Structural relationships of actin, myosin, and tropomyosin revealed by cryo-electron microscopy

We have calculated three-dimensional maps from images of myosin subfragment-1 (S1)-decorated thin filaments and S1-decorated actin filaments preserved in frozen solution. By averaging many data sets we obtained highly reproducible maps that can be interpreted simply to provide a model for the native structure of decorated filaments. From our results we have made the following conclusions. The bulk of the actin monomer is approximately 65 X 40 X 40 A and is composed of two domains. In the filaments the monomers are strongly connected along the genetic helix with weaker connections following the long pitch helix. The long axis of the monomer lies roughly perpendicular to the filament axis. The myosin head (S1) approaches the actin filament tangentially and binds to a single actin, the major interaction being with the outermost domain of actin. In the map the longest chord of S1 is approximately 130 A. The region of S1 closest to actin is of high density, whereas the part furthest away is poorly defined and may be disordered. By comparing maps from decorated thin filaments with those from decorated actin, we demonstrate that tropomyosin is bound to the inner domain of actin just in front of the myosin binding site at a radius of approximately 40 A. A small change in the azimuthal position of tropomyosin, as has been suggested by others to occur during Ca2+-mediated regulation in vertebrate striated muscle, appears to be insufficient to eclipse totally the major site of interaction between actin and myosin.     

Mediation of pinocytosis in cultured arterial smooth muscle and endothelial cells by platelet-derived growth factor

Pinocytosis was measured in monkey aortic smooth muscle cells (SMC), bovine aortic endothelial cells, and Swiss 3T3 cells in culture as cellular uptake of [U-(14)C]sucrose and horseradish peroxidase (HRP) from the tissue culture medium. Monkey arterial SMC and Swiss 3T3 cells were maintained in a quiescent state of growth at low cells density in medium containing 5 percent monkey plasma-derived serum (PDS). Replacement of PDS with 5 percent monkey whole blood serum (WBS) from the same donor, or addition to PDS of partially purified platelet-derived growth factor(s) (PF), resulted in a marked stimulation of pinocytosis as well as of cellular proliferation. In SMC, enhancement of the rate of pinocytosis occurred 4-6 h after exposure to WBS or PF, and the rate was up to twofold higher than the rate in medium containing PDS. In contrast, [(3)H]thymidine uptake by SMC did not increase until 12-16 h after exposure to PF. In endothelial cells the presence of PF or WBS did not enhance either the rate of pinocytosis or the rate of proliferation over that in PDS. Thus, endothelial cells did not become quiescent at subconfluent densities in PDS but maintained rates of proliferation and pinocytosis that were equivalent to those in WBS. By autoradiography, the fraction of labeled nuclei in SMC cultures 24 h after change of medium increased from 0.061 +/- 0.004 in quiescent cultures to 0.313 +/- 0.028 after exposure to WBS or PF. In contrast, labeling indices of endothelial cells were similar for cultures grown in PDS, WBS, or PF at any single time point after change of medium. These findings suggest that the rate of pinocytosis maybe be coupled in some fashion to growth regulation, which may be mediated in part by specific growth factors, such as that derived from the thrombocyte.     

Deprescribing in Frail Older People: A Randomised Controlled Trial

Objectives

Deprescribing has been proposed as a way to reduce polypharmacy in frail older people. We aimed to reduce the number of medicines consumed by people living in residential aged care facilities (RACF). Secondary objectives were to explore the effect of deprescribing on survival, falls, fractures, hospital admissions, cognitive, physical, and bowel function, quality of life, and sleep.

Methods

Ninety-five people aged over 65 years living in four RACF in rural mid-west Western Australia were randomised in an open study. The intervention group (n = 47) received a deprescribing intervention, the planned cessation of non-beneficial medicines. The control group (n = 48) received usual care. Participants were monitored for twelve months from randomisation. Primary outcome was change in the mean number of unique regular medicines. All outcomes were assessed at baseline, six, and twelve months.

Results

Study participants had a mean age of 84.3±6.9 years and 52% were female. Intervention group participants consumed 9.6±5.0 and control group participants consumed 9.5±3.6 unique regular medicines at baseline. Of the 348 medicines targeted for deprescribing (7.4±3.8 per person, 78% of regular medicines), 207 medicines (4.4±3.4 per person, 59% of targeted medicines) were successfully discontinued. The mean change in number of regular medicines at 12 months was -1.9±4.1 in intervention group participants and +0.1±3.5 in control group participants (estimated difference 2.0±0.9, 95%CI 0.08, 3.8, p = 0.04). Twelve intervention participants and 19 control participants died within 12 months of randomisation (26% versus 40% mortality, p = 0.16, HR 0.60, 95%CI 0.30 to 1.22) There were no significant differences between groups in other secondary outcomes. The main limitations of this study were the open design and small participant numbers.

Conclusions

Deprescribing reduced the number of regular medicines consumed by frail older people living in residential care with no significant adverse effects on survival or other clinical outcomes.

Trial Registration

Australian New Zealand Clinical Trials Registry ACTRN12611000370909     

Is formation of visible channels in a phospholipid bilayer by botulinum neurotoxin type B sensitive to its disulfide?

Botulinum neurotoxin, produced by Clostridium botulinum as a approximately 150-kDa single-chain protein, is nicked proteolytically either endogenously or exogenously. The approximately 50- and approximately 100-kDa chains of the dichain molecule remain held together by an interchain disulfide bridge and noncovalent interactions. The neurotoxin binds to receptors of the target cell and is internalized by endocytosis. Thereafter, a portion of the neurotoxin, the approximately 50-kDa chain, escapes to the cytosol, where it blocks neurotransmitter release. Botulinum neurotoxin serotype B is released by the bacteria primarily as an unnicked single chain. We reduced this unnicked protein and used its binding to ganglioside in a lipid layer to produce helical tubular crystals of unnicked botulinum neurotoxin type B in its disulfide-reduced state. The helical arrangement of the neurotoxin allowed determination of the structure of the molecule using cryo-electron microscopy and image processing. The resulting model reveals that neurotoxin molecules formed loops extending out from the surface of the bilayer and bending toward a neighboring loop. Although channels have been seen with disulfide-linked neurotoxin (Schmid, Robinson, and DasGupta (1993) Direct visualization of botulinum neurotoxin-induced channels in phospholipid vesicles, Nature 364, 827-830), no channels were seen here, a finding which suggests that the reduced, unnicked neurotoxin is incapable of forming a visible channel.     

A human monoclonal IgE antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, Phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain

Almost 90% of grass pollen-allergic patients are sensitized against group 5 grass pollen allergens. We isolated a monoclonal human IgE Fab out of a combinatorial library prepared from lymphocytes of a grass pollen-allergic patient and studied its interaction with group 5 allergens. The IgE Fab cross-reacted with group 5A isoallergens from several grass and corn species. By allergen gene fragmentation we mapped the binding site of the IgE Fab to a 11.2-kDa N-terminal fragment of the major timothy grass pollen allergen Phl p 5A. The IgE Fab-defined Phl p 5A fragment was expressed in Escherichia coli and purified to homogeneity. Circular dichroism analysis revealed that the rPhl p 5A domain, as well as complete rPhl p 5A, assumed a folded conformation consisting predominantly of an alpha helical secondary structure, and exhibited a remarkable refolding capacity. It reacted with serum IgE from 76% of grass pollen-allergic patients and revealed an extremely high allergenic activity in basophil histamine release as well as skin test experiments. Thus, the rPhl p 5A domain represents an important allergen domain containing several IgE epitopes in a configuration optimal for efficient effector cell activation. We suggest the rPhl p 5A fragment and the corresponding IgE Fab as paradigmatic tools to explore the structural requirements for highly efficient effector cell activation and, perhaps later, for the development of generally applicable allergen-specific therapy strategies.     

Control of stem cell fate by engineering their micro and nanoenvironment

Stem cells are capable of long-term self-renewal and differentiation into specialised cell types, making them an ideal candidate for a cell source for regenerative medicine. The control of stem cell fate has become a major area of interest in the field of regenerative medicine and therapeutic intervention. Conventional methods of chemically inducing stem cells into specific lineages is being challenged by the advances in biomaterial technology, with evidence highlighting that material properties are capable of driving stem cell fate. Materials are being designed to mimic the clues stem cells receive in their in vivo stem cell niche including topographical and chemical instructions. Nanotopographical clues that mimic the extracellular matrix(ECM) in vivo have shown to regulate stem cell differentiation. The delivery of ECM components on biomaterials in the form of short peptides sequences has also proved successful in directing stem cell lineage. Growth factors responsible for controlling stem cell fate in vivo have also been delivered via biomaterials to provide clues to determine stem cell differentiation. An alternative approach to guide stem cells fate is to provide genetic clues including delivering DNA plasmids and small interfering RNAs via scaffolds. This review, aims to provide an overview of the topographical, chemical and molecular clues that biomaterials can provide to guide stem cell fate. The promising features and challenges of such approaches will be highlighted, to provide directions for future advancements in this exciting area of stem cell translation for regenerative medicine.     

A cluster-randomised trial of staff education to improve the quality of life of people with dementia living in residential care: the DIRECT study

Background

The Dementia In Residential care: EduCation intervention Trial (DIRECT) was conducted to determine if delivery of education designed to meet the perceived need of GPs and care staff improves the quality of life of participants with dementia living in residential care.

Methodology/Principal Findings

This cluster-randomised controlled trial was conducted in 39 residential aged care facilities in the metropolitan area of Perth, Western Australia. 351 care facility residents aged 65 years and older with Mini-Mental State Examination ≤24, their GPs and facility staff participated. Flexible education designed to meet the perceived needs of learners was delivered to GPs and care facility staff in intervention groups. The primary outcome of the study was self-rated quality of life of participants with dementia, measured using the QOL-Alzheimer''s Disease Scale (QOL-AD) at 4 weeks and 6 months after the conclusion of the intervention. Analysis accounted for the effect of clustering by using multi-level regression analysis. Education of GPs or care facility staff did not affect the primary outcome at either 4 weeks or 6 months. In a post hoc analysis excluding facilities in which fewer than 50% of staff attended an education session, self-rated QOL-AD scores were 6.14 points (adjusted 95%CI 1.14, 11.15) higher at four-week follow-up among residents in facilities randomly assigned to the education intervention.

Conclusion

The education intervention directed at care facilities or GPs did not improve the quality of life ratings of participants with dementia as a group. This may be explained by the poor adherence to the intervention programme, as participants with dementia living in facilities where staff participated at least minimally seemed to benefit.

Trial Registration

ANZCTR.org.au ACTRN12607000417482     

Genetic covariance between indices of body condition and immunocompetence in a passerine bird

Background  

Condition-dependence is a ubiquitous feature of animal life histories and has important implications for both natural and sexual selection. Mate choice, for instance, is typically based on condition-dependent signals. Theory predicts that one reason why condition-dependent signals may be special is that they allow females to scan for genes that confer high parasite resistance. Such explanations require a genetic link between immunocompetence and body condition, but existing evidence is limited to phenotypic associations. It remains unknown, therefore, whether females selecting males with good body condition simply obtain a healthy mate, or if they acquire genes for their offspring that confer high immunocompetence.     

Older men who use computers have lower risk of dementia

Objective

To determine if older men who use computers have lower risk of developing dementia.

Methods

Cohort study of 5506 community-dwelling men aged 69 to 87 years followed for up to 8.5 years. Use of computers measured as daily, weekly, less than weekly and never. Participants also reported their use of email, internet, word processors, games or other computer activities. The primary outcome was the incidence of ICD-10 diagnosis of dementia as recorded by the Western Australian Data Linkage System.

Results

1857/5506 (33.7%) men reported using computers and 347 (6.3%) received a diagnosis of dementia during an average follow up of 6.0 years (range: 6 months to 8.5 years). The hazard ratio (HR) of dementia was lower among computer users than non-users (HR = 0.62, 95%CI = 0.47–0.81, after adjustment for age, educational attainment, size of social network, and presence of depression or of significant clinical morbidity). The HR of dementia appeared to decrease with increasing frequency of computer use: 0.68 (95%CI = 0.41–1.13), 0.61 (95%CI = 0.39–0.94) and 0.59 (95%CI = 0.40–0.87) for less than weekly, at least weekly and daily. The HR of dementia was 0.66 (95%CI = 0.50–0.86) after the analysis was further adjusted for baseline cognitive function, as measured by the Mini-Mental State Examination.

Conclusion

Older men who use computers have lower risk of receiving a diagnosis of dementia up to 8.5 years later. Randomised trials are required to determine if the observed associations are causal.