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.

Characterization and functional analysis of Toxoplasma Golgi-associated proteins identified by proximity labelling.

Toxoplasma gondii possesses a highly polarized secretory pathway that contains both broadly conserved eukaryotic organelles and unique apicomplexan organelles which play essential roles in the parasite's lytic cycle. As in other eukaryotes, the T. gondii Golgi apparatus sorts and modifies proteins prior to their distribution to downstream organelles. Many of the typical trafficking factors found involved in these processes are missing from apicomplexan genomes, suggesting that these parasites have evolved unique proteins to fill these roles. Here we identify a novel Golgi-localizing protein (ULP1) which contains structural homology to the eukaryotic trafficking factor p115/Uso1. We demonstrate that depletion of ULP1 leads to a dramatic reduction in parasite fitness and replicative ability. Using ULP1 as bait for TurboID proximity labelling and immunoprecipitation, we identify eleven more novel Golgi-associated proteins and demonstrate that ULP1 interacts with the T. gondii COG complex. These proteins include both conserved trafficking factors and parasite-specific proteins. Using a conditional knockdown approach, we assess the effect of each of these eleven proteins on parasite fitness. Together, this work reveals a diverse set of novel T. gondii Golgi-associated proteins that play distinct roles in the secretory pathway. As several of these proteins are absent outside of the Apicomplexa, they represent potential targets for the development of novel therapeutics against these parasites. Importance: Apicomplexan parasites such as Toxoplasma gondii infect a large percentage of the world's population and cause substantial human disease. These widespread pathogens use specialized secretory organelles to infect their host cells, modulate host cell functions, and cause disease. While the functions of the secretory organelles are now better understood, the Golgi apparatus of the parasite remains largely unexplored, particularly regarding parasite-specific innovations that may help direct traffic intracellularly. In this work, we characterize ULP1, a protein that is unique to parasites but shares structural similarity to the eukaryotic trafficking factor p115/Uso1. We show that ULP1 plays an important role in parasite replication and demonstrate that it interacts with the conserved oligomeric Golgi (COG) complex. We then use ULP1 proximity labelling to identify eleven additional Golgi-associated proteins which we functionally analyze via conditional knockdown. This work expands our knowledge of the Toxoplasma Golgi apparatus and identifies potential targets for therapeutic intervention.

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.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma's ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

The Toxoplasma subpellicular network is highly interconnected and defines parasite shape for efficient motility and replication.

Apicomplexan parasites possess several specialized structures to invade their host cells and replicate successfully. One of these is the inner membrane complex (IMC), a peripheral membrane-cytoskeletal system underneath the plasma membrane. It is composed of a series of flattened, membrane-bound vesicles and a cytoskeletal subpellicular network (SPN) comprised of intermediate filament-like proteins called alveolins. While the alveolin proteins are conserved throughout the Apicomplexa and the broader Alveolata, their precise functions and interactions remain poorly understood. Here, we describe the function of one of these alveolin proteins, TgIMC6. Disruption of IMC6 resulted in striking morphological defects that led to aberrant motility, invasion, and replication. Deletion analyses revealed that the alveolin domain alone is largely sufficient to restore localization and partially sufficient for function. As this highlights the importance of the IMC6 alveolin domain, we implemented unnatural amino acid photoreactive crosslinking to the alveolin domain and identified multiple binding interfaces between IMC6 and two other cytoskeletal proteins - IMC3 and ILP1. To our knowledge, this provides the first direct evidence of protein-protein interactions in the alveolin domain and supports the long-held hypothesis that the alveolin domain is responsible for filament formation. Collectively, our study features the conserved alveolin proteins as critical components that maintain the parasite's structural integrity and highlights the alveolin domain as a key mediator of SPN architecture.

Toxoplasma gondii encodes an array of TBC-domain containing proteins including an essential regulator that targets 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. We then use an auxin-inducible degron approach to demonstrate that the protozoan-specific TgTBC9 protein that localizes to the ER is essential for parasite survival. Knockdown of TgTBC9 results in parasite growth arrest and affects the organization of the ER and Golgi apparatus. 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 P. falciparum orthologue of TgTBC9 can rescue the lethal knockdown. We additionally show by immunoprecipitation and yeast two hybrid analyses that TgTBC9 directly binds Rab2, indicating that this TBC-Rab pair controls ER to Golgi traffic in the parasite. Together, these studies identify the first essential TBC protein described in any protozoan, provide new insight into intracellular vesicle trafficking in T. gondii , and reveal promising targets for the design of novel therapeutics that can specifically target apicomplexan parasites.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii 's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with Δ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon gamma (IFN-γ) in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-κB complex. While GRA15 similarly regulates NF-κB, infection with Δ gra83/ Δ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden. Importance: Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma' s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

IMC29 Plays an Important Role in Toxoplasma Endodyogeny and Reveals New Components of the Daughter-Enriched IMC Proteome.

The Toxoplasma inner membrane complex (IMC) is a unique organelle that plays critical roles in parasite motility, invasion, egress, and replication. The IMC is delineated into the apical, body, and basal regions, defined by proteins that localize to these distinct subcompartments. The IMC can be further segregated by proteins that localize specifically to the maternal IMC, the daughter bud IMC, or both. While the function of the maternal IMC has been better characterized, the precise roles of most daughter IMC components remain poorly understood. Here, we demonstrate that the daughter protein IMC29 plays an important role in parasite replication. We show that Δimc29 parasites exhibit severe replication defects, resulting in substantial growth defects and loss of virulence. Deletion analyses revealed that IMC29 localization is largely dependent on the N-terminal half of the protein containing four predicted coiled-coil domains while IMC29 function requires a short C-terminal helical region. Using proximity labeling, we identify eight novel IMC proteins enriched in daughter buds, significantly expanding the daughter IMC proteome. We additionally report four novel proteins with unique localizations to the interface between two parasites or to the outer face of the IMC, exposing new subregions of the organelle. Together, this work establishes IMC29 as an important early daughter bud component of replication and uncovers an array of new IMC proteins that provides important insights into this organelle. IMPORTANCE The inner membrane complex (IMC) is a conserved structure across the Apicomplexa phylum, which includes obligate intracellular parasites that cause toxoplasmosis, malaria, and cryptosporidiosis. The IMC is critical for the parasite to maintain its intracellular lifestyle, particularly in providing a scaffold for daughter bud formation during parasite replication. While many IMC proteins in the later stages of division have been identified, components of the early stages of division remain unknown. Here, we focus on the early daughter protein IMC29, demonstrating that it is crucial for faithful parasite replication and identifying specific regions of the protein that are important for its localization and function. We additionally use proximity labeling to reveal a suite of daughter-enriched IMC proteins, which represent promising candidates to further explore this IMC subcompartment.

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.

Selective plasticity of callosal neurons in the adult contralesional cortex following murine traumatic brain injury.

Traumatic brain injury (TBI) results in deficits that are often followed by recovery. The contralesional cortex can contribute to this process but how distinct contralesional neurons and circuits respond to injury remains to be determined. To unravel adaptations in the contralesional cortex, we used chronic in vivo two-photon imaging. We observed a general decrease in spine density with concomitant changes in spine dynamics over time. With retrograde co-labeling techniques, we showed that callosal neurons are uniquely affected by and responsive to TBI. To elucidate circuit connectivity, we used monosynaptic rabies tracing, clearing techniques and histology. We demonstrate that contralesional callosal neurons adapt their input circuitry by strengthening ipsilateral connections from pre-connected areas. Finally, functional in vivo two-photon imaging demonstrates that the restoration of pre-synaptic circuitry parallels the restoration of callosal activity patterns. Taken together our study thus delineates how callosal neurons structurally and functionally adapt following a contralateral murine TBI.

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.

Reassessing the multiple values of lowland British floodplains.

Ecosystem services provided by lowland British floodplains respectively under semi-natural conditions and converted for intensive maize production were assessed. Floodplains across lowland Britain have been extensively disconnected from river channels, depleting habitat for wildlife and other beneficial ecosystem services. Conservation measures are often regarded as costly constraints on economic and development freedoms whilst, conversely, conversion for intensive agricultural production is rewarded by markets despite many often-overlooked externalities. Maize growing has increased in Britain since the 1970s, initially for feedlot production of livestock and now increasingly for grant-aided biofuel production for anaerobic digestion. Comparative literature-based ecosystem service assessments using the RAWES (Rapid Assessment of Wetland Ecosystem Services) approach reveal that lowland British floodplains in semi-natural condition provide a wider range of provisioning services than those converted for monocultural intensive production of maize, in addition to a diversity of regulating, cultural and supporting service benefits that are lost or transformed into disservices when floodplains are converted for intensive maize growth. Benefits and disbenefits of floodplains managed under the two scenarios (semi-natural versus monocultural maize) are presented graphically as an intuitive means to support decision-makers. Monetisation of benefits would be risky, not merely due to uncertainties but as this may skew conclusions and subsequent decision-making towards maximisation of marketed or near-market services, consequently misrepresenting the diversity of values of whole socioecological floodplain systems. Management solutions protective of the societal values provided by floodplain ecosystem may include buffer zoning as a mitigation measure, but a more strategic solution may be zonation of land use based on suitability not only for crop production but recognising the full spectrum of societally beneficial ecosystem services demonstrated by RAWES assessment. A variety of drivers for a changing approach to floodplain farming - statutory, fiscal and self-beneficial - are highlighted, and are generically applicable beyond Britain with context-specific modification.

Efficient Gene Knockout and Knockdown Systems in Neospora caninum Enable Rapid Discovery and Functional Assessment of Novel Proteins.

The development of molecular genetics has greatly enhanced the study of the biology and pathology associated with parasites of the phylum Apicomplexa. While the molecular tools are highly developed for the apicomplexan Toxoplasma gondii, the closely related parasite Neospora caninum lacks efficient tools for genetic manipulation. To enable efficient homologous recombination in N. caninum, we targeted the Ku heterodimer DNA repair mechanism in the genomic reference strain, Nc-Liverpool (NcLiv), and show that deletion of Ku80 results in a destabilization and loss of its partner Ku70. Disruption of Ku80 generated parasites in which genes are efficiently epitope tagged and only short homology regions are required for gene knockouts. We used this improved strain to target novel nonessential genes encoding dense granule proteins that are unique to N. caninum or conserved in T. gondii. To expand the utility of this strain for essential genes, we developed the auxin-inducible degron system for N. caninum using parasite-specific promoters. As a proof of concept, we knocked down a novel nuclear factor in both N. caninum and T. gondii and showed that it is essential for survival of both parasites. Together, these efficient knockout and knockdown technologies will enable the field to unravel specific gene functions in N. caninum, which is likely to aid in the identification of targets responsible for the phenotypic differences observed between these two closely related apicomplexan parasites. IMPORTANCE Neospora caninum is a parasite with veterinary relevance, inducing severe disease in dogs and reproductive disorders in ruminants, especially cattle, leading to major losses. The close phylogenetic relationship to Toxoplasma gondii and the lack of pathogenicity in humans drives an interest of the scientific community toward using N. caninum as a model to study the pathogenicity of T. gondii. To enable this comparison, it is important to develop efficient molecular tools for N. caninum, to gain accuracy and save time in genetic manipulation protocols. Here, we have developed base strains and protocols using the genomic reference strain of N. caninum to enable efficient knockout and knockdown assays in this model. We demonstrate that these tools are effective in targeting known and previously unexplored genes. Thus, these tools will greatly improve the study of this protozoan, as well as enhance its ability to serve as a model to understand other apicomplexan parasites.

A Novel Toxoplasma Inner Membrane Complex Suture-Associated Protein Regulates Suture Protein Targeting and Colocalizes with Membrane Trafficking Machinery.

The cytoskeleton of Toxoplasma gondii is composed of the inner membrane complex (IMC) and an array of underlying microtubules that provide support at the periphery of the parasite. Specific subregions of the IMC carry out distinct roles in replication, motility, and host cell invasion. Building on our previous in vivo biotinylation (BioID) experiments of the IMC, we identified here a novel protein that localizes to discrete puncta that are embedded in the parasite's cytoskeleton along the IMC sutures. Gene knockout analysis showed that loss of the protein results in defects in cytoskeletal suture protein targeting, cytoskeletal integrity, parasite morphology, and host cell invasion. We then used deletion analyses to identify a domain in the N terminus of the protein that is critical for both localization and function. Finally, we used the protein as bait for in vivo biotinylation, which identified several other proteins that colocalize in similar spot-like patterns. These putative interactors include several proteins that are implicated in membrane trafficking and are also associated with the cytoskeleton. Together, these data reveal an unexpected link between the IMC sutures and membrane trafficking elements of the parasite and suggest that the suture puncta are likely a portal for trafficking cargo across the IMC. IMPORTANCE The inner membrane complex (IMC) is a peripheral membrane and cytoskeletal system that is organized into intriguing rectangular plates at the periphery of the parasite. The IMC plates are delimited by an array of IMC suture proteins that are tethered to both the membrane and the cytoskeleton and are thought to provide structure to the organelle. Here, we identified a protein that forms discrete puncta that are embedded in the IMC sutures, and we show that it is important for the proper sorting of a group of IMC suture proteins as well as maintaining parasite shape and IMC cytoskeletal integrity. Intriguingly, proximity labeling experiments identified several proteins that are involved in membrane trafficking or endocytosis, suggesting that the IMC puncta provide a gateway for transporting molecules across the structure.

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.

Identification and Molecular Dissection of IMC32, a Conserved Toxoplasma Inner Membrane Complex Protein That Is Essential for Parasite Replication.

The inner membrane complex (IMC) is a unique organelle of apicomplexan parasites that plays critical roles in parasite motility, host cell invasion, and replication. Despite the common functions of the organelle, relatively few IMC proteins are conserved across the phylum and the precise roles of many IMC components remain to be characterized. Here, we identify a novel component of the Toxoplasma gondii IMC (IMC32) that localizes to the body portion of the IMC and is recruited to developing daughter buds early during endodyogeny. IMC32 is essential for parasite survival, as its conditional depletion results in a complete collapse of the IMC that is lethal to the parasite. We demonstrate that localization of IMC32 is dependent on both an N-terminal palmitoylation site and a series of C-terminal coiled-coil domains. Using deletion analyses and functional complementation, we show that two conserved regions within the C-terminal coiled-coil domains play critical roles in protein function during replication. Together, this work reveals an essential component of parasite replication that provides a novel target for therapeutic intervention of T. gondii and related apicomplexan parasites.IMPORTANCE The IMC is an important organelle that apicomplexan parasites use to maintain their intracellular lifestyle. While many IMC proteins have been identified, only a few central players that are essential for internal budding have been described and even fewer are conserved across the phylum. Here, we identify IMC32, a novel component of the Toxoplasma gondii IMC that localizes to very early daughter buds, indicating a role in the early stages of parasite replication. We then demonstrate that IMC32 is essential for parasite survival and pinpoint conserved regions within the protein that are important for membrane association and daughter cell formation. As IMC32 is unique to these parasites and not present in their mammalian hosts, it serves as a new target for the development of drugs that exclusively affect these important intracellular pathogens.

Multivalent Interactions Drive the Toxoplasma AC9:AC10:ERK7 Complex To Concentrate ERK7 in the Apical Cap.

The Toxoplasma inner membrane complex (IMC) is a specialized organelle that is crucial for the parasite to establish an intracellular lifestyle and ultimately cause disease. The IMC is composed of both membrane and cytoskeletal components, further delineated into the apical cap, body, and basal subcompartments. The apical cap cytoskeleton was recently demonstrated to govern the stability of the apical complex, which controls parasite motility, invasion, and egress. While this role was determined by individually assessing the apical cap proteins AC9, AC10, and the mitogen-activated protein kinase ERK7, how the three proteins collaborate to stabilize the apical complex is unknown. In this study, we use a combination of deletion analyses and yeast two-hybrid experiments to establish that these proteins form an essential complex in the apical cap. We show that AC10 is a foundational component of the AC9:AC10:ERK7 complex and demonstrate that the interactions among them are critical to maintaining the apical complex. Importantly, we identify multiple independent regions of pairwise interaction between each of the three proteins, suggesting that the AC9:AC10:ERK7 complex is organized by multivalent interactions. Together, these data support a model in which multiple interacting domains enable the oligomerization of the AC9:AC10:ERK7 complex and its assembly into the cytoskeletal IMC, which serves as a structural scaffold that concentrates ERK7 kinase activity in the apical cap. IMPORTANCE The phylum Apicomplexa consists of obligate, intracellular parasites, including the causative agents of toxoplasmosis, malaria, and cryptosporidiosis. Hallmarks of these parasites are the IMC and the apical complex, both of which are unique structures that are conserved throughout the phylum and required for parasite survival. The apical cap portion of the IMC has previously been shown to stabilize the apical complex. Here, we expand on those studies to determine the precise protein-protein interactions of the apical cap complex that confer this essential function. We describe the multivalent nature of these interactions and show that the resulting protein oligomers likely tether ERK7 in the apical cap. This study represents the first description of the architecture of the apical cap at a molecular level, expanding our understanding of the unique cell biology that drives Toxoplasma infections.

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'.

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.