RESUMEN
Mitochondrial cristae architecture is crucial for optimal respiratory function of the organelle. Cristae shape is maintained in part by the mitochondrial contact site and cristae organizing system (MICOS) complex. While MICOS is required for normal cristae morphology, the precise mechanistic role of each of the seven human MICOS subunits, and how the complex coordinates with other cristae-shaping factors, has not been fully determined. Here, we examine the MICOS complex in Schizosaccharomyces pombe, a minimal model whose genome only encodes for four core subunits. Using an unbiased proteomics approach, we identify a poorly characterized inner mitochondrial membrane protein that interacts with MICOS and is required to maintain cristae morphology, which we name Mmc1. We demonstrate that Mmc1 works in concert with MICOS to promote normal mitochondrial morphology and respiratory function. Mmc1 is a distant relative of the dynamin superfamily of proteins (DSPs), GTPases, which are well established to shape and remodel membranes. Similar to DSPs, Mmc1 self-associates and forms high-molecular-weight assemblies. Interestingly, however, Mmc1 is a pseudoenzyme that lacks key residues required for GTP binding and hydrolysis, suggesting that it does not dynamically remodel membranes. These data are consistent with the model that Mmc1 stabilizes cristae architecture by acting as a scaffold to support cristae ultrastructure on the matrix side of the inner membrane. Our study reveals a new class of proteins that evolved early in fungal phylogeny and is required for the maintenance of cristae architecture. This highlights the possibility that functionally analogous proteins work with MICOS to establish cristae morphology in metazoans.
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Membranas Mitocondriales , Proteínas Mitocondriales , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Dinaminas/metabolismo , Dinaminas/genética , Mitocondrias/metabolismo , Membranas Asociadas a MitocondriasRESUMEN
INTRODUCTION: Pulmonary hypertension in children is associated with high rates of adverse events under anesthesia. In children who have failed medical therapy, a posttricuspid shunt such as a Potts shunt can offload the right ventricle and possibly delay or replace the need for lung transplantation. Intraoperative management of this procedure, during which an anastomosis between the pulmonary artery and the descending aorta is created, is complex and requires a deep understanding of the pathophysiology of acute and chronic right ventricular failure. This retrospective case review describes the intraoperative management of children undergoing surgical creation of a Potts shunt at a single center. METHODS: A retrospective case review of all patients under the age of 18 who underwent Potts shunt between April 2013 and June 2022. Medical records were examined, and clinical data of demographics, intraoperative vital signs, anesthetic management, and postoperative outcomes were extracted. RESULTS: Twenty-nine children with medically refractory pulmonary hypertension underwent surgical Potts shunts with a median age of 12 years (range 4 months to 17.4 years). Nineteen Potts shunts (65%) were placed via thoracotomy and 10 (35%) were placed via median sternotomy with use of cardiopulmonary bypass. Ketamine was the most frequently utilized induction agent (17 out of 29, 59%), and the majority of patients were initiated on vasopressin prior to intubation (20 out of 29, 69%). Additional inotropic support with epinephrine (45%), milrinone (28%), norepinephrine (17%), and dobutamine (14%) was used prior to shunt placement. Following opening of the Potts shunt, hemodynamic support was continued with vasopressin (66%), epinephrine (62%), milrinone (59%), dobutamine (14%), and norepinephrine (10%). Major intraoperative complications included severe hypoxemia (21 out of 29, 72%) and hypotension requiring boluses of epinephrine (10 out of 29, 34.5%) but no patient suffered intraoperative cardiac arrest. There were four in-hospital mortalities. DISCUSSION: A Potts shunt offers another palliative option for children with medically refractory pulmonary hypertension. General anesthesia in these children carries high risk for pulmonary hypertensive crises. Anesthesiologists must understand underlying physiological mechanisms responsble for acute hemodynaic decompensation during acute pulmonary hypertneisve crises. Severe physiological perturbations imposed by thoracic surgery and use of cardiopulmonay bypass can be mitigated by aggresive heodynamic support of ventricle function and maintainence of systemic vascular resistance. Early use of vasopressin, before or immidiately after anesthesia induction, in combination with other inotropes is a useful agent during the perioperative care of thes. Early use of vasopressin during anesthesia induction, and aggressive inotropic support of right ventricular function can help mitigate effects of induction and intubation, single-lung ventilation, and cardiopulmonary bypass. CONCLUSIONS: Our single center expereince shows that the Potts shunt surgery, despite high short-term mortaility, may offer another option for palliation in children with medically refractory pulmonary hypertension.
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Anestésicos , Hipertensión Pulmonar , Niño , Humanos , Lactante , Hipertensión Pulmonar/diagnóstico , Estudios Retrospectivos , Dobutamina , Milrinona , Anestesia General , Norepinefrina , Epinefrina , VasopresinasRESUMEN
Mitochondrial cristae architecture is crucial for optimal respiratory function of the organelle. Cristae shape is maintained in part by the mitochondrial inner membrane-localized MICOS complex. While MICOS is required for normal cristae morphology, the precise mechanistic role of each of the seven human MICOS subunits, and how the complex coordinates with other cristae shaping factors, has not been fully determined. Here, we examine the MICOS complex in Schizosaccharomyces pombe, a minimal model whose genome only encodes for four core subunits. Using an unbiased proteomics approach, we identify a poorly characterized inner mitochondrial membrane protein that interacts with MICOS and is required to maintain cristae morphology, which we name Mmc1. We demonstrate that Mmc1 works in concert with MICOS complexes to promote normal mitochondrial morphology and respiratory function. Bioinformatic analyses reveal that Mmc1 is a distant relative of the Dynamin-Related Protein (DRP) family of GTPases, which are well established to shape and remodel membranes. We find that, like DRPs, Mmc1 self-associates and forms high molecular weight assemblies. Interestingly, however, Mmc1 is a pseudoenzyme that lacks key residues required for GTP binding and hydrolysis, suggesting it does not dynamically remodel membranes. These data are consistent with a model in which Mmc1 stabilizes cristae architecture by acting as a scaffold to support cristae ultrastructure on the matrix side of the inner membrane. Our study reveals a new class of proteins that evolved early in fungal phylogeny and is required for the maintenance of cristae architecture. This highlights the possibility that functionally analogous proteins work with MICOS to establish cristae morphology in metazoans.
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Accurate cellular replication balances the biogenesis and turnover of complex structures. In the apicomplexan parasite Toxoplasma gondii, daughter cells form within an intact mother cell, creating additional challenges to ensuring fidelity of division. The apical complex is critical to parasite infectivity and consists of apical secretory organelles and specialized cytoskeletal structures. We previously identified the kinase ERK7 as required for maturation of the apical complex in Toxoplasma. Here, we define the Toxoplasma ERK7 interactome, including a putative E3 ligase, CSAR1. Genetic disruption of CSAR1 fully suppresses loss of the apical complex upon ERK7 knockdown. Furthermore, we show that CSAR1 is normally responsible for turnover of maternal cytoskeleton during cytokinesis, and that its aberrant function is driven by mislocalization from the parasite residual body to the apical complex. These data identify a protein homeostasis pathway critical for Toxoplasma replication and fitness and suggest an unappreciated role for the parasite residual body in compartmentalizing processes that threaten the fidelity of parasite development.
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Quinasas MAP Reguladas por Señal Extracelular , Proteínas Protozoarias , Toxoplasma , División Celular , Citocinesis , Citoesqueleto/metabolismo , Orgánulos/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasma/genética , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The apical complex is a specialized collection of cytoskeletal and secretory machinery in apicomplexan parasites, which include the pathogens that cause malaria and toxoplasmosis. Its structure and mechanism of motion are poorly understood. We used cryo-FIB-milling and cryo-electron tomography to visualize the 3D-structure of the apical complex in its protruded and retracted states. Averages of conoid-fibers revealed their polarity and unusual nine-protofilament arrangement with associated proteins connecting and likely stabilizing the fibers. Neither the structure of the conoid-fibers nor the architecture of the spiral-shaped conoid complex change during protrusion or retraction. Thus, the conoid moves as a rigid body, and is not spring-like and compressible, as previously suggested. Instead, the apical-polar-rings (APR), previously considered rigid, dilate during conoid protrusion. We identified actin-like filaments connecting the conoid and APR during protrusion, suggesting a role during conoid movements. Furthermore, our data capture the parasites in the act of secretion during conoid protrusion.
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Neospora , Toxoplasma , Toxoplasma/citología , Toxoplasma/ultraestructura , Neospora/citología , Neospora/ultraestructura , Tomografía con Microscopio Electrónico , Tubulina (Proteína)/ultraestructura , Citoesqueleto/ultraestructura , Membrana Celular/ultraestructuraRESUMEN
Reversible phosphorylation by protein kinases is one of the core mechanisms by which biological signals are propagated and processed. Mitogen-activated protein kinases, or MAPKs, are conserved throughout eukaryotes where they regulate cell cycle, development, and stress response. Here, we review advances in our understanding of the function and biochemistry of MAPK signaling in apicomplexan parasites. As expected for well-conserved signaling modules, MAPKs have been found to have multiple essential roles regulating both Toxoplasma tachyzoite replication and sexual differentiation in Plasmodium. However, apicomplexan MAPK signaling is notable for the lack of the canonical kinase cascade that normally regulates the networks, and therefore must be regulated by a distinct mechanism. We highlight what few regulatory relationships have been established to date, and discuss the challenges to the field in elucidating the complete MAPK signaling networks in these parasites.
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Madres , Toxoplasma , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Transducción de Señal , Toxoplasma/metabolismoRESUMEN
Apicomplexan parasites like Toxoplasma gondii grow and replicate within a specialized organelle called the parasitophorous vacuole. The vacuole is decorated with parasite proteins that integrate into the membrane after trafficking through the parasite secretory system as soluble, chaperoned complexes. A regulator of this process is an atypical protein kinase called WNG1. Phosphorylation by WNG1 appears to serve as a switch for membrane integration. However, like its substrates, WNG1 is secreted from the parasite dense granules, and its activity must, therefore, be tightly regulated until the correct membrane is encountered. Here, we demonstrate that, while another member of the WNG family can adopt multiple multimeric states, WNG1 is monomeric and therefore not regulated by multimerization. Instead, we identify two phosphosites on WNG1 that are required for its kinase activity. Using a combination of in vitro biochemistry and structural modeling, we identify basic residues that are also required for WNG1 activity and appear to recognize the activating phosphosites. Among these coordinating residues are the 'HRD' Arg, which recognizes activation loop phosphorylation in canonical kinases. WNG1, however, is not phosphorylated on its activation loop, but rather on atypical phosphosites on its C-lobe. We propose a simple model in which WNG1 is activated by increasing ATP concentration above a critical threshold once the kinase traffics to the parasitophorous vacuole.
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Proteínas Protozoarias , Toxoplasma , Adenosina Trifosfato/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/fisiología , Vacuolas/metabolismoRESUMEN
Schistosomes cause untold disease and disability in the developing world. Here, we introduce SchistoCyte Atlas, a web-based platform for exploring gene expression at single-cell resolution in adult Schistosoma mansoni. Similar resources accessible to non-specialists across the globe will expedite our ability to understand the biology of these devastating parasites.
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Proteínas del Helminto/genética , Schistosoma mansoni/genética , Transcriptoma , Animales , Proteínas del Helminto/metabolismo , Estadios del Ciclo de Vida/genética , Schistosoma mansoni/metabolismoRESUMEN
Toxoplasma gondii is a useful model for intracellular parasitism given its ease of culture in the laboratory and genomic resources. However, as for many other eukaryotes, the T. gondii genome contains hundreds of sequence gaps owing to repetitive and/or unclonable sequences that disrupt the assembly process. Here, we use the Oxford Nanopore Minion platform to generate near-complete de novo genome assemblies for multiple strains of T. gondii and its near relative, N. caninum We significantly improved T. gondii genome contiguity (average N50 of â¼6.6 Mb) and added â¼2 Mb of newly assembled sequence. For all of the T. gondii strains that we sequenced (RH, ME49, CTG, II×III progeny clones CL13, S27, S21, S26, and D3X1), the largest contig ranged in size between 11.9 and 12.1 Mb in size, which is larger than any previously reported T. gondii chromosome, and found to be due to a consistent fusion of Chromosomes VIIb and VIII. These data were validated by mapping existing T. gondii ME49 Hi-C data to our assembly, providing parallel lines of evidence that the T. gondii karyotype consists of 13, rather than 14, chromosomes. By using this technology, we also resolved hundreds of tandem repeats of varying lengths, including in well-known host-targeting effector loci like rhoptry protein 5 (ROP5) and ROP38 Finally, when we compared T. gondii with N. caninum, we found that although the 13-chromosome karyotype was conserved, extensive, previously unappreciated chromosome-scale rearrangements had occurred in T. gondii and N. caninum since their most recent common ancestry.
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Toxoplasma , Variaciones en el Número de Copia de ADN , Genoma , Cariotipo , Análisis de Secuencia de ADN , Toxoplasma/genéticaRESUMEN
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.
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Toxoplasma , Toxoplasmosis , Humanos , Toxoplasma/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasmosis/parasitología , Membrana Celular/metabolismo , Citoesqueleto/metabolismoRESUMEN
BACKGROUND: Cerebrospinal fluid (CSF) analysis is a sensitive tool for evaluating patients with neurologic diseases but is rarely specific. Biomarkers can be measured from any bodily fluid and can be useful indicators for the presence, severity, and prognosis of diseases. OBJECTIVES: This study was designed to evaluate if CSF lactate can be used as a biomarker in dogs with central nervous system disease. METHODS: Peripheral venous blood and CSF were collected from 49 dogs with various intracranial diseases to evaluate correlations between blood and CSF lactate levels. Total nucleated cell count (TNCC) and CSF protein concentrations were also evaluated. All samples obtained were divided into normal (NG) and abnormal (AG) dogs based on a TNCC of ≤5 and >5 cells/µL and a protein concentration of ≤25 and >25 mg/dL, respectively. The AG dogs were further subdivided into those having <100 TNCCs/µL (AGL) and those having >100 TNCCs/µL (AGH). They were also subdivided into groups based on seizure activity (AGS), and inflammatory (AGI), or neoplastic diseases (AGN), and the respective lactate concentrations were then compared. RESULTS: Lactate concentrations were significantly increased in CSF and venous blood samples in the AG compared with the NG dogs, but no differences were found among the individual disease processes. In all dogs, CSF lactate concentrations were higher than venous blood lactate levels; however, no direct correlation between CSF and blood lactate concentrations was identified. CONCLUSIONS: Cerebrospinal fluid lactate can be used as a biomarker in clinical settings as it can be measured via a commercially available lactometer immediately after collection without the need for special instrumentation or laboratory personnel.
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Enfermedades del Sistema Nervioso Central , Enfermedades de los Perros , Animales , Enfermedades del Sistema Nervioso Central/diagnóstico , Enfermedades del Sistema Nervioso Central/veterinaria , Enfermedades de los Perros/diagnóstico , Perros , Ácido LácticoRESUMEN
Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis.IMPORTANCEToxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.
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Ciclo Celular , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Protozoarias/metabolismo , Toxoplasma/citología , Toxoplasma/enzimología , Centrosoma/metabolismo , Replicación del ADN , Humanos , Estadios del Ciclo de Vida , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteínas Protozoarias/genética , Toxoplasma/genética , Toxoplasma/crecimiento & desarrollo , Toxoplasmosis/parasitologíaRESUMEN
Schistosomiasis is a neglected tropical disease that infects 240 million people. With no vaccines and only one drug available, new therapeutic targets are needed. The causative agents, schistosomes, are intravascular flatworm parasites that feed on blood and lay eggs, resulting in pathology. The function of the parasite's various tissues in successful parasitism are poorly understood, hindering identification of therapeutic targets. Using single-cell RNA sequencing (RNA-seq), we characterize 43,642 cells from the adult schistosome and identify 68 distinct cell populations, including specialized stem cells that maintain the parasite's blood-digesting gut. These stem cells express the gene hnf4, which is required for gut maintenance, blood feeding, and pathology in vivo. Together, these data provide molecular insights into the organ systems of this important pathogen and identify potential therapeutic targets.
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Sangre/parasitología , Proteínas del Helminto/fisiología , Factor Nuclear 4 del Hepatocito/fisiología , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/sangre , Esquistosomiasis mansoni/parasitología , Animales , Atlas como Asunto , Femenino , Expresión Génica , Proteínas del Helminto/genética , Factor Nuclear 4 del Hepatocito/genética , Masculino , Interferencia de ARN , RNA-Seq , Schistosoma mansoni/genética , Análisis de la Célula Individual , Células Madre/metabolismoRESUMEN
Host resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite's protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including 'avirulent' ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.
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Linfocitos T CD8-positivos/inmunología , Inflamasomas/inmunología , Interferón gamma/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas Protozoarias/metabolismo , Transducción de Señal , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Linfocitos T CD8-positivos/parasitología , Femenino , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas Protozoarias/genética , Toxoplasmosis Animal/parasitología , Vacuolas/inmunología , Vacuolas/metabolismo , Vacuolas/parasitología , Virulencia/inmunologíaRESUMEN
This study compared water-heated mats (WM) and electric-heated mats (EM) with heat lamps (HL) as supplemental heat sources for suckling piglets. Forty-two litters were studied in 3 trials. In all trials, behavior of piglets was video-recorded on day 1, 3, 7, 14, and 21 postpartum. Videos were scan-sampled to register postures (lying and standing) and locations (on or away from mat) to assess piglet use of heat sources. Litter size and weight at birth and weaning, and pre-weaning mortality were recorded. Data were analyzed using Glimmix Procedures of SAS. Piglets spent more time on WM than under HL (67.5% vs. 51.0%, p = 0.002). No difference in piglet performance between WM and HL was observed, except mortality tended to be higher in WM (22.9% vs. 8.9%; p = 0.06). Piglet performance and use of the heat source were comparable for HL and EM. When comparing WM with EM, piglets provided WM spent more time on the mat compared to those provided EM (21.8% vs. 17.1%; p = 0.02). No difference in pre-weaning mortality, litter weight, and individual daily gain was observed between WM and EM group. These results suggest EM and HL were comparable to maintain performance and postural behaviors of piglets.
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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.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Fibroblastos/parasitología , Biogénesis de Organelos , Proteínas Protozoarias/metabolismo , Toxoplasma/patogenicidad , Toxoplasmosis/patología , Quinasas MAP Reguladas por Señal Extracelular/química , Quinasas MAP Reguladas por Señal Extracelular/genética , Humanos , Fosforilación , Conformación Proteica , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Transducción de Señal , Toxoplasmosis/metabolismo , Toxoplasmosis/parasitologíaRESUMEN
Primary cilia are important organizing centers that control diverse cellular processes. Apicomplexan parasites like Toxoplasma gondii have a specialized cilium-like structure called the conoid that organizes the secretory and invasion machinery critical for the parasites' lifestyle. The proteins that initiate the biogenesis of this structure are largely unknown. We identified the Toxoplasma orthologue of the conserved kinase ERK7 as essential to conoid assembly. Parasites in which ERK7 has been depleted lose their conoids late during maturation and are immotile and thus unable to invade new host cells. This is the most severe phenotype to conoid biogenesis yet reported, and is made more striking by the fact that ERK7 is not a conoid protein, as it localizes just basal to the structure. ERK7 has been recently implicated in ciliogenesis in metazoan cells, and our data suggest that this kinase has an ancient and central role in regulating ciliogenesis throughout Eukaryota.
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Cilios/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Toxoplasma/enzimología , Cilios/fisiología , Cilios/ultraestructura , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/fisiología , Toxoplasma/metabolismo , Toxoplasma/fisiologíaRESUMEN
Previous decades have seen an explosion in our understanding of protein kinase function in human health and disease. Hundreds of unique kinase structures have been solved, allowing us to create generalized rules for catalysis, assign roles of communities within the catalytic core, and develop specific drugs for targeting various pathways. Although our understanding of intracellular kinases has developed at a fast rate, our exploration into extracellular kinases has just begun. In this review, we will cover the secreted protein kinase families found in humans, bacteria, and parasites. © 2019 IUBMB Life, 71(6):749-759, 2019.
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Transporte Biológico/genética , Fosforilación/genética , Proteínas Quinasas/genética , Animales , Bacterias/enzimología , Humanos , Mamíferos/genética , Parásitos/enzimología , Proteínas Quinasas/clasificación , Especificidad por SustratoRESUMEN
BACKGROUND: After diagnosis, a substantial number of people with HIV disease fall out of care. Effective interventions are needed for this priority population. METHODS: The "Peers Keep It Real" study aimed to help adults who were disengaged from HIV treatment. Peers, lay individuals living with HIV, facilitated intervention sessions. Participants were randomized to immediately receive the peer-facilitated intervention or were wait-listed. RESULTS: Considerable attrition occurred in the control group. Pre-/postanalyses showed that among participants (n = 23) who received the intervention, 65% had viral load suppression and 100% remained in care at 12 months postintervention. Impact on viral load was significant ( P = .0326), suggesting that peers are effective change agents who positively impacted outcomes for individuals struggling with adherence to HIV treatment. CONCLUSION: Future endeavors should consider providing all individuals from this priority population with an active peer intervention from the onset to enhance retention and adherence.