Alveolar proteins stabilize cortical microtubules in Toxoplasma gondii

Single-celled protists use elaborate cytoskeletal structures, including arrays of microtubules at the cell periphery, to maintain polarity and rigidity. The obligate intracellular parasite Toxoplasma gondii has unusually stable cortical microtubules beneath the alveoli, a network of flattened membrane vesicles that subtends the plasmalemma. However, anchoring of microtubules along alveolar membranes is not understood. Here, we show that GAPM1a, an integral membrane protein of the alveoli, plays a role in maintaining microtubule stability. Degradation of GAPM1a causes cortical microtubule disorganisation and subsequent depo-lymerisation. These changes in the cytoskeleton lead to parasites becoming shorter and rounder, which is accompanied by a decrease in cellular volume. Extended GAPM1a depletion leads to severe defects in division, reminiscent of the effect of disrupting other alveolar proteins. We suggest that GAPM proteins link the cortical microtubules to the alveoli and are required to maintain the shape and rigidity of apicomplexan zoites

Managing early succession for biodiversity and long-term productivity of conifer forests in southwestern Oregon

Early-successional stages have been truncated and altered in many western U.S. forest landscapes by planting conifers, controlling competing vegetation, suppressing fire, and focusing on maintaining late-seral species and undisturbed riparian zones. Declining area of early-successional stages may be reducing resilience and sustainability on landscapes that experience elevated disturbance related to future climate changes. In this study, two post-harvest early-successional treatments were compared to each other and to two mature-forest treatments using 20 years of evidence from replicated 7-ha experimental units in a southwestern Oregon forest dominated by Douglas-fir (Pseudotsuga menziesii Mirb. Franco). One early-successional treatment (Douglas-fir plantation) planted Douglas-fir and was followed by a brushing to reduce hardwood competition to move quickly to the conifer stem-exclusion stage; the other (Early-seral plantation) favored natural sprouting and regeneration of hardwood shrubs and trees and planted scattered knobcone pines (Pinus attenuata Lemmon) and Douglas-fir. Plant diversity in the Early-seral plantation was 56% (year 2) and 26% (year 6) higher than in the Douglas-fir plantation. Both early-successional treatments far exceeded plant diversity in Unaltered and Thinned mature stands. Fifteen years of growth of shrubs and hardwood trees in the Early-seral plantation was remarkable, resulting in total aboveground biomass increment (18 Mg ha⁻¹ yr⁻¹) double that of the Douglas-fir plantations. Important process effects related to primary productivity were noted: losses of soil organic matter from the B horizon in young Douglas-fir, and, after wildfire, increases in N₂-fixing plant cover in Early-seral plantation. The burl-sprouting and deep rooting of many hardwoods also created opportunities for nutrient retention and release from primary minerals as well as deep-profile water supply. Recognizing the importance of intentionally managing for shrubs and hardwood trees is particularly relevant at this site, because stand reconstruction and historical records indicate these species, along with knobcone pine, dominated the site for 40 years before the current mature Douglas-fir forest started gaining dominance. In contrast, the prolific natural regeneration of Douglas-fir after recent harvest and wildfire suggests that what comes back ‘‘naturally’’ in modern times will not allow this history to be repeated.Keywords: N₂ fixation, Early-seral, Long-term productivity, Biodiversity, Early successionKeywords: N₂ fixation, Early-seral, Long-term productivity, Biodiversity, Early successio

A Method for Generation of Bone Marrow-Derived Macrophages from Cryopreserved Mouse Bone Marrow Cells

The broad use of transgenic and gene-targeted mice has established bone marrow-derived macrophages (BMDM) as important mammalian host cells for investigation of the macrophages biology. Over the last decade, extensive research has been done to determine how to freeze and store viable hematopoietic human cells; however, there is no information regarding generation of BMDM from frozen murine bone marrow (BM) cells. Here, we establish a highly efficient protocol to freeze murine BM cells and further generate BMDM. Cryopreserved murine BM cells maintain their potential for BMDM differentiation for more than 6 years. We compared BMDM obtained from fresh and frozen BM cells and found that both are similarly able to trigger the expression of CD80 and CD86 in response to LPS or infection with the intracellular bacteria Legionella pneumophila. Additionally, BMDM obtained from fresh or frozen BM cells equally restrict or support the intracellular multiplication of pathogens such as L. pneumophila and the protozoan parasite Leishmania (L.) amazonensis. Although further investigation are required to support the use of the method for generation of dendritic cells, preliminary experiments indicate that bone marrow-derived dendritic cells can also be generated from cryopreserved BM cells. Overall, the method described and validated herein represents a technical advance as it allows ready and easy generation of BMDM from a stock of frozen BM cells

Service engagement in interventions for street-connected children and young people: a summary of evidence supplementing a recent Cochrane–Campbell review

Abstract Background This paper builds on a Cochrane–Campbell systematic review of interventions that reduce harms and promote reintegration in street-connected children and young people focusing on intervention outcomes. The aim of the present analysis is to explore questions raised in the systematic review over the potential role of service engagement in mediating outcomes of relevant interventions. Objective The paper summarises engagement-related findings from quantitative intervention evaluations with street-connected populations of children and young people, as reported by study authors. It seeks to contribute to theoretical and methodological understandings of service engagement with street-connected youth populations and to highlight gaps in current knowledge. Methods Drawing on the original search for the Cochrane–Campbell review, we rescreened search results in our database and included quantitative findings if relevant to our current research questions, regardless of study design. Additionally, we sought new study publications from authors whose work was included in the original systematic review. The discussion explores relevant data from five studies included in the original systematic review, ten studies excluded from the review, and two studies published after the completion of the review. Results The measures of service engagement in the included studies focused on treatment attendance, ‘level of engagement’, and service satisfaction. Evidence on the impact of service engagement on other outcomes in interventions for street-connected children and young people was limited. Available data on the predictors and impact of service engagement were mixed and appear not to provide robust support for common hypotheses in the relevant context

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion

EGFRvIII upregulates DNA mismatch repair resulting in increased temozolomide sensitivity of MGMT promoter methylated glioblastoma

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR

A Novel Family of Toxoplasma IMC Proteins Displays a Hierarchical Organization and Functions in Coordinating Parasite Division

Apicomplexans employ a peripheral membrane system called the inner membrane complex (IMC) for critical processes such as host cell invasion and daughter cell formation. We have identified a family of proteins that define novel sub-compartments of the Toxoplasma gondii IMC. These IMC Sub-compartment Proteins, ISP1, 2 and 3, are conserved throughout the Apicomplexa, but do not appear to be present outside the phylum. ISP1 localizes to the apical cap portion of the IMC, while ISP2 localizes to a central IMC region and ISP3 localizes to a central plus basal region of the complex. Targeting of all three ISPs is dependent upon N-terminal residues predicted for coordinated myristoylation and palmitoylation. Surprisingly, we show that disruption of ISP1 results in a dramatic relocalization of ISP2 and ISP3 to the apical cap. Although the N-terminal region of ISP1 is necessary and sufficient for apical cap targeting, exclusion of other family members requires the remaining C-terminal region of the protein. This gate-keeping function of ISP1 reveals an unprecedented mechanism of interactive and hierarchical targeting of proteins to establish these unique sub-compartments in the Toxoplasma IMC. Finally, we show that loss of ISP2 results in severe defects in daughter cell formation during endodyogeny, indicating a role for the ISP proteins in coordinating this unique process of Toxoplasma replication

TgICMAP1 Is a Novel Microtubule Binding Protein in Toxoplasma gondii

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction

Distance travelled : Outcomes and evidence in flexible learning options

Flexible learning options (FLOs) provide individualised learning pathways for disengaged young people with strong emphasis on inclusivity and wellbeing support. Amidst a rapid expansion of Australia’s flexible learning sector, service providers are under increasing pressure to substantiate participant outcomes. This paper stems from a national study of the value of FLOs to young people and the broader Australian community. The study enumerates the outcomes valued by flexible learning practitioners, as well as the various evidence forms they cite to substantiate participant outcomes. Framing success as ‘distance travelled’ (i.e. an individual’s progress relative to his or her own starting point), practitioners demonstrate critical awareness of the social and structural mechanisms by which young people are marginalised from mainstream schooling. Holistic assessment practices also reveal practitioners’ efforts to expand the terms of reference by which educational outcomes may be validated in alternative education settings

SAS6-like protein in Plasmodium indicates that conoid-associated apical complex proteins persist in invasive stages within the mosquito vector

The SAS6-like (SAS6L) protein, a truncated paralogue of the ubiquitous basal body/centriole protein SAS6, has been characterised recently as a flagellum protein in trypanosomatids, but associated with the conoid in apicomplexan Toxoplasma. The conoid has been suggested to derive from flagella parts, but is thought to have been lost from some apicomplexans including the malaria-causing genus Plasmodium. Presence of SAS6L in Plasmodium, therefore, suggested a possible role in flagella assembly in male gametes, the only flagellated stage. Here, we have studied the expression and role of SAS6L throughout the Plasmodium life cycle using the rodent malaria model P. berghei. Contrary to a hypothesised role in flagella, SAS6L was absent during gamete flagellum formation. Instead, SAS6L was restricted to the apical complex in ookinetes and sporozoites, the extracellular invasive stages that develop within the mosquito vector. In these stages SAS6L forms an apical ring, as we show is also the case in Toxoplasma tachyzoites. The SAS6L ring was not apparent in blood-stage invasive merozoites, indicating that the apical complex is differentiated between the different invasive forms. Overall this study indicates that a conoid-associated apical complex protein and ring structure is persistent in Plasmodium in a stage-specific manner