Systematic characterization of all Toxoplasma gondii TBC domain-containing proteins identifies an essential regulator of Rab2 in the secretory pathway.

Toxoplasma gondii resides in its intracellular niche by employing a series of specialized secretory organelles that play roles in invasion, host cell manipulation, and parasite replication. Rab GTPases are major regulators of the parasite's secretory traffic that function as nucleotide-dependent molecular switches to control vesicle trafficking. While many of the Rab proteins have been characterized in T. gondii, precisely how these Rabs are regulated remains poorly understood. To better understand the parasite's secretory traffic, we investigated the entire family of Tre2-Bub2-Cdc16 (TBC) domain-containing proteins, which are known to be involved in vesicle fusion and secretory protein trafficking. We first determined the localization of all 18 TBC domain-containing proteins to discrete regions of the secretory pathway or other vesicles in the parasite. Second, we use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein, which localizes to the endoplasmic reticulum (ER), is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and mitochondrial morphology. TgTBC9 knockdown also results in the formation of large lipid droplets (LDs) and multi-membranous structures surrounded by ER membranes, further indicating a disruption of ER functions. We show that the conserved dual-finger active site in the TBC domain of the protein is critical for its GTPase-activating protein (GAP) function and that the Plasmodium falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast 2 hybrid analyses that TgTBC9 preferentially binds Rab2, indicating that the TBC9-Rab2 pair controls ER morphology and vesicular trafficking in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan and provide new insight into intracellular vesicle trafficking in T. gondii.

BCC0 collaborates with IMC32 and IMC43 to form the Toxoplasma gondii essential daughter bud assembly complex.

Toxoplasma gondii divides by endodyogeny, in which two daughter buds are formed within the cytoplasm of the maternal cell using the inner membrane complex (IMC) as a scaffold. During endodyogeny, components of the IMC are synthesized and added sequentially to the nascent daughter buds in a tightly regulated manner. We previously showed that the early recruiting proteins IMC32 and IMC43 form an essential daughter bud assembly complex which lays the foundation of the daughter cell scaffold in T. gondii. In this study, we identify the essential, early recruiting IMC protein BCC0 as a third member of this complex by using IMC32 as bait in both proximity labeling and yeast two-hybrid screens. We demonstrate that BCC0's localization to daughter buds depends on the presence of both IMC32 and IMC43. Deletion analyses and functional complementation studies reveal that residues 701-877 of BCC0 are essential for both its localization and function and that residues 1-899 are sufficient for function despite minor mislocalization. Pairwise yeast two-hybrid assays additionally demonstrate that BCC0's essential domain binds to the coiled-coil region of IMC32 and that BCC0 and IMC43 do not directly interact. This data supports a model for complex assembly in which an IMC32-BCC0 subcomplex initially recruits to nascent buds via palmitoylation of IMC32 and is locked into the scaffold once bud elongation begins by IMC32 binding to IMC43. Together, this study dissects the organization and function of a complex of three early recruiting daughter proteins which are essential for the proper assembly of the IMC during endodyogeny.

Identification of IMC43, a novel IMC protein that collaborates with IMC32 to form an essential daughter bud assembly complex in Toxoplasma gondii.

The inner membrane complex (IMC) of Toxoplasma gondii is essential for all phases of the parasite's life cycle. One of its most critical roles is to act as a scaffold for the assembly of daughter buds during replication by endodyogeny. While many daughter IMC proteins have been identified, most are recruited after bud initiation and are not essential for parasite fitness. Here, we report the identification of IMC43, a novel daughter IMC protein that is recruited at the earliest stages of daughter bud initiation. Using an auxin-inducible degron system we show that depletion of IMC43 results in aberrant morphology, dysregulation of endodyogeny, and an extreme defect in replication. Deletion analyses reveal a region of IMC43 that plays a role in localization and a C-terminal domain that is essential for the protein's function. TurboID proximity labelling and a yeast two-hybrid screen using IMC43 as bait identify 30 candidate IMC43 binding partners. We investigate two of these: the essential daughter protein IMC32 and a novel daughter IMC protein we named IMC44. We show that IMC43 is responsible for regulating the localization of both IMC32 and IMC44 at specific stages of endodyogeny and that this regulation is dependent on the essential C-terminal domain of IMC43. Using pairwise yeast two-hybrid assays, we determine that this region is also sufficient for binding to both IMC32 and IMC44. As IMC43 and IMC32 are both essential proteins, this work reveals the existence of a bud assembly complex that forms the foundation of the daughter IMC during endodyogeny.

IMC10 and LMF1 mediate mitochondrial morphology through mitochondrion-pellicle contact sites in Toxoplasma gondii.

The single mitochondrion of Toxoplasma gondii is highly dynamic, being predominantly in a peripherally distributed lasso-shape in intracellular parasites and collapsed in extracellular parasites. The peripheral positioning of the mitochondrion is associated with apparent contacts between the mitochondrion membrane and the parasite pellicle. The outer mitochondrial membrane-associated protein LMF1 is critical for the correct positioning of the mitochondrion. Intracellular parasites lacking LMF1 fail to form the lasso-shaped mitochondrion. To identify other proteins that tether the mitochondrion of the parasite to the pellicle, we performed a yeast two-hybrid screen for LMF1 interactors. We identified 70 putative interactors localized in different cellular compartments, such as the apical end of the parasite, mitochondrial membrane and the inner membrane complex (IMC), including with the pellicle protein IMC10. Using protein-protein interaction assays, we confirmed the interaction of LMF1 with IMC10. Conditional knockdown of IMC10 does not affect parasite viability but severely affects mitochondrial morphology in intracellular parasites and mitochondrial distribution to the daughter cells during division. In effect, IMC10 knockdown phenocopies disruption of LMF1, suggesting that these two proteins define a novel membrane tether between the mitochondrion and the IMC in Toxoplasma. This article has an associated First Person interview with the first author of the paper.

Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis.

Apicomplexan parasites use a specialized cilium structure called the apical complex to organize their secretory organelles and invasion machinery. The apical complex is integrally associated with both the parasite plasma membrane and an intermediate filament cytoskeleton called the inner-membrane complex (IMC). While the apical complex is essential to the parasitic lifestyle, little is known about the regulation of apical complex biogenesis. Here, we identify AC9 (apical cap protein 9), a largely intrinsically disordered component of the Toxoplasma gondii IMC, as essential for apical complex development, and therefore for host cell invasion and egress. Parasites lacking AC9 fail to successfully assemble the tubulin-rich core of their apical complex, called the conoid. We use proximity biotinylation to identify the AC9 interaction network, which includes the kinase extracellular signal-regulated kinase 7 (ERK7). Like AC9, ERK7 is required for apical complex biogenesis. We demonstrate that AC9 directly binds ERK7 through a conserved C-terminal motif and that this interaction is essential for ERK7 localization and function at the apical cap. The crystal structure of the ERK7-AC9 complex reveals that AC9 is not only a scaffold but also inhibits ERK7 through an unusual set of contacts that displaces nucleotide from the kinase active site. ERK7 is an ancient and autoactivating member of the mitogen-activated kinase (MAPK) family and its regulation is poorly understood in all organisms. We propose that AC9 dually regulates ERK7 by scaffolding and concentrating it at its site of action while maintaining it in an "off" state until the specific binding of a true substrate.

A photoactivatable crosslinking system reveals protein interactions in the Toxoplasma gondii inner membrane complex.

The Toxoplasma gondii inner membrane complex (IMC) is an important organelle involved in parasite motility and replication. The IMC resides beneath the parasite's plasma membrane and is composed of both membrane and cytoskeletal components. Although the protein composition of the IMC is becoming better understood, the protein-protein associations that enable proper functioning of the organelle remain largely unknown. Determining protein interactions in the IMC cytoskeletal network is particularly challenging, as disrupting the cytoskeleton requires conditions that disrupt protein complexes. To circumvent this problem, we demonstrate the application of a photoreactive unnatural amino acid (UAA) crosslinking system to capture protein interactions in the native intracellular environment. In addition to identifying binding partners, the UAA approach maps the binding interface of the bait protein used for crosslinking, providing structural information of the interacting proteins. We apply this technology to the essential IMC protein ILP1 and demonstrate that distinct regions of its C-terminal coiled-coil domain crosslink to the alveolins IMC3 and IMC6, as well as IMC27. We also show that the IMC3 C-terminal domain and the IMC6 N-terminal domain are necessary for binding to ILP1, further mapping interactions between ILP1 and the cytoskeleton. Together, this study develops a new approach to study protein-protein interactions in Toxoplasma and provides the first insight into the architecture of the cytoskeletal network of the apicomplexan IMC.

Care delivery and self-management strategies for children with epilepsy.

BACKGROUND: Epilepsy is a neurological disorder affecting both children and adults. Epileptic seizures are the result of excessive and abnormal cortical cell electrical activity in the brain. In response to criticism that epilepsy care for children has little impact on long-term outcomes, healthcare professionals and administrators have developed various service models and strategies to address perceived inadequacies. This is an updated version of a Cochrane Review previously published in 2018. OBJECTIVES: To assess the effects of any specialised or dedicated intervention for epilepsy versus usual care in children and adolescents with epilepsy and their families. SEARCH METHODS: We searched the following databases on 14 January 2020: the Cochrane Register of Studies (CRS Web), MEDLINE (Ovid, 1946 to 13 January 2020), PsycINFO (1887 to 14 January 2020), CINAHL Plus (1937 to 14 January 2020), ClinicalTrials.gov, and the World Health Organization International Clinical Trials Registry Platform. The Cochrane Register of Studies (CRS Web) includes the Cochrane Epilepsy Group Specialised Register and the Cochrane Central Register of Controlled Trials (CENTRAL). We also contacted experts in the field seeking information on unpublished and ongoing studies and checked the websites of epilepsy organisations and the reference lists of included studies. SELECTION CRITERIA: We included randomised controlled trials recruiting children and adolescents with epilepsy. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials for inclusion and extracted the relevant data. We assessed the following outcomes: 1. Seizure frequency and severity; 2. Appropriateness and volume of medication prescribed (including evidence of drug toxicity); 3. Participants' reported knowledge of information and advice received from professionals; 4. Participants' reports of health and quality of life; 5. Objective measures of general health status; 6. Objective measures of social or psychological functioning (including the number of days spent on sick leave/absence from school or work, and employment status); and 7. Costs of care or treatment. The results of the data extraction and quality assessment for each study were presented in structured tables and as a narrative summary. All summary statistics were extracted for each outcome. MAIN RESULTS: We included nine studies of eight interventions in the review, reporting on seven distinct self-management programmes for educating or counselling children with epilepsy and their parents, and one new model of care. Based largely on self-reported outcomes, each programme showed some benefits for the well-being of children with epilepsy; however, all of the included studies had methodological flaws. No single programme was evaluated with different study samples, and in no instance was the same outcome measured and reported in the same way across studies, precluding any possible meta-analysis, even if the interventions were considered sufficiently similar to include in meta-analysis.  We chose the outcomes for which data might be important for decisions about the interventions as per guidance in the Cochrane Handbook for Systematic Reviews of Interventions. We found moderate certainty evidence that one of the educational interventions reduced seizure frequency. There was low certainty evidence that two other educational interventions reduced seizure severity, seizure control, and seizure cure rates. The evidence for all other outcomes (drug adherence, knowledge, self-efficacy and self-perception of epilepsy on quality of life) was mixed. AUTHORS' CONCLUSIONS: Whilst each of the programmes evaluated in this review showed some benefit to children with epilepsy, their impact was extremely variable. No programme showed benefits across the full range of outcomes, and all studies had methodological problems. There is currently insufficient evidence in favour of any single programme. Further evidence from randomised controlled trials using validated measures and considering clinical meaningfulness as well as statistical significance of results is required.

Elastic-plastic properties of mesoscale electrodeposited LIGA nickel alloy films: microscopy and mechanics.

The elastic-plastic properties of mesoscale electrodeposited LIGA Ni alloy specimens are investigated as a function of specimen size, strain rate, and material composition. Two material compositions are studied: a high-strength fine-grained Ni-Fe alloy and a high-ductility coarse-grained Ni-Co alloy. The specimens have thicknesses of approximately 200 µm and gauge widths ranging from 75 µm to 700 µm. Tensile tests are conducted at strain rates of 0.001/s and 1/s using tabletop loading apparatuses and digital image correlation (DIC) for strain measurement. For each test condition, the apparent Young's modulus, yield strength, ultimate tensile strength, and strain hardening exponent and strength coefficient are extracted from the tensile tests. The true strains to failure are also assessed from fractography. Size, rate, and composition effects are discussed. For most properties, the statistical scatter represented by the standard deviation exceeds the measurement uncertainty; the notable exceptions to these observations are the apparent Young's modulus and yield strength, where large measurement uncertainties are ascribed to common experimental factors and material microplasticity.

Skin integrity-the perpetual challenge.

In an Essity-sponsored symposium, entitled 'Skin integrity-the perpetual challenge', three key opinion leaders discussed the importance of recognising and managing skin vulnerability in a patient-centred way. Peter Bradley summarises the main points they presented at the EWMA 2020 Virtual Conference This article offers an introduction to the symposium, Skin integrity-the perpetual challenge, held on 18 November 2020, as part of the EWMA 2020 Virtual Conference. There were three speakers. Dimitri Beeckman, Professor of Skin Integrity and Clinical Nursing, Ghent University, Belgium, focused on moisture lesions or MASD (moisture-associated skin damage). Karen Campbell, Consultant, Primacare Living Solutions and Adjunct Professor, MClScWH, Western University, London, Ontario, Canada, focused on the concepts related to skin vulnerability. She aimed to identify shared risk factors for skin conditions and ways to promote skin integrity, formulating a synergistic prevention approach to break down barriers in practice. The third speaker was Alessandro Corsi, Wound Care Consultant and Surgeon, Director of Wound Care Unit, IRCCS San Raffaele Hospital, Milan. He looked in detail at the dressings available in this area, detailing how he and his team had successfully used the Essity line of Skin Sensitive silicone dressings in their hospital.

Antimicrobial resistance-is wound care prepared?

In an Essity-sponsored symposium, entitled 'Antimicrobial resistance-is wound care prepared?', four key opinion leaders discussed the difficult journey to managing antimicrobial resistance within wound care at the global and local levels, backed up by the latest research. Peter Bradley summarises the main points they presented at the EWMA 2020 Virtual Conference. This article offers an introduction to the symposium, Antimicrobial resistance-is wound care prepared?, held on 18 November 2020, as part of the EWMA 2020 Virtual Conference. There were four speakers. Claire Kilpatrick, Director, S3 Global and Consultant to the World Health Organization, spoke on 'Preventing infections and managing antibiotic use-tackling a crisis for the health of nations'. The topic of Karen Ousey, Professor of Skin Integrity, Director for the Institute of Skin Integrity and Infection Prevention, University of Huddersfield, was 'Antimicrobial stewardship in wound management-less words more action'. Finally, Hadar Lev-Tov, Assistant Professor, and Irena Pastar, Associate Professor, both of the Dr Phillip Frost Department of Dermatology and Cutaneous Surgery, University of Miami, gave a talk on 'Silver-resistant bacteria in venous leg ulcers? First results of an exploratory study'.

Modelling the test methods used to determine material compatibility for hydrogen pressure vessel service.

This work aims to create finite element models to simulate the three ISO 11114-4 test methods applicable to hydrogen gas cylinders, coupled with calibrated constitutive models, to predict the deformation response of each. Experimental measurements are used to calibrate a monotonic constitutive model and a constitutive model of cyclic deformation. Six finite element solid models are discussed: monotonic tensile test of dog bone-shaped specimens, strain-controlled fatigue test of dog bone-shaped specimens, ISO test Method A, ISO test Method B, and ISO Method C (from ISO 11114-4), and a gas cylinder. Each finite element solid model is paired with the appropriate constitutive model based upon loading conditions. The modeling results are then combined with a new damage parameter in an attempt to compare each of the test methods to the others, as well as to in-service conditions. It is shown that the proposed damage parameter may be used to correlate all test methods considered (except for ISO Method A, a burst-disc test) as well as in-service conditions. The calibrated damage parameter may be coupled with any geometry, loading condition, and boundary condition modeled within a finite element package to predict the onset of critical damage in the material for which the coupled constitutive model is calibrated to. Parametric modelling study results provide estimated cycles to the onset of crack extension for DOT 3AA cylinders having varying sizes of internal thumbnail-shaped cracks. This work provides the baseline for measurements and models in air, with similar work in hydrogen to follow.

Novel insights into the composition and function of the Toxoplasma IMC sutures.

The Toxoplasma inner membrane complex (IMC) is a specialized organelle underlying the parasite's plasma membrane that consists of flattened rectangular membrane sacs that are sutured together and positioned atop a supportive cytoskeleton. We have previously identified a novel class of proteins localizing to the transverse and longitudinal sutures of the IMC, which we named IMC sutures components (ISCs). Here, we have used proximity-dependent biotin identification at the sutures to better define the composition of this IMC subcompartment. Using ISC4 as bait, we demonstrate biotin-dependent labeling of the sutures and have uncovered two new ISCs. We also identified five new proteins that exclusively localize to the transverse sutures that we named transverse sutures components (TSCs), demonstrating that components of the IMC sutures consist of two groups: those that localize to the transverse and longitudinal sutures (ISCs) and those residing only in the transverse sutures (TSCs). In addition, we functionally analyze the ISC protein ISC3 and demonstrate that ISC3-null parasites have morphological defects and reduced fitness in vitro. Most importantly, Δisc3 parasites exhibit a complete loss of virulence in vivo. These studies expand the known composition of the IMC sutures and highlight the contribution of ISCs to the ability of the parasite to proliferate and cause disease.

Care delivery and self-management strategies for children with epilepsy.

BACKGROUND: In response to criticism that epilepsy care for children has little impact, healthcare professionals and administrators have developed various service models and strategies to address perceived inadequacies. OBJECTIVES: To assess the effects of any specialised or dedicated intervention for epilepsy versus usual care in children with epilepsy and in their families. SEARCH METHODS: We searched the Cochrane Epilepsy Group Specialized Register (27 September 2016), the Cochrane Central Register of Controlled Trials (CENTRAL; 2016, Issue 9) in the Cochrane Library, MEDLINE (1946 to 27 September 2016), Embase (1974 to 27 September 2016), PsycINFO (1887 to 27 September 2016) and CINAHL Plus (1937 to 27 September 2016). In addition, we also searched clinical trials registries for ongoing or recently completed trials, contacted experts in the field to seek information on unpublished and ongoing studies, checked the websites of epilepsy organisations and checked the reference lists of included studies. SELECTION CRITERIA: We included randomised controlled trials (RCTs), cohort studies or other prospective studies with a (matched or unmatched) control group (controlled before-and-after studies), or time series studies. DATA COLLECTION AND ANALYSIS: We used standard methodological procedures expected by Cochrane. MAIN RESULTS: Our review included six interventions reported through seven studies (of which five studies were designed as RCTs). They reported on different education and counselling programmes for children and parents; teenagers and parents; or children, adolescents and their parents. Each programme showed some benefits for the well-being of children with epilepsy, but all had methodological flaws (e.g. in one of the studies designed as an RCT, randomisation failed), no single programme was independently evaluated with different study samples and no interventions were sufficiently homogeneous enough to be included in a meta-analysis,. AUTHORS' CONCLUSIONS: While each of the programmes in this review showed some benefit to children with epilepsy, their impacts were extremely variable. No programme showed benefits across the full range of outcomes, and all studies had major methodological problems. At present there is insufficient evidence in favour of any single programme.

Wound Exudate.

Tina Chambers, Independent Tissue Viability Consultant.

RON5 is critical for organization and function of the Toxoplasma moving junction complex.

Apicomplexans facilitate host cell invasion through formation of a tight-junction interface between parasite and host plasma membranes called the moving junction (MJ). A complex of the rhoptry neck proteins RONs 2/4/5/8 localize to the MJ during invasion where they are believed to provide a stable anchoring point for host penetration. During the initiation of invasion, the preformed MJ RON complex is injected into the host cell where RON2 spans the host plasma membrane while RONs 4/5/8 localize to its cytosolic face. While much attention has been directed toward an AMA1-RON2 interaction supposed to occur outside the cell, little is known about the functions of the MJ RONs positioned inside the host cell. Here we provide a detailed analysis of RON5 to resolve outstanding questions about MJ complex organization, assembly and function during invasion. Using a conditional knockdown approach, we show loss of RON5 results in complete degradation of RON2 and mistargeting of RON4 within the parasite secretory pathway, demonstrating that RON5 plays a key role in organization of the MJ RON complex. While RON8 is unaffected by knockdown of RON5, these parasites are unable to invade new host cells, providing the first genetic demonstration that RON5 plays a critical role in host cell penetration. Although invasion is not required for injection of rhoptry effectors into the host cytosol, parasites lacking RON5 also fail to form evacuoles suggesting an intact MJ complex is a prerequisite for secretion of rhoptry bulb contents. Additionally, while the MJ has been suggested to function in egress, disruption of the MJ complex by RON5 depletion does not impact this process. Finally, functional complementation of our conditional RON5 mutant reveals that while proteolytic separation of RON5 N- and C-terminal fragments is dispensable, a portion of the C-terminal domain is critical for RON2 stability and function in invasion.

Care delivery and self management strategies for adults with epilepsy.

BACKGROUND: Researchers have criticised epilepsy care for adults for its lack of impact, stimulating the development of various service models and strategies to respond to perceived inadequacies. OBJECTIVES: To assess the effects of any specialised or dedicated intervention beyond that of usual care in adults with epilepsy. SEARCH METHODS: For the latest update of this review, we searched the Cochrane Epilepsy Group Specialized Register (9 December 2013), the Cochrane Central Register of Controlled Trials (CENTRAL) (2013, Issue 11), MEDLINE (1946 to June 2013), EMBASE (1988 to June 2013), PsycINFO (1887 to December 2013) and CINAHL (1937 to December 2013). In addition, we contacted experts in the field to seek information on unpublished and ongoing studies, checked the websites of epilepsy organisations and checked the reference lists of included studies. SELECTION CRITERIA: We included randomised controlled trials, controlled or matched trials, cohort studies or other prospective studies with a control group, and time series studies. DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies, extracted all data, and assessed the quality of all included studies. MAIN RESULTS: Our review included 18 different studies of 16 separate interventions, which we classified into seven distinct groups. Most of the studies have methodological weaknesses, and many results from other analyses within studies need to be interpreted with caution because of study limitations. Consequently, there is currently limited evidence for the effectiveness of interventions to improve the health and quality of life in people with epilepsy. It was not possible to combine study results in a meta-analysis because of the heterogeneity of outcomes, study populations, interventions and time scales across the studies. AUTHORS' CONCLUSIONS: Two intervention types, the specialist epilepsy nurse and self management education, have some evidence of benefit. However, we did not find clear evidence that other service models substantially improve outcomes for adults with epilepsy. It is also possible that benefits are situation specific and may not apply to other settings. These studies included only a small number of service providers whose individual competence or expertise may have had a significant impact on outcomes. At present it is not possible to advocate any single model of service provision.

The inner membrane complex sub-compartment proteins critical for replication of the apicomplexan parasite Toxoplasma gondii adopt a pleckstrin homology fold.

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation.

A Toxoplasma palmitoyl acyl transferase and the palmitoylated armadillo repeat protein TgARO govern apical rhoptry tethering and reveal a critical role for the rhoptries in host cell invasion but not egress.

Apicomplexans are obligate intracellular parasites that actively penetrate their host cells to create an intracellular niche for replication. Commitment to invasion is thought to be mediated by the rhoptries, specialized apical secretory organelles that inject a protein complex into the host cell to form a tight-junction for parasite entry. Little is known about the molecular factors that govern rhoptry biogenesis, their subcellular organization at the apical end of the parasite and subsequent release of this organelle during invasion. We have identified a Toxoplasma palmitoyl acyltransferase, TgDHHC7, which localizes to the rhoptries. Strikingly, conditional knockdown of TgDHHC7 results in dispersed rhoptries that fail to organize at the apical end of the parasite and are instead scattered throughout the cell. While the morphology and content of these rhoptries appears normal, failure to tether at the apex results in a complete block in host cell invasion. In contrast, attachment and egress are unaffected in the knockdown, demonstrating that the rhoptries are not required for these processes. We show that rhoptry targeting of TgDHHC7 requires a short, highly conserved C-terminal region while a large, divergent N-terminal domain is dispensable for both targeting and function. Additionally, a point mutant lacking a key residue predicted to be critical for enzyme activity fails to rescue apical rhoptry tethering, strongly suggesting that tethering of the organelle is dependent upon TgDHHC7 palmitoylation activity. We tie the importance of this activity to the palmitoylated Armadillo Repeats-Only (TgARO) rhoptry protein by showing that conditional knockdown of TgARO recapitulates the dispersed rhoptry phenotype of TgDHHC7 knockdown. The unexpected finding that apicomplexans have exploited protein palmitoylation for apical organelle tethering yields new insight into the biogenesis and function of rhoptries and may provide new avenues for therapeutic intervention against Toxoplasma and related apicomplexan parasites.

Small-molecule inhibition of a depalmitoylase enhances Toxoplasma host-cell invasion.

Although there have been numerous advances in our understanding of how apicomplexan parasites such as Toxoplasma gondii enter host cells, many of the signaling pathways and enzymes involved in the organization of invasion mediators remain poorly defined. We recently performed a forward chemical-genetic screen in T. gondii and identified compounds that markedly enhanced infectivity. Although molecular dissection of invasion has benefited from the use of small-molecule inhibitors, the mechanisms underlying induction of invasion by small-molecule enhancers have never been described. Here we identify the Toxoplasma ortholog of human APT1, palmitoyl protein thioesterase-1 (TgPPT1), as the target of one class of small-molecule enhancers. Inhibition of this uncharacterized thioesterase triggered secretion of invasion-associated organelles, increased motility and enhanced the invasive capacity of tachyzoites. We demonstrate that TgPPT1 is a bona fide depalmitoylase, thereby establishing an important role for dynamic and reversible palmitoylation in host-cell invasion by T. gondii.

STAT3 promotes corticospinal remodelling and functional recovery after spinal cord injury.

If and how neurons remodel their connections after CNS injury critically influences recovery of function. Here, we investigate the role of the growth-initiating transcription factor STAT3 during remodelling of the injured corticospinal tract (CST). Endogenous STAT3 expression in lesioned cortical projection neurons is transient but can be sustained by viral gene transfer. Sustained activation of STAT3 enhances remodelling of lesioned CST fibres and induces de novo formation of collaterals from unlesioned CST fibres. In a unilateral pyramidotomy paradigm, this recruitment of unlesioned fibres leads to the formation of midline crossing circuits that establish ipsilateral forelimb activation and functional recovery.