RESUMO
BACKGROUND: Mentor MemoryGel Xtra breast implants (Mentor Worldwide LLC, Irvine, CA) were designed to maintain the soft, natural feel of MemoryGel implants while increasing fullness and projection and minimizing wrinkling, rippling, and related complications through optimization of shell gel-fill. OBJECTIVES: To measure 3-year safety and effectiveness of MemoryGel Xtra breast implants in the Mentor MemoryGel and MemoryShape Combined Cohort Clinical Study. METHODS: Participants were implanted with MemoryGel Xtra breast implants in a prospective, multicenter clinical trial. Rates of complications and reoperations were analyzed to assess device safety and BREAST-Q was employed to assess device effectiveness. RESULTS: Two hundred eighty-seven females receiving MemoryGel Xtra breast implants were enrolled. Complication rates in the primary augmentation cohort included rates of 1.5% for implant-related reoperation, 2.3% for explantation, and 1.5% for Baker grade III or IV capsular contracture. For the revisional augmentation cohort, these rates were 2.8% for implant-related reoperation, 4.3% for explantation, and 3.0% for capsular contracture. For the primary reconstruction cohort, these rates were 12.0% for implant-related reoperation, 12.3% for explantation, and 7.3% for capsular contracture. For the revisional reconstruction cohort, these rates were 7.1% for capsular contracture, with zero implant-related reoperations or explantations. There were no reports of infection or implant malposition or displacement in any of these cohorts. Each cohort showed significantly improved satisfaction with breasts and psychosocial and sexual well-being at 1 year following the primary procedure. CONCLUSIONS: These data are consistent with legacy clinical data for MemoryGel and provide the first published safety and effectiveness data regarding the use of MemoryGel Xtra breast implants for breast augmentation and reconstruction.
Assuntos
Implante Mamário , Implantes de Mama , Contratura , Feminino , Humanos , Implante Mamário/métodos , Implantes de Mama/efeitos adversos , Contratura/complicações , Seguimentos , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Estudos Prospectivos , Desenho de Prótese , Reoperação/efeitos adversos , Géis de SiliconeRESUMO
Toxoplasma gondii utilizes specialized secretory organelles called rhoptries to invade and hijack its host cell. Many rhoptry proteins are proteolytically processed at a highly conserved SΦXE site to remove organellar targeting sequences that may also affect protein activity. We have studied the trafficking and biogenesis of a secreted rhoptry metalloprotease with homology to insulysin that we named toxolysin-1 (TLN1). Through genetic ablation and molecular dissection of TLN1, we have identified the smallest rhoptry targeting domain yet reported and expanded the consensus sequence of the rhoptry pro-domain cleavage site. In addition to removal of its pro-domain, TLN1 undergoes a C-terminal cleavage event that occurs at a processing site not previously seen in Toxoplasma rhoptry proteins. While pro-domain cleavage occurs in the nascent rhoptries, processing of the C-terminal region precedes commitment to rhoptry targeting, suggesting that it is mediated by a different maturase, and we have identified residues critical for proteolysis. We have additionally shown that both pieces of TLN1 associate in a detergent-resistant complex, formation of which is necessary for trafficking of the C-terminal portion to the rhoptries. Together, these studies reveal novel processing and trafficking events that are present in the protein constituents of this unusual secretory organelle.
Assuntos
Metaloendopeptidases/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Transporte Proteico/fisiologia , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Substituição de Aminoácidos/fisiologia , Domínio Catalítico/genética , Clonagem Molecular , DNA Complementar/genética , Precursores Enzimáticos/metabolismo , Técnicas de Inativação de Genes , Insulisina , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/genética , Anotação de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína/fisiologia , Proteólise , Proteômica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Vacúolos/metabolismo , Virulência/fisiologiaRESUMO
Like most intracellular pathogens, Toxoplasma synthesizes and secretes an arsenal of proteins to successfully invade its host cell and hijack host functions for intracellular survival. The rhoptries are key secretory organelles that inject proteins into the host cell where they are positioned to co-opt host processes, although little is known regarding how these proteins exert their functions. We show here that the rhoptry protein ROP13 is synthesized as a pre-pro-protein that is processed in the parasite. Processing occurs at a conserved SphiXE cleavage site as mutagenesis of glutamic acid to alanine at the P1 position disrupts ROP13 maturation. We also demonstrate that processing of the prodomain is not necessary for rhoptry targeting and secretion. While gene disruption reveals that ROP13 is not essential for growth in fibroblasts in vitro or for virulence in vivo, we find that ROP13 is a soluble effector protein that can access the cytoplasm of host cells. Exogenously expressed ROP13 in human cells remains cytosolic but also appears toxic, suggesting that over-expression of this effector protein is disrupting some function within the host cell.
Assuntos
Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Fatores de Virulência/metabolismo , Substituição de Aminoácidos/genética , Animais , Modelos Animais de Doenças , Fibroblastos/parasitologia , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Toxoplasma/patogenicidade , Toxoplasmose/parasitologia , Toxoplasmose/patologia , VirulênciaRESUMO
Toxoplasma gondii is an obligate intracellular parasite that resides in the cytoplasm of its host in a unique membrane-bound vacuole known as the parasitophorous vacuole (PV). The membrane surrounding the parasite is remodeled by the dense granules, secretory organelles that release an array of proteins into the vacuole and to the PV membrane (PVM). Only a small portion of the protein constituents of the dense granules have been identified, and little is known regarding their roles in infection or how they are trafficked within the infected host cell. In this report, we identify a novel secreted dense granule protein, GRA14, and show that it is targeted to membranous structures within the vacuole known as the intravacuolar network and to the vacuolar membrane surrounding the parasite. We disrupted GRA14 and exploited the knockout strain to show that GRA14 can be transferred between vacuoles in a coinfection experiment with wild-type parasites. We also show that GRA14 has an unexpected topology in the PVM with its C terminus facing the host cytoplasm and its N terminus facing the vacuolar lumen. These findings have important implications both for the trafficking of GRA proteins to their ultimate destinations and for expectations of functional domains of GRA proteins at the host-parasite interface.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Toxoplasmose/metabolismo , Vacúolos/química , Animais , Sequência de Bases , Western Blotting , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/parasitologia , Fibroblastos/parasitologia , Humanos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transporte Proteico/fisiologia , Toxoplasma/metabolismo , Toxoplasma/patogenicidade , Vacúolos/parasitologia , Vacúolos/ultraestruturaRESUMO
Intracellular pathogens have evolved a wide array of mechanisms to invade and co-opt their host cells for intracellular survival. Apicomplexan parasites such as Toxoplasma gondii employ the action of unique secretory organelles named rhoptries for internalization of the parasite and formation of a specialized niche within the host cell. We demonstrate that Toxoplasma gondii also uses secretion from the rhoptries during invasion to deliver a parasite-derived protein phosphatase 2C (PP2C-hn) into the host cell and direct it to the host nucleus. Delivery to the host nucleus does not require completion of invasion, as evidenced by the fact that parasites blocked in the initial stages of invasion with cytochalasin D are able to target PP2C-hn to the host nucleus. We have disrupted the gene encoding PP2C-hn and shown that PP2C-hn-knockout parasites exhibit a mild growth defect that can be rescued by complementation with the wild-type gene. The delivery of parasite effector proteins via the rhoptries provides a novel mechanism for Toxoplasma to directly access the command center of its host cell during infection by the parasite.
Assuntos
Núcleo Celular/enzimologia , Fosfoproteínas Fosfatases/metabolismo , Toxoplasma/patogenicidade , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Membrana Celular/metabolismo , Núcleo Celular/parasitologia , Células Cultivadas , Interações Hospedeiro-Parasita , Humanos , Metais/química , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/genética , Proteína Fosfatase 2C , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Homologia de Sequência de Aminoácidos , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/imunologia , Toxoplasmose AnimalRESUMO
Oxidative stress is a key player in a variety of neurodegenerative disorders including Parkinson's disease. Widely used as a parkinsonian mimetic, 6-hydroxydopamine (6-OHDA) generates reactive oxygen species (ROS) as well as coordinated changes in gene transcription associated with the unfolded protein response (UPR) and apoptosis. Whether 6-OHDA-induced UPR activation is dependent on ROS has not yet been determined. The present study used molecular indicators of oxidative stress to place 6-OHDA-generated ROS upstream of the appearance of UPR markers such as activating transcription factor 3 (ATF3) and phosphorylated stress-activated protein kinase (SAPK/JNK) signaling molecules. Antioxidants completely blocked 6-OHDA-mediated UPR activation and rescued cells from toxicity. Moreover, cytochrome c release from mitochondria was observed after the appearance of early UPR markers, suggesting that cellular stress pathways are responsible for its release. Mechanistically, the 6-OHDA-induced UPR was independent of intracellular calcium changes. Rather, evidence of protein oxidation was observed before the expression of UPR markers, suggesting that the rapid accumulation of damaged proteins triggered cell stress/UPR. Taken together, 6-OHDA-mediated cell death in dopaminergic cells proceeds via ROS-dependent UPR up-regulation which leads to an interaction with the intrinsic mitochondrial pathway and downstream caspase activation.
Assuntos
Morte Celular/fisiologia , Mesencéfalo/fisiologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Imuno-Histoquímica , Mesencéfalo/citologia , Mesencéfalo/fisiopatologia , Camundongos , Camundongos Endogâmicos , Oxidopamina/farmacologia , Transtornos Parkinsonianos , Superóxidos/metabolismoRESUMO
The parkinsonian mimetic 6-hydroxydopamine (6-OHDA) has been shown to cause transcriptional changes associated with cellular stress and the unfolded protein response. As these cellular sequelae depend on upstream signaling events, the present study used functional genomics and proteomic approaches to aid in deciphering toxin-mediated regulatory pathways. Microarray analysis of RNA collected from multiple time points following 6-OHDA treatment was combined with data mining and clustering techniques to identify distinct functional subgroups of genes. Notably, stress-induced transcription factors such as ATF3, ATF4, CHOP, and C/EBP beta were robustly up-regulated, yet exhibited unique kinetic patterns. Genes involved in the synthesis and modification of proteins (various tRNA synthetases), protein degradation (e.g., ubiquitin, Herpud1, Sqstm1), and oxidative stress (Hmox1, Por) could be subgrouped into distinct kinetic profiles as well. Realtime PCR and/or two-dimensional electrophoresis combined with western blotting validated data derived from microarray analyses. Taken together, these data support the notion that oxidative stress and protein dysfunction play a role in Parkinson's disease, as well as provide a time course for many of the molecular events associated with 6-OHDA neurotoxicity.