sought advice from GUM, and 83/103 (81%) referred gonorrhoea nucleic acid amplification test (NAAT)-positive patients. Conclusion There is scope for improvement of STIs management in primary care to ensure that patients are optimally investigated, treated, and referred

The crystal structure of PD1, a Haemophilus surface fibril domain

The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel `hairpin-like' architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the `hairpin-like' hypothesis.Peer reviewe

Resummation Methods at Finite Temperature: The Tadpole Way

We examine several resummation methods for computing higher order corrections to the finite temperature effective potential, in the context of a scalar $\phi^4$ theory. We show by explicit calculation to four loops that dressing the propagator, not the vertex, of the one-loop tadpole correctly counts ``daisy'' and ``super-daisy'' diagrams.Comment: 18 pages, LaTeX, CALT-68-1858, HUTP-93-A011, EFI-93-2

Human Skeletal myopathy myosin mutations disrupt myosin head sequestration

Myosin heavy chains encoded by MYH7 and MYH2 are abundant in human skeletal muscle, and important for muscle contraction. However, it is unclear how mutations in these genes disrupt myosin structure and function leading to skeletal muscle myopathies termed myosinopathies. Here, we used multiple approaches to analyse the effects of common MYH7 and MYH2 mutations in the light meromyosin region of myosin (LMM). Analyses of expressed and purified MYH7 and MYH2 LMM mutant proteins combined with in-silico modelling showed that myosin coiled-coil structure and packing of filaments in vitro are commonly disrupted. Using muscle biopsies from patients, and Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, together with X-ray diffraction measurements to estimate myosin head order we found that basal myosin ATP consumption was increased and the myosin super-relaxed state was decreased in vivo. In addition, myofibre mechanics experiments to investigate contractile function showed myofibre contractility was not affected. These findings indicate that the structural remodelling associated with LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help design future therapies for myosinopathies

Corrections to the Electroweak Effective Action at Finite Temperature

We calculate contributions to the finite temperature effective action for the electroweak phase transition (EWPT) at \O(g^4), {\it i.e.} at second order in (g^2 T/\M) and all orders in (g^2 T^2/\M^2). This requires plasma-mass corrections in the calculation of the effective potential, inclusion of the ``lollipop'' diagram, and an estimate of derivative corrections. We find the EWPT remains too weakly first-order to drive baryogenesis. We calculate some one loop kinetic energy corrections using both functional and diagrammatic methods; these may be important for saddlepoint configurations such as the bounce or sphaleron.Comment: LaTeX, 6 figures available by email, CALT-68-1795, HUTP-92-A027, EFI-92-2

Immunodominance of HIV-1 Specific CD8+ T-Cell Responses Is Related to Disease Progression Rate in Vertically Infected Adolescents

BACKGROUND: HIV-1 vertically infected children in the USA are living into adolescence and beyond with the widespread use of antiretroviral drugs. These patients exhibit striking differences in the rate of HIV-1 disease progression which could provide insights into mechanisms of control. We hypothesized that differences in the pattern of immunodomination including breadth, magnitude and polyfunctionality of HIV-1 specific CD8+ T cell response could partially explain differences in progression rate. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we mapped, quantified, and assessed the functionality of these responses against individual HIV-1 Gag peptides in 58 HIV-1 vertically infected adolescents. Subjects were divided into two groups depending upon the rate of disease progression: adolescents with a sustained CD4%≥25 were categorized as having no immune suppression (NS), and those with CD4%≤15 categorized as having severe immune suppression (SS). We observed differences in the area of HIV-1-Gag to which the two groups made responses. In addition, subjects who expressed the HLA- B*57 or B*42 alleles were highly likely to restrict their immunodominant response through these alleles. There was a significantly higher frequency of naïve CD8+ T cells in the NS subjects (p = 0.0066) compared to the SS subjects. In contrast, there were no statistically significant differences in any other CD8+ T cell subsets. The differentiation profiles and multifunctionality of Gag-specific CD8+ T cells, regardless of immunodominance, also failed to demonstrate meaningful differences between the two groups. CONCLUSIONS/SIGNIFICANCE: Together, these data suggest that, at least in vertically infected adolescents, the region of HIV-1-Gag targeted by CD8+ T cells and the magnitude of that response relative to other responses may have more importance on the rate of disease progression than their qualitative effector functions