Emerging Roles of Viroporins Encoded by DNA Viruses: Novel Targets for Antivirals?

Studies have highlighted the essential nature of a group of small, highly hydrophobic, membrane embedded, channel-forming proteins in the life cycles of a growing number of RNA viruses. These viroporins mediate the flow of ions and a range of solutes across cellular membranes and are necessary for manipulating a myriad of host processes. As such they contribute to all stages of the virus life cycle. Recent discoveries have identified proteins encoded by the small DNA tumor viruses that display a number of viroporin like properties. This review article summarizes the recent developments in our understanding of these novel viroporins; describes their roles in the virus life cycles and in pathogenesis and speculates on their potential as targets for anti-viral therapeutic intervention

Microtubule organization within mitotic spindles revealed by serial block face scanning electron microscopy and image analysis

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus; and fourth, volumetric analysis of kinetochores. Our workflow also includes new computational tools for exploring the spatial arrangement of microtubules within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle

Defining endogenous TACC3–chTOG–clathrin–GTSE1 interactions at the mitotic spindle using induced relocalization

A multiprotein complex containing TACC3, clathrin and other proteins has been implicated in mitotic spindle stability. To disrupt this complex in an anti-cancer context, we need to understand its composition and how it interacts with microtubules. Induced relocalization of proteins in cells is a powerful way to analyze protein–protein interactions and, additionally, monitor where and when these interactions occur. We used CRISPR/Cas9 gene editing to add tandem FKBP–GFP tags to each complex member. The relocalization of endogenous tagged protein from the mitotic spindle to mitochondria and assessment of the effect on other proteins allowed us to establish that TACC3 and clathrin are core complex members and that chTOG (also known as CKAP5) and GTSE1 are ancillary to the complex, binding respectively to TACC3 and clathrin, but not each other. We also show that PIK3C2A, a clathrin-binding protein that was proposed to stabilize the TACC3–chTOG–clathrin–GTSE1 complex during mitosis, is not a member of the complex. This work establishes that targeting the TACC3–clathrin interface or their microtubule-binding sites are the two strategies most likely to disrupt spindle stability mediated by this multiprotein complex

A genetically encoded reporter of synaptic activity in vivo

To image synaptic activity within neural circuits, we tethered the genetically encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detected the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: it identified the locations of synapses and had a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicated that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrated that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum and graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses

A systematic review of biomarkers for disease progression in Parkinson's disease

Peer reviewedPublisher PD

Brain age predicts mortality

Age-associated disease and disability are placing a growing burden on society. However, ageing does not affect people uniformly. Hence, markers of the underlying biological ageing process are needed to help identify people at increased risk of age-associated physical and cognitive impairments and ultimately, death. Here, we present such a biomarker, ‘brain-predicted age’, derived using structural neuroimaging. Brain-predicted age was calculated using machine-learning analysis, trained on neuroimaging data from a large healthy reference sample (N=2001), then tested in the Lothian Birth Cohort 1936 (N=669), to determine relationships with age-associated functional measures and mortality. Having a brain-predicted age indicative of an older-appearing brain was associated with: weaker grip strength, poorer lung function, slower walking speed, lower fluid intelligence, higher allostatic load and increased mortality risk. Furthermore, while combining brain-predicted age with grey matter and cerebrospinal fluid volumes (themselves strong predictors) not did improve mortality risk prediction, the combination of brain-predicted age and DNA-methylation-predicted age did. This indicates that neuroimaging and epigenetics measures of ageing can provide complementary data regarding health outcomes. Our study introduces a clinically-relevant neuroimaging ageing biomarker and demonstrates that combining distinct measurements of biological ageing further helps to determine risk of age-related deterioration and death

The Mitotic Arrest Deficient Protein MAD2B Interacts with the Clathrin Light Chain A during Mitosis

Contains fulltext : 87811.pdf (publisher's version ) (Open Access)BACKGROUND: Although the mitotic arrest deficient protein MAD2B (MAD2L2) is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1 (FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the human clathrin light chain A (CLTA) as a novel MAD2B binding protein. A direct interaction was established in mammalian cells via GST pull-down and endogenous co-immunoprecipitation during the G2/M phase of the cell cycle. Through subsequent confocal laser scanning microscopy we found that MAD2B and CLTA co-localize at the mitotic spindle. Clathrin forms a trimeric structure, i.e., the clathrin triskelion, consisting of three heavy chains (CLTC), each with an associated light chain. This clathrin structure has previously been shown to be required for the function of the mitotic spindle through stabilization of kinetochore fibers. Upon siRNA-mediated MAD2B depletion, we found that CLTA was no longer concentrated at the mitotic spindle but, instead, diffusely distributed throughout the cell. In addition, we found a marked increase in the percentage of misaligned chromosomes. CONCLUSIONS/SIGNIFICANCE: Previously, we identified MAD2B as an interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and a concomitant failure to shuttle MAD2B to the nucleus. Our current data show that MAD2B interacts with CLTA during the G2/M phase of the cell cycle and that depletion of MAD2B leads to a marked increase in the percentage of misaligned chromosomes and a redistribution of CLTA during mitosis

Cell Cycle Regulation and Cytoskeletal Remodelling Are Critical Processes in the Nutritional Programming of Embryonic Development

Many mechanisms purport to explain how nutritional signals during early development are manifested as disease in the adult offspring. While these describe processes leading from nutritional insult to development of the actual pathology, the initial underlying cause of the programming effect remains elusive. To establish the primary drivers of programming, this study aimed to capture embryonic gene and protein changes in the whole embryo at the time of nutritional insult rather than downstream phenotypic effects. By using a cross-over design of two well established models of maternal protein and iron restriction we aimed to identify putative common “gatekeepers” which may drive nutritional programming

Clathrin Is Spindle-Associated but Not Essential for Mitosis

Clathrin is a multimeric protein involved in vesicle coat assembly. Recently clathrin distribution was reported to change during the cell cycle and was found to associate with the mitotic spindle. Here we test whether the recruitment of clathrin to the spindle is indicative of a critical functional contribution to mitosis.Previously a chicken pre-B lymphoma cell line (DKO-R) was developed in which the endogenous clathrin heavy chain alleles were replaced with the human clathrin heavy chain under the control of a tetracycline-regulatable promoter. Receptor-mediated and fluid-phase endocytosis were significantly inhibited in this line following clathrin knockout, and we used this to explore the significance of clathrin heavy chain expression for cell cycle progression. We confirmed using confocal microscopy that clathrin colocalised with tubulin at mitotic spindles. Using a propidium iodide flow cytometric assay we found no statistical difference in the cell cycle distribution of the knockout cells versus the wild-type. Additionally, we showed that the ploidy and the recovery kinetics following cell cycle arrest with nocodazole were unchanged by repressing clathrin heavy chain expression.We conclude that the association of clathrin with the mitotic spindle and the contribution of clathrin to endocytosis are evolutionarily conserved. However we find that the contribution of clathrin to mitosis is less robust and dependent on cellular context. In other cell-lines silencing RNA has been used by others to knockdown clathrin expression resulting in an increase in the mitotic index of the cells. We show an effect on the G2/M phase population of clathrin knockdown in HEK293 cells but show that repressing clathrin expression in the DKO-R cell-line has no effect on the size of this population. Consequently this work highlights the need for a more detailed molecular understanding of the recruitment and function of clathrin at the spindle, since the localisation but not the impact of clathrin on mitosis appears to be robust in plants, mammalian and chicken B-cells

Halloween, Organization, and the Ethics of Uncanny Celebration

This article examines the relationship between organizational ethics, the uncanny, and the annual celebration of Halloween. We begin by exploring the traditional and contemporary organizational function of Halloween as ‘tension-management ritual’ (Etzioni, Sociol Theory 18(1):44–59, 2000) through which collective fears, anxieties, and fantasies are played out and given material expression. Combining the uncanny with the folkloric concept of ostension, we then examine an incident in which UK supermarket retailers made national news headlines for selling offensive Halloween costumes depicting ‘escaped mental patients’. Rather than treating this incident as a problem of moral hygiene—in which products are removed, apologies made, and lessons learned—we consider the value of Halloween as a unique and disruptive ethical encounter with the uncanny Other. Looking beyond its commercial appeal and controversy, we reflect on the creative, generous, and disruptive potential of Halloween as both tension-management ritual and unique organizational space of hospitality through which to receive and embrace alterity and so discover the homely within the unheimlich