1,880 research outputs found
Clutching at Guidance Cues: The Integrin–FAK Axis Steers Axon Outgrowth
No full textIntegrin receptors are essential contributors to neurite outgrowth and axon elongation. Activated integrins engage components of the extracellular matrix, enabling the growth cone to form point contacts, which connect the extracellular substrate to dynamic intracellular protein complexes. These adhesion complexes facilitate efficient growth cone migration and neurite extension. Major signalling pathways mediated by the adhesion complex are instigated by focal adhesion kinase (FAK), whilst axonal guidance molecules present in vivo promote growth cone turning or retraction by local modulation of FAK activity. Activation of FAK is marked by phosphorylation following integrin engagement, and this activity is tightly regulated during neurite outgrowth. FAK inhibition slows neurite outgrowth by reducing point contact turnover; however, mutant FAK constructs with enhanced activity stimulate aberrant outgrowth. Importantly, FAK is a major structural component of maturing adhesion sites, which provide the platform for actin polymerisation to drive leading edge advance. In this review, we discuss the coordinated signalling of integrin receptors and FAK, as well as their role in regulating neurite outgrowth and axon elongation. We also discuss the importance of the integrin–FAK axis in vivo, as integrin expression and activation are key determinants of successful axon regeneration following injury
Advancements in antimicrobial nanoscale materials and self-assembling systems
No full textAntimicrobial resistance is directly responsible for more deaths per year than either HIV/AIDS or malaria and is predicted to incur a cumulative societal financial burden of at least $100 trillion between 2014 and 2050. Already heralded as one of the greatest threats to human health, the onset of the coronavirus pandemic has accelerated the prevalence of antimicrobial resistant bacterial infections due to factors including increased global antibiotic/antimicrobial use. Thus an urgent need for novel therapeutics to combat what some have termed the ‘silent pandemic’ is evident. This review acts as a repository of research and an overview of the novel therapeutic strategies being developed to overcome antimicrobial resistance, with a focus on self-assembling systems and nanoscale materials. The fundamental mechanisms of action, as well as the key advantages and disadvantages of each system are discussed, and attention is drawn to key examples within each field. As a result, this review provides a guide to the further design and development of antimicrobial systems, and outlines the interdisciplinary techniques required to translate this fundamental research towards the clinic
Characterising a Novel Interaction between Rap1b and Rhea sheds light on new Mechanisms for Focal Adhesion Assembly
No full textFor the first time, we reveal a direct interaction between Rap1b and the fly homolog of talin, Rhea. Using a combination of biochemical and biophysical techniques, the Rap1 binding site on Rhea has been successfully mapped. Additionally, we reveal that an acidic-to-basic K17E substitution, on Rhea, completely abolishes Rap1 binding. Our collaborators have shown that this mutation results in non-viable embryos and our data links the Rap1:Rhea interaction to this lethal phenotype. The implications of our findings support currently proposed mechanisms of RIAM-independent integrin activation, that would challenge our understanding of focal adhesion formation.
Furthermore, we propose a double-dependent Rap1 integrin-activation pathway, involving Rap1 directly interacting with the FERM domain, alongside the known Rap1-dependent recruitment of talin.
Optimisations have allowed us to express both the wild-type and mutant Rhea F0 domain in E.coli BL21(DE3) cells. Efficient purification via Ni-NTA-based affinity chromatography results in yields of ~50-60 mg/litre being obtained. Using circular dichroism, it is shown that substitution of the K17 residue does not interfere with the structural integrity of Rhea; both proteins have identical full spectrum measurements and Tm values.
Optimal expression of the conserved G-domain of mouse Rap1b was achieved in the CK600K cell line. This region is highly conserved to that in fly (90% identical). NMR was used to show direct interaction between drosophila Rhea F0 and Rap1b; whilst additionally confirming that Rap1b was unable to induce chemical shifts in the F0-K17E mutant. Triple resonance NMR experiments revealed the location of the Rap1 binding site on the wild-type Rhea F0, with V15, K17, T18, K37 and E40 being highlighted at the centre of this interaction. Structural models of Rap1:Rhea F0 binding agree with our findings, with the 5 highlighted residues seen to make close contact with the Rap1 switch I domain.
Together this work confirms a direct interaction between Rhea and Rap1 whilst providing biochemical validation for the lethal phenotypes observed in mutant flies. It also provides further insight into new mechanisms of focal adhesion formation and integrin activation
Developing an innovative approach to identify novel talin-binding ligands in the brain
No full textCell adhesion, a process underlying the maintenance and development of multicellularity, is fundamentally dependent on the activity of talin as a mechanosensitive signalling nexus. Talin couples integrin-mediated cell-to-matrix adhesions to the actin cytoskeleton, facilitating the formation of integrin adhesion complexes, which act as sophisticated information processing centres. Variation of talin rod domain conformation, following adhesion, promotes different talin-mediated recruitment and signalling outputs, enabling the modification of a cell's internal programming. The recently published MeshCODE theory describes how information in the brain may be stored binarily via the mechanicalistic alteration of talin rod binary switches, highly enriched at every synapse. To validate this theory and discover a connection between neuronal activity and talin-mediated mechanical signalling, an innovative method, termed the talin 'fishing' rod experiment, was developed to identify novel talin-binding ligands in murine neuronal extract. Many proteins were identified as promising neuronal talin-binding ligands, including dynamin-1, synaptojanin-1, and myosin-Va, all of which exhibit particular significance to many aspects of synaptic transmission, particularly in the regulation of synaptic vesicle endo- and exocytosis. A bioinformatics pipeline and subsequent scoring system was also developed to accompany this experimental approach, enabling the identification of probable talin-binding sites in known synaptic vesicle proteins through I/LD motif evaluation. Whilst only weak binding was demonstrated between several talin rod domains and the proteins selected through bioinformatics analysis, it implies that both the bioinformatics pipeline and scoring system is effective in identifying promising talin-binding I/LD motifs. The identification of adhesome proteins, prior to this project, was restricted to cell lines and fibroblasts, limiting our knowledge of talin-binding ligands outside of these cell types. However, the talin 'fishing' rod experiment, developed in this project, can be utilised to help fill this current knowledge gap. Validation of the promising interactors, identified in this study, will support the concepts presented in the MeshCODE theory, potentially enabling the scientific community to achieve understanding regarding the enigma of memory storage at long last
Public worship and practical theology in the work of Benjamin Keach (1640-1704)
No full textThe late seventeenth century was a critical and fruitful period
for the Particular Baptists of England. Severely persecuted following
the Restoration, toleration in 1689 brought its own perils.
Particular Baptists were fortunate in having several strong leaders,
especially the London trio of Hanserd Knollys, William Kiffin, and
Benjamin Keach. Such a small and severely persecuted group as the
Baptists could afford little time for academic pursuits, thus of
necessity most of their theology was practical in nature.
Benjamin Keach (1640-1704) was the most outstanding practical
theologian among the English Particular Baptists of the late
seventeenth century. This dissertation is a study of Keach, in
particular his writings on public worship and practical theology.
Although Keach was a prolific author, he has been almost completely
neglected by scholars.
After a biographical sketch of Keach, this study considers his
writings on public worship and practical theology. In the area of
worship, Keach made two outstanding contributions: First, he was the
most vocal apologist for Baptist views on Baptism of his period.
Secondly, and more importantly, his hymn writing and defense of hymn
singing broke new ground, not just for Baptists, but for English
Protestantism, in general. In addition to his contributions in these
areas, he also dealt with the laying on of hands and the sabbath day
worship controversy.
Keach's contributions to practical theology fall into two main
groups: his writings that concern religious education and those that
deal with polity. In addition to these, Keach's vigorous advocacy of
a high Calvinist soteriology are also considered under the rubric of
practical theology. Keach's most important (although not his most
positive) contribution in this area were his soteriological writings.
Although well within the bounds of orthodoxy, some of the tendencies
in Keach's soteriology were taken up by the following generation of
Baptist leaders and developed into a stultifying hyper-Calvinism that
handicapped Baptist evangelism and missions.
In the conclusion, Keach's contributions to a theory of practical
theology are considered
Next-generation protein-based materials capture and preserve projectiles from supersonic impacts
No full textExtreme energy-dissipating materials are essential for a range of applications. The military and police force require ballistic armour to ensure the safety of their personnel, while the aerospace industry requires materials that enable the capture, preservation and study of hypervelocity projectiles. However, current industry standards display at least one inherent limitation, such as weight, breathability, stiffness, durability and failure to preserve captured projectiles. To resolve these limitations, we have turned to nature, using proteins that have evolved over millennia to enable effective energy dissipation. Specifically, a recombinant form of the mechanosensitive protein talin was incorporated into a monomeric unit and crosslinked, resulting in a talin shock-absorbing material (TSAM). When subjected to 1.5 km s−1 supersonic shots, TSAMs were shown to absorb the impact and capture and preserve the projectile
The Mechanical Basis of Memory – the MeshCODE Theory
No full textOne of the major unsolved mysteries of biological science concerns the question of where and in what form information is stored in the brain. I propose that memory is stored in the brain in a mechanically encoded binary format written into the conformations of proteins found in the cell-extracellular matrix (ECM) adhesions that organise each and every synapse. The MeshCODE framework outlined here represents a unifying theory of data storage in animals, providing read-write storage of both dynamic and persistent information in a binary format. Mechanosensitive proteins that contain force-dependent switches can store information persistently, which can be written or updated using small changes in mechanical force. These mechanosensitive proteins, such as talin, scaffold each synapse, creating a meshwork of switches that together form a code, the so-called MeshCODE. Large signalling complexes assemble on these scaffolds as a function of the switch patterns and these complexes would both stabilise the patterns and coordinate synaptic regulators to dynamically tune synaptic activity. Synaptic transmission and action potential spike trains would operate the cytoskeletal machinery to write and update the synaptic MeshCODEs, thereby propagating this coding throughout the organism. Based on established biophysical principles, such a mechanical basis for memory would provide a physical location for data storage in the brain, with the binary patterns, encoded in the information-storing mechanosensitive molecules in the synaptic scaffolds, and the complexes that form on them, representing the physical location of engrams. Furthermore, the conversion and storage of sensory and temporal inputs into a binary format would constitute an addressable read-write memory system, supporting the view of the mind as an organic supercomputer
Mechanical activation of vinculin binding to talin locks talin in an unfolded conformation
No full textThe force-dependent interaction between talin and vinculin plays a crucial role in the initiation and growth of focal adhesions. Here we use magnetic tweezers to characterise the mechano-sensitive compact N-terminal region of the talin rod, and show that the three helical bundles R1-R3 in this region unfold in three distinct steps consistent with the domains unfolding independently. Mechanical stretching of talin R1-R3 enhances its binding to vinculin and vinculin binding inhibits talin refolding after force is released. Mutations that stabilize R3 identify it as the initial mechano-sensing domain in talin, unfolding at ~5 pN, suggesting that 5 pN is the force threshold for vinculin binding and adhesion progression
The structure of an integrin/talin complex reveals the basis of inside-out signal transduction
No full textFundamental to cell adhesion and migration, integrins are large heterodimeric membrane proteins that uniquely mediate inside-out signal transduction, whereby adhesion to the extracellular matrix is activated from within the cell by direct binding of talin to the cytoplasmic tail of the beta integrin subunit. Here, we report the first structure of talin bound to an authentic full-length beta integrin tail. Using biophysical and whole cell measurements, we show that a specific ionic interaction between the talin F3 domain and the membrane-proximal helix of the beta tail disrupts an integrin alpha/beta salt bridge that helps maintain the integrin inactive state. Second, we identify a positively charged surface on the talin F2 domain that precisely orients talin to disrupt the heterodimeric integrin transmembrane (TM) complex. These results show key structural features that explain the ability of talin to mediate inside-out TM signalling.Nicholas J. Anthis, Kate L. Wegener, Feng Ye, Chungho Kim, Benjamin T. Goult, Edward D. Lowe, Ioannis Vakonakis, Neil Bate, David R. Critchley, Mark H. Ginsberg and Iain D. Campbel
Sharp ill-posedness result for the periodic Benjamin-Ono equation (ERRATUM : PAPER WITHDRAWN)
No full textERRATUM : This paper has been withdrawn by the author since there were errors in the calculus of the defect coefficient in Page 11. The corrected calculus gives actually zero which do not lead to a contradiction on the continuity of the flow-map of the Benjamin-Ono equation. The author warmly thank Professor Patrick Gérard for having pointing out this error to him.ERRATUM : This paper has been withdrawn by the author since there were errors in the calculus of the defect coefficient in Page 11. The corrected calculus gives actually zero which do not lead to a contradiction on the continuity of the flow-map of the Benjamin-Ono equation. The author warmly thank Professor Patrick Gérard for having pointing out this error to him. (We prove the discontinuity for the weak L^2(\T) -topology of the flow-map associated with the periodic Benjamin-Ono equation. This ensures that this equation is ill-posed in H^s(\T) as soon as and thus completes exactly the well-posedness result obtained by the author.
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