The role of the flagellum attachment zone in Leishmania mexicana flagellar pocket architecture

tle:16338bd7-9cdf-4eaf-8a36-c4a07e0a00f0:d6bd9758-527a-46cd-bfe2-c433766e8fca:1Leishmania spp are flagellate protozoan parasites, with a digenetic life cycle, alternating between a mammalian host and insect vector. Within both the host and vector Leishmania adopts different morphologies and cell types, which are adapted to that ecological niche. One of the key architectural features in the determination of cell morphology is the flagellar pocket, which is the sole site of endo/exocytosis. It was shown that a large cytoskeletal structure, the flagellum attachment zone (FAZ), which connects the flagellum to the cell body, was an important contributor to the maintenance of the flagellar pocket architecture and its function. However, a poor understanding of FAZ molecular organisation and the specific roles of its constituent proteins remains. Here, 28 FAZ proteins were discovered by the endogenous tagging of the orthologs of T. brucei FAZ proteins identified through TrypTag. These proteins were categorised into five classes based on their localisation patterns. The classes sit within the three structural domains, the flagellum, intracellular and cell body of the FAZ structure. A deletion screen of these proteins revealed functional groups responsible for flagellum attachment and cell morphogenesis, with the specific function of these proteins connected to their exact location within the FAZ. Analyses of the flagellum domain proteins FAZ27 and FAZ34 revealed their importance for flagellum attachment. Moreover, the cell body domain CC2D was shown to be required for flagellum attachment and anterior cell tip morphogenesis and was dependent on FAZ2 for assembly. Importantly, the analysis of these proteins defined the assembly hierarchy of the three FAZ structural domains, with the cell body domain required for the assembly of the intracellular and flagellum domains and FAZ2 of the cell body domain being a key foundational protein. Together these results have provided crucial insights into the molecular organisation and function of the FAZ in Leishmania

Subcellular protein localisation of Trypanosoma brucei bloodstream form-upregulated proteins maps stage-specific adaptations

Background: Genome-wide subcellular protein localisation in Trypanosoma brucei, through our TrypTag project, has comprehensively dissected the molecular organisation of this important pathogen. Powerful as this resource is, T. brucei has multiple developmental forms and we previously only analysed the procyclic form. This is an insect life cycle stage, leaving the mammalian bloodstream form unanalysed. The expectation is that between life stages protein localisation would not change dramatically (completely unchanged or shifting to analogous stage-specific structures). However, this has not been specifically tested. Similarly, which organelles tend to contain proteins with stage-specific expression can be predicted from known stage specific adaptations but has not been comprehensively tested. Methods: We used endogenous tagging with mNG to determine the sub-cellular localisation of the majority of proteins encoded by transcripts significantly upregulated in the bloodstream form, and performed comparison to the existing localisation data in procyclic forms. Results: We have confirmed the localisation of known and identified the localisation of novel stage-specific proteins. This gave a map of which organelles tend to contain stage specific proteins: the mitochondrion for the procyclic form, and the endoplasmic reticulum, endocytic system and cell surface in the bloodstream form. Conclusions: This represents the first genome-wide map of life cycle stage-specific adaptation of organelle molecular machinery in T. brucei

Pharmacologically antagonizing the CXCR4-CXCL12 chemokine pathway with AMD3100 inhibits sunlight-induced skin cancer

One way sunlight causes skin cancer is by suppressing anti-tumor immunity. A major mechanism involves altering mast cell migration via the C-X-C motif chemokine receptor 4–C-X-C motif chemokine ligand 12 (CXCR4-CXCL12) chemokine pathway. We have discovered that pharmacologically blocking this pathway with the CXCR4 antagonist AMD3100 prevents both UV radiation-induced immune suppression and skin cancer. The majority of control mice receiving UV-only developed histopathologically confirmed squamous cell carcinomas. In contrast, skin tumor incidence and burden was significantly lower in AMD3100-treated mice. Perhaps most striking was that AMD3100 completely prevented the outgrowth of latent tumors that occurred once UV irradiation ceased. AMD3100 protection from UV immunosuppression and skin cancer was associated with reduced mast cell infiltration into the skin, draining lymph nodes, and the tumor itself. Thus a major target of CXCR4 antagonism was the mast cell. Our results indicate that interfering with UV-induced CXCL12 by antagonizing CXCR4 significantly inhibits skin tumor development by blocking UV-induced effects on mast cells. Hence, the CXCR4-CXCL12 chemokine pathway is a novel therapeutic target in the prevention of UV-induced skin cancer

Role for the flagellum attachment zone in Leishmania anterior cell tip morphogenesis

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum. These are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenesis of the anterior cell tip. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption to the membrane organisation at the anterior cell tip, resulting in cells that were connected to each other by a membranous bridge structure between their flagella. Moreover, the FAZ2 null mutant was unable to develop and proliferate in sand flies and had a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division

Reframing “participation” and “inclusion” in public health policy and practice to address health inequalities: Evidence from a major resident-led neighbourhood improvement initiative

There is a need for greater conceptual clarity in place‐based initiatives that seek to give residents of disadvantaged neighbourhoods more control over action to address the social determinants of health inequalities at a local level. In this article, we address this issue as it relates to the concepts of participation and inclusion. We draw on qualitative data generated during the first phase of the Communities in Control Study, a longitudinal multisite independent evaluation of the impact of Big Local on the social determinants of health and health inequalities. Big Local is a resident‐led area improvement initiative in England, funded by the UK Big Lottery Fund. Initiatives focused on community empowerment are increasingly prominent in public health policy and practice globally. Approaches emphasise the promotion of greater control over decisions and action among individuals, groups, and communities, particularly those living in disadvantaged circumstances. However, when it comes to participation and inclusion in taking action and making decisions, the field is characterised by conceptual confusion. This risks undermining the impact of these initiatives. While participation and inclusion are necessary conditions for empowerment and collective control, they are not necessarily sufficient. Sufficiency requires attention to the breadth of participation (i.e., to inclusion) and to the depth of participation (i.e., the extent to which it is experienced as empowering and ultimately enables the exercise of collective control over decisions and actions). In observing how different Big Local resident‐led partnerships across England are tackling the day‐to‐day challenges of engaging with their communities, we reveal the potential for policy and practice of reframing, and therefore clarifying (to highlight the different roles they have) the concepts of participation and inclusion in terms of depth and breadth

Cellular landmarks of Trypanosoma brucei and Leishmania mexicana

The kinetoplastids Trypanosoma brucei and Leishmania mexicana are eukaryotes with a highly structured cellular organisation that is reproduced with great fidelity in each generation. The pattern of signal from a fluorescently tagged protein can define the specific structure/organelle that this protein localises to, and can be extremely informative in phenotype analysis in experimental perturbations, life cycle tracking, post-genomic assays and functional analysis of organelles. Using the vast coverage of protein subcellular localisations provided by the TrypTag project, an ongoing project to determine the localisation of every protein encoded in the T. brucei genome, we have generated an inventory of reliable reference organelle markers for both parasites that combines epifluorescence images with a detailed description of the key features of each localisation. We believe this will be a useful comparative resource that will enable researchers to quickly and accurately pinpoint the localisation of their proteins of interest and will provide cellular markers for many types of cell biology studies. We see this as another important step in the post-genomic era analyses of these parasites, in which ever expanding datasets generate numerous candidates to analyse. Adoption of these reference proteins by the community is likely to enhance research studies and enable better comparison of data

Patterns of debate in tertiary level asynchronous text-based conferencing

Argumentation can be defined at different levels and serve different purposes, but its role in knowledge understanding and construction has given it a central place in education, particularly at tertiary level. The advent of computer-supported text-based conferences has created new sites where such educational dialogues can take place, but the quality of the interaction and whether it is serving its educational purpose is still uncertain. This paper reports on a framework of analysis that has been developed to illuminate the arguing process within an asynchronous electronic conferencing environment, showing how it is both similar to, and different from, argumentation in the more traditional forums of multi-party, face-to-face discussion and traditional written essays. The framework develops earlier work by the authors and is applied to two electronic conferences within the same postgraduate course, comparing overall patterns of argumentation. Findings are presented on the extent to which the technology of electronic conferencing shapes and supports students’ participation in academic literacy practices relating to argumentation, proposing, at the same time, that the teaching strategy adopted by the lecturer is also an important variable

Power, control, communities and health inequalities III: participatory spaces-an English case.

This article-third in a series of three-uses theoretical frameworks described in Part 1, and empirical markers reported in Part 2, to present evidence on how power dynamics shifted during the early years of a major English community empowerment initiative. We demonstrate how the capabilities disadvantaged communities require to exercise collective control over decisions/actions impacting on their lives and health (conceptualized as emancipatory power) and the exercise of power over these communities (conceptualized as limiting power) were shaped by the characteristics of participatory spaces created by and/or associated with this initiative. Two main types of participatory spaces were identified: governance and sense-making. Though all forms of emancipatory power emerged in all spaces, some were more evident in particular spaces. In governance spaces, the development and enactment of 'power to' emerged as residents made formal decisions on action, allocated resources and managed accountability. Capabilities for alliance building-power with-were more likely to emerge in these spaces, as was residents' resistance to the exercise of institutional power over them. In contrast, in sense-making spaces residents met informally and 'made sense' of local issues and their ability to influence these. These processes led to the development of power within capabilities and power to resist stigmatizing forms of productive power. The findings highlight the importance of designing community initiatives that: nurture diverse participatory spaces; attend to connectivity between spaces; and identify and act on existing power dynamics undermining capabilities for collective control in disadvantaged communities

The Importance of Brain Banks for Molecular Neuropathological Research: The New South Wales Tissue Resource Centre Experience

New developments in molecular neuropathology have evoked increased demands for postmortem human brain tissue. The New South Wales Tissue Resource Centre (TRC) at The University of Sydney has grown from a small tissue collection into one of the leading international brain banking facilities, which operates with best practice and quality control protocols. The focus of this tissue collection is on schizophrenia and allied disorders, alcohol use disorders and controls. This review highlights changes in TRC operational procedures dictated by modern neuroscience, and provides examples of applications of modern molecular techniques to study the neuropathogenesis of many different brain disorders

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis

Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18–24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks