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Background: Delayed anterior cruciate ligament (ACL) reconstruction has been associated with an increased risk of meniscal tears. However, studies comparing early versus delayed ACL reconstruction have not clearly demonstrated that meniscal tears diagnosed arthroscopically are new injuries as opposed to concomitant injuries sustained during ACL rupture. Purpose: To determine whether and how delay of ACL reconstruction is associated with risk of "new" meniscal tears (defined as those visualized arthroscopically that had not been detected on magnetic resonance imaging [MRI]) in adult and pediatric patients. Study Design: Cohort study; Level of evidence, 3. Methods: We retrospectively identified patients who underwent primary ACL reconstruction between 2013 and 2022 at our institution. To ensure that MRI reflected initial intra-articular pathology, we included only patients who had an MRI scan within 3 weeks after injury (173 pediatric and 369 adult patients). Multivariate Poisson regression was performed to calculate the adjusted relative risk (ARR) of new meniscal tears after delayed (≥8 weeks from injury) operative treatment. Results: The mean (± SD) time from injury to MRI was 1.0 ± 0.8 weeks for pediatric patients and 1.1 ± 0.7 weeks for adults. Less than half of the meniscal tears observed arthroscopically had been absent on initial MRI. New medial meniscal tears occurred in 15% of pediatric patients and 16% of adults. New lateral meniscal tears occurred in 48% of pediatric patients and 34% of adults. Among pediatric patients, delayed ACL reconstruction was associated with higher risk of new medial tears (ARR, 3.9; 95% CI, 1.5-10) but not lateral tears (ARR, 0.8; 95% CI, 0.4-1.5). In contrast, adults had no significant increase in risk of meniscal tears associated with operative delay. Conclusion: Delayed ACL reconstruction may be acceptable in adults, who may be less active and less injury-prone than children and adolescents.
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Salmonella Typhimurium drives uptake into non-phagocytic host cells by injecting effector proteins that reorganize the actin cytoskeleton. The host actin regulator N-WASP has been implicated in bacterial entry, but its precise role is not clear. We demonstrate that Cdc42-dependent N-WASP activation, instigated by the Cdc42-activating effector SopE2, strongly impedes Salmonella uptake into host cells. This inhibitory pathway is predominant later in invasion, with the ubiquitin ligase activity of the effector SopA specifically interfering with negative Cdc42-N-WASP signaling at early stages. The cell therefore transitions from being susceptible to invasion, into a state almost completely recalcitrant to bacterial uptake, providing a mechanism to limit the number of internalized Salmonella. Our work raises the possibility that Cdc42-N-WASP, known to be activated by numerous bacterial and viral species during infection and commonly assumed to promote pathogen uptake, is used to limit the entry of multiple pathogens.
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BACKGROUND: The imperative to identify patients at risk of medication-related harm has never been greater. Hospital clinicians cannot easily predict risk of readmission or harm. Candidate variables associated with medication-related harm derived from the literature or significantly represented in a complex patient cohort have been previously described by PHarmacie-4. With a focus on polypharmacy and high-risk medicines in vulnerable patient cohorts, PHarmacie-4 was easy to use and highlighted risks. However it over-estimated risk, reducing its usefulness in stratifying risk of readmission. OBJECTIVE: Develop a risk prediction tool built into a smart phone app, enabling clinicians to identify and refer high-risk patients for an early post-discharge medicines review. Demonstrate usability, real world application and validity in an independent dataset. METHODS: A retrospective, observational study was conducted with 1201 randomly selected patients admitted to Sir Charles Gairdner Hospital between June 1, 2016 to December 31, 2016. Patient characteristics and outcomes of interest were reported, including unplanned hospital utilisation at 30, 60 and 90 days post-discharge. Using multivariable logistic regression modelling, an algorithm was developed, built into a smart phone app and used and validated in an independent dataset. RESULTS: 738 patients (61%) were included in the derivation sample. The best predictive performance was achieved by PHarmacie-R (C-statistic 0.72, 95% CI 0.68-0.75) which included PHarmacie-4 risk variables, a non-linear effect of age, unplanned hospital utilisation in the preceding six months and gender. The independent validation dataset had a C-statistic of 0.64 (95% CI 0.56-0.72). CONCLUSION: PHarmacie-R is the first readmission risk prediction tool, built into a smart phone app, focussing on polypharmacy and high-risk medicines in vulnerable patients. It can assist clinical pharmacists to identify medical inpatients who may benefit from early post-discharge medication management services. External validation is needed to enable application in other clinical settings.
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Readmisión del Paciente , Farmacias , Cuidados Posteriores , Humanos , Lactante , Alta del Paciente , Estudios Retrospectivos , Factores de RiesgoRESUMEN
The p21-activated kinase (PAK) family regulate a multitude of cellular processes, including actin cytoskeleton remodelling. Numerous bacterial pathogens usurp host signalling pathways that regulate actin reorganisation in order to promote Infection. Salmonella and pathogenic Escherichia coli drive actin-dependent forced uptake and intimate attachment respectively. We demonstrate that the pathogen-driven generation of both these distinct actin structures relies on the recruitment and activation of PAK. We show that the PAK kinase domain is dispensable for this actin remodelling, which instead requires the GTPase-binding CRIB and the central poly-proline rich region. PAK interacts with and inhibits the guanine nucleotide exchange factor ß-PIX, preventing it from exerting a negative effect on cytoskeleton reorganisation. This kinase-independent function of PAK may be usurped by other pathogens that modify host cytoskeleton signalling and helps us better understand how PAK functions in normal and diseased eukaryotic cells.
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Actinas/química , Citoesqueleto/química , Infecciones por Salmonella/microbiología , Salmonella enterica/fisiología , Quinasas p21 Activadas/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosforilación , Factores de Intercambio de Guanina Nucleótido Rho/genética , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/patología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Quinasas p21 Activadas/genéticaRESUMEN
BACKGROUND: The rotator cable (RCa) is an important articular-sided structure of the cuff capsular complex that helps prevent suture pull out during rotator cuff repairs (RCRs) and plays a role in force transmission. Yet, the RCa cannot be located during bursal-sided RCRs. The purpose of this study is to develop a method to locate the RCa in the subacromial space and compare its bursal- and articular-sided dimensions. METHODS: In 20 fresh-frozen cadaveric specimens, the RCa was found from the articular side, outlined with stitches, and then evaluated from the bursal side using an easily identifiable reference point, the intersection of a line bisecting the supraspinatus (SS) tendon and posterior SS myotendinous junction (MTJ). Four bursal-sided lengths were measured on the SS-bisecting line as well as the RCa's outside anteroposterior base. For the articular-sided measurements, the rotator cuff capsular complex was detached from bone and optically scanned creating 3D solid models. Using the 3D models, 4 articular-sided lengths were made, including the RCa's inside and outside anteroposterior base. RESULTS: The RCa's medial arch was located 9.9 ± 5.6 mm from the reference point in 10 intact specimens and 4.1 ± 2.4 mm in 10 torn specimens (P = .007). The RCa's width was 10.9 ± 2.1 mm, and the distance from the lateral edge of the RCa to the lateral SS insertion was 13.9 ± 4.8 mm. The bursal- and articular-sided outside anteroposterior base measured 48.1 ± 6.4 mm and 49.6 ± 6.5 mm, respectively (P = .268). The average inside anteroposterior base measurement was 37.3 ± 5.9 mm. DISCUSSION: The medial arch of the RCa can be reliably located during subacromial arthroscopy using the reference point, analogous to the posterior SS MTJ. The RCa is located 10 mm in intact and 4 mm in torn tendons (P = .007) from the posterior SS MTJ. If the above 6-mm shift in location of the RCa is not taken into consideration during rotator cuff suture placement, it could negatively affect time zero repair strength. The inside anteroposterior base of the RCa measures on average 37 mm; therefore, rotator cuff tears measuring >37 mm are at risk of rupturing part or all of the RCa's 2 humeral attachments, which if not recognized and addressed could impact postoperative function.
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Artroscopía , Lesiones del Manguito de los Rotadores , Bolsa Sinovial/cirugía , Humanos , Manguito de los Rotadores/cirugía , Lesiones del Manguito de los Rotadores/cirugía , TendonesRESUMEN
BACKGROUND: Partial avulsions of the short and/or long head of the distal biceps tendon cause pain and loss of strength. The goal of the present study was to quantify the loss of supination and flexion strength following a series of surgical releases designed to simulate partial and complete short and long head traumatic avulsions. METHODS: Mechanical testing was performed to measure supination moment arms and flexion force efficiency on 18 adult fresh-frozen specimens in pronation, neutral, and supination. The distal biceps footprint length was divided into 4 equal segments. In 9 specimens (the distal-first group), the tendon was partially cut starting distally by releasing 25%, 50%, and 75% of the insertion site. In the other 9 specimens (the proximal-first group), the releases started proximally. Mechanical testing was performed before and after each release. RESULTS: Significant decreases in the supination moment arm occurred after a 75% release in the distal-first release group; the decrease was 24% in pronation (p = 0.003) and 10% in neutral (p = 0.043). No significant differences in the supination moment arm (p ≥ 0.079) or in flexion force efficiency (p ≥ 0.058) occurred in the proximal-first group. CONCLUSIONS: A simulated complete short head avulsion significantly decreased the supination moment arm and therefore supination strength. CLINICAL RELEVANCE: A mechanical case can be made for repair of partial distal biceps tendon avulsions when the rupture involves ≥75% of the distal insertion site.
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Fuerza Muscular/fisiología , Músculo Esquelético/lesiones , Rotura/complicaciones , Supinación/fisiología , Traumatismos de los Tendones/complicaciones , Adulto , Brazo , Fenómenos Biomecánicos , Cadáver , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/fisiopatología , Pronación/fisiología , Distribución Aleatoria , Rango del Movimiento Articular/fisiología , Rotura/fisiopatología , Traumatismos de los Tendones/fisiopatologíaRESUMEN
Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that tightly adheres to host cells by forming "actin pedestals" beneath the bacteria, a critical step in pathogenesis. EPEC injects effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. We have recently shown that one such effector, EspG, hijacks p21-activated kinase (PAK) and sustains its activated state to drive the cytoskeletal changes necessary for attachment of the pathogen to target cells. This EspG subversion of PAK required active Rho family small GTPases in the host cell. Here we show that EPEC itself promotes the activation of Rho GTPases by recruiting Frabin, a host guanine nucleotide exchange factor (GEF) for the Rho GTPase Cdc42. Cells devoid of Frabin showed significantly lower EPEC-induced PAK activation, pedestal formation, and bacterial attachment. Frabin recruitment to sites of EPEC attachment was driven by EspG and required localized enrichment of phosphatidylinositol 4,5-bisphosphate (PIP2) and host Arf6. Our findings identify Frabin as a key target for EPEC to ensure the activation status of cellular GTPases required for actin pedestal formation.IMPORTANCE Enteropathogenic Escherichia coli (EPEC) is a leading cause of diarrhea in children, especially in the developing world. EPEC initiates infection by attaching to cells in the host intestine, triggering the formation of actin-rich "pedestal" structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, which work together to hijack host signaling pathways to drive pedestal production. Here we show how EPEC hijacks a host protein, Frabin, which creates the conditions in the cell necessary for the pathogen to manipulate a specific pathway that promotes pedestal formation. This provides new insights into this essential early stage in disease caused by EPEC.
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Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Escherichia coli Enteropatógena/fisiología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Interacciones Huésped-Patógeno , Proteínas de Microfilamentos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Modelos Biológicos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de ProteínasRESUMEN
The small GTPase ADP-ribosylation factor 6 (Arf6) anchors at the plasma membrane to orchestrate key functions, such as membrane trafficking and regulating cortical actin cytoskeleton rearrangement. A number of studies have identified key players that interact with Arf6 to regulate actin dynamics in diverse cell processes, yet it is still unknown whether Arf6 can directly signal to the wave regulatory complex to mediate actin assembly. By reconstituting actin dynamics on supported lipid bilayers, we found that Arf6 in co-ordination with Rac1(Ras-related C3 botulinum toxin substrate 1) can directly trigger actin polymerization by recruiting wave regulatory complex components. Interestingly, we demonstrated that Arf6 triggers actin assembly at the membrane directly without recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO (ARF nucleotide-binding site opener), which is able to activate Arf1 to enable WRC-dependent actin assembly. Furthermore, using labelled E. coli, we demonstrated that actin assembly by Arf6 also contributes towards efficient phagocytosis in THP-1 macrophages. Taken together, this study reveals a mechanism for Arf6-driven actin polymerization.
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Factores de Ribosilacion-ADP/metabolismo , Actinas/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factor 6 de Ribosilación del ADP , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Modelos Biológicos , Fagocitosis/inmunología , Unión Proteica , Células THP-1RESUMEN
Enteropathogenic Escherichia coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) are extracellular pathogens that reorganize the host cell cytoskeleton to form "actin pedestals" beneath the tightly adherent bacteria, a critical step in pathogenesis. EPEC and EHEC inject effector proteins that manipulate host cell signaling cascades to trigger pedestal assembly. One such effector, EspG, has been reported to bind and activate p21-activated kinase (PAK), a key cytoskeletal regulator, but the function of this interaction and whether it impacts pedestal assembly are unknown. Here, we demonstrate that deletion of espG significantly impairs pedestal formation and attachment by both EPEC and EHEC. This role of EspG is shown to be dependent on its interaction with PAK. Unexpectedly, EspG was able to subvert PAK only in the presence of Rho family small GTPases, which function to both concentrate PAK at the membrane and stimulate PAK activation. Our findings reveal a novel mechanism by which EspG hijacks PAK and sustains its active state to drive bacterial attachment to host cells.IMPORTANCE Enteropathogenic E. coli and enterohemorrhagic E. coli (EPEC and EHEC, respectively) remain a significant global health problem. Both EPEC and EHEC initiate infection by attaching to cells in the host intestine, triggering the formation of actin-rich "pedestal" structures directly beneath the adherent pathogen. These bacteria inject their own receptor into host cells, which upon binding to a protein on the pathogen surface triggers pedestal formation. Multiple other proteins are also delivered into the cells of the host intestine, but how they contribute to disease is often less clear. Here, we show how one of these injected proteins, EspG, hijacks a host signaling pathway for pedestal production. This provides new insights into this essential early stage in EPEC and EHEC disease.
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Actinas/metabolismo , Escherichia coli Enterohemorrágica/metabolismo , Escherichia coli Enteropatógena/metabolismo , GTP Fosfohidrolasas/metabolismo , Quinasas p21 Activadas/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Proteínas Portadoras , Línea Celular , Citoesqueleto/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The Arf and Rho subfamilies of small GTPases are nucleotide-dependent molecular switches that act as master regulators of vesicular trafficking and the actin cytoskeleton organization. Small GTPases control cell processes with high fidelity by acting through distinct repertoires of binding partners called effectors. While we understand a great deal about how these GTPases act individually, relatively little is known about how they cooperate, especially in the control of effectors. This review highlights how Arf GTPases collaborate with Rac1 to regulate actin cytoskeleton dynamics at the membrane via recruiting and activating the Wave Regulatory Complex (WRC), a Rho effector that underpins lamellipodia formation and macropinocytosis. This provides insight into Arf regulation of the actin cytoskeleton, while putting the spotlight on small GTPase cooperation with emerging evidence of its importance in fundamental cell biology and interactions with pathogenic bacteria.
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Factores de Ribosilacion-ADP/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Animales , Escherichia coli/metabolismo , Humanos , Salmonella/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Salmonella causes disease in humans and animals ranging from mild self-limiting gastroenteritis to potentially life-threatening typhoid fever. Salmonellosis remains a considerable cause of morbidity and mortality globally, and hence imposes a huge socio-economic burden worldwide. A key property of all pathogenic Salmonella strains is the ability to invade non-phagocytic host cells. The major determinant of this invasiveness is a Type 3 Secretion System (T3SS), a molecular syringe that injects virulence effector proteins directly into target host cells. These effectors cooperatively manipulate multiple host cell signaling pathways to drive pathogen internalization. Salmonella does not only rely on these injected effectors, but also uses several other T3SS-independent mechanisms to gain entry into host cells. This review summarizes our current understanding of the methods used by Salmonella for cell invasion, with a focus on the host signaling networks that must be coordinately exploited for the pathogen to achieve its goal.
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Interacciones Huésped-Patógeno/fisiología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad , Sistemas de Secreción Tipo III/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Islas Genómicas/genética , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Salmonella typhimurium/metabolismo , VirulenciaRESUMEN
To establish infections, Salmonella injects virulence effectors that hijack the host actin cytoskeleton and phosphoinositide signaling to drive pathogen invasion. How effectors reprogram the cytoskeleton network remains unclear. By reconstituting the activities of the Salmonella effector SopE, we recapitulated Rho GTPase-driven actin polymerization at model phospholipid membrane bilayers in cell-free extracts and identified the network of Rho-recruited cytoskeleton proteins. Knockdown of network components revealed a key role for myosin VI (MYO6) in Salmonella invasion. SopE triggered MYO6 localization to invasion foci, and SopE-mediated activation of PAK recruited MYO6 to actin-rich membranes. We show that the virulence effector SopB requires MYO6 to regulate the localization of PIP3 and PI(3)P phosphoinositides and Akt activation. SopE and SopB target MYO6 to coordinate phosphoinositide production at invasion foci, facilitating the recruitment of cytoskeleton adaptor proteins to mediate pathogen uptake.
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Interacciones Huésped-Patógeno/fisiología , Cadenas Pesadas de Miosina/metabolismo , Fosfatidilinositol 3-Quinasa/metabolismo , Salmonella typhimurium/patogenicidad , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/microbiología , Células HeLa , Humanos , Proteínas de Microfilamentos/metabolismo , Cadenas Pesadas de Miosina/genética , Fosfatidilinositoles/metabolismo , Salmonella typhimurium/metabolismo , Transducción de Señal , Factores de Virulencia/metabolismoRESUMEN
UNLABELLED: To establish intracellular infections, Salmonella bacteria trigger host cell membrane ruffling and invasion by subverting cellular Arf guanine nucleotide exchange factors (GEFs) that activate Arf1 and Arf6 GTPases by promoting GTP binding. A family of cellular Arf GTPase-activating proteins (GAPs) can downregulate Arf signaling by stimulating GTP hydrolysis, but whether they do this during infection is unknown. Here, we uncovered a remarkable role for distinct Arf GAP family members in Salmonella invasion. The Arf6 GAPs ACAP1 and ADAP1 and the Arf1 GAP ASAP1 localized at Salmonella-induced ruffles, which was not the case for the plasma membrane-localized Arf6 GAPs ARAP3 and GIT1 or the Golgi-associated Arf1 GAP1. Surprisingly, we found that loss of ACAP1, ADAP1, or ASAP1 impaired Salmonella invasion, revealing that GAPs cannot be considered mere terminators of cytoskeleton remodeling. Salmonella invasion was restored in Arf GAP-depleted cells by expressing fast-cycling Arf derivatives, demonstrating that Arf GTP/GDP cycles facilitate Salmonella invasion. Consistent with this view, both constitutively active and dominant-negative Arf derivatives that cannot undergo GTP/GDP cycles inhibited invasion. Furthermore, we demonstrated that Arf GEFs and GAPs colocalize at invading Salmonella and collaborate to drive Arf1-dependent pathogen invasion. This study revealed that Salmonella bacteria exploit a remarkable interplay between Arf GEFs and GAPs to direct cycles of Arf GTPase activation and inactivation. These cycles drive Salmonella cytoskeleton remodeling and enable intracellular infections. IMPORTANCE: To initiate infections, the Salmonella bacterial pathogen remodels the mammalian actin cytoskeleton and invades host cells by subverting host Arf GEFs that activate Arf1 and Arf6 GTPases. Cellular Arf GAPs deactivate Arf GTPases and negatively regulate cell processes, but whether they target Arfs during infection is unknown. Here, we uncovered an important role for the Arf GAP family in Salmonella invasion. Surprisingly, we found that Arf1 and Arf6 GAPs cooperate with their Arf GEF counterparts to facilitate cycles of Arf GTPase activation and inactivation, which direct pathogen invasion. This report illustrates that GAP proteins promote actin-dependent processes and are not necessarily restricted to negatively regulating cellular signaling. It uncovers a remarkable interplay between Arf GEFs and GAPs that is exploited by Salmonella to establish infection and expands our understanding of Arf GTPase-regulated cytoskeleton remodeling.
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Proteínas Activadoras de GTPasa/metabolismo , Familia de Multigenes , Infecciones por Salmonella/enzimología , Salmonella typhimurium/fisiología , Citoesqueleto/enzimología , Citoesqueleto/microbiología , Proteínas Activadoras de GTPasa/genética , Interacciones Huésped-Patógeno , Humanos , Infecciones por Salmonella/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genéticaRESUMEN
The phage shock protein (Psp) systems found in bacteria, archaea and higher plants respond to extracytoplasmic stresses that damage the cytoplasmic membrane and enable cells to repair their membranes. The conserved membrane-associated effector protein PspA has four α-helical domains (HD1-HD4) and helps to repair the membrane as a high-order oligomer. In enterobacteria, under non-stress conditions, PspA as a low-order assembly directly inhibits its cognate transcription activator PspF. Here we show that N-terminal amphipathic helices ahA and ahB in PspA HD1 are functional determinants involved in negative gene control and stress signal perception and its transduction via interactions with the PspBC membrane stress sensors and the inner membrane (IM). The amphipathic helices enable PspA to switch from a low-order gene regulator into an IM-bound high-order effector complex under membrane stress. Conserved residue proline 25 is involved in sequential use of the amphipathic helices and ahA-IM interaction. Single molecule imaging of eGFP-PspA and its amphipathic helices variants in live Escherichia coli cells show distinct spatial and temporal organisations of PspA corresponding to its negative control and effector functions. These findings inform studies on the role of the Psp system in persister cell formation and cell envelope protection in bacterial pathogens and provide a basis for exploring the specialised roles of PspA homologues such as YjfJ, LiaH and Vipp1.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Regulación Bacteriana de la Expresión Génica , Estructura Secundaria de ProteínaRESUMEN
ADP ribosylation factor (Arf) 6 anchors to the plasma membrane, where it coordinates membrane trafficking and cytoskeleton remodelling, but how it assembles actin filaments is unknown. By reconstituting membrane-associated actin assembly mediated by the WASP family veroprolin homolog (WAVE) regulatory complex (WRC), we recapitulated an Arf6-driven actin polymerization pathway. We show that Arf6 is divergent from other Arf members, as it was incapable of directly recruiting WRC. We demonstrate that Arf6 triggers actin assembly at the membrane indirectly by recruiting the Arf guanine nucleotide exchange factor (GEF) ARNO that activates Arf1 to enable WRC-dependent actin assembly. The pathogen Salmonella usurped Arf6 for host cell invasion by recruiting its canonical GEFs EFA6 and BRAG2. Arf6 and its GEFs facilitated membrane ruffling and pathogen invasion via ARNO, and triggered actin assembly by generating an Arf1-WRC signaling hub at the membrane in vitro and in cells. This study reconstitutes Arf6-dependent actin assembly to reveal a mechanism by which related Arf GTPases orchestrate distinct steps in the WRC cytoskeleton remodelling pathway.
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Factores de Ribosilacion-ADP/metabolismo , Actinas/metabolismo , Complejos Multiproteicos/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella/metabolismo , Transducción de Señal , Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Actinas/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Células HeLa , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Complejos Multiproteicos/genética , Salmonella/patogenicidad , Infecciones por Salmonella/genética , Sulfotransferasas/genética , Sulfotransferasas/metabolismoRESUMEN
Tetrabenazine is effective in the treatment of the chorea associated with Huntington disease and other hyperkinetic movement disorders. Following oral administration, tetrabenazine is hepatically transformed into 2 active metabolites that are CYP2D6 substrates. There are 4 CYP2D6 genotypes: poor metabolizers, intermediate metabolizers, extensive metabolizers, and ultrarapid metabolizers. CYP2D6 genotyping was performed on sequential subjects treated with tetrabenazine, but results were not known at the time of titration. Duration of titration to a stable dose, total daily dose, response rating scores, and adverse events were retrospectively collected and subsequently analyzed. Of 127 patients, the majority (n = 100) were categorized as extensive metabolizers, 14 as intermediate metabolizers, 11 as poor metabolizers, and 2 as ultrarapid metabolizers. Ultrarapid metabolizer patients needed a longer titration (8 vs 3.3, 4.4, and 3 weeks, respectively; P < .01) to achieve optimal benefit and required a higher average daily dose than the other patients, but this difference did not reach statistical significance. The treatment response was less robust in the intermediate metabolizer group when compared with the extensive metabolizer patients (P = .013), but there were no statistically significant differences between the various groups with regard to adverse effects. Our findings demonstrate that, aside from the need for a longer titration in the ultrarapid metabolizers, there are no distinguishing features of patients with various CYP2D6 genotypes, and therefore the current recommendation to systematically genotype all patients prescribed more than 50 mg/day of tetrabenazine should be reconsidered.
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Antidiscinéticos/uso terapéutico , Citocromo P-450 CYP2D6/genética , Trastornos del Movimiento/tratamiento farmacológico , Trastornos del Movimiento/genética , Tetrabenazina/uso terapéutico , Adulto , Antidiscinéticos/efectos adversos , Antidiscinéticos/farmacocinética , Femenino , Genotipo , Humanos , Enfermedad de Huntington/tratamiento farmacológico , Enfermedad de Huntington/genética , Hipercinesia/tratamiento farmacológico , Hipercinesia/genética , Masculino , Persona de Mediana Edad , Mioclonía/tratamiento farmacológico , Mioclonía/genética , Farmacogenética , Fenotipo , Agitación Psicomotora/tratamiento farmacológico , Agitación Psicomotora/genética , Tetrabenazina/efectos adversos , Tetrabenazina/farmacocinética , Síndrome de Tourette/tratamiento farmacológico , Síndrome de Tourette/genética , Resultado del TratamientoRESUMEN
The WAVE regulatory complex (WRC) drives the polymerisation of actin filaments located beneath the plasma membrane to generate lamellipodia that are pivotal to cell architecture and movement. By reconstituting WRC-dependent actin assembly at the membrane, we recently discovered that several classes of Arf family GTPases directly recruit and activate WRC in cell extracts, and that Arf cooperates with Rac1 to trigger actin polymerisation. Here, we demonstrate that the Class 1 Arf1 homologue Arf79F colocalises with the WRC at dynamic lamellipodia. We report that Arf79F is required for lamellipodium formation in Drosophila S2R+ cells, which only express one Arf isoform for each class. Impeding Arf function either by dominant-negative Arf expression or by Arf double-stranded RNA interference (dsRNAi)-mediated knockdown uncovered that Arf-dependent lamellipodium formation was specific to Arf79F, establishing that Class 1 Arfs, but not Class 2 or Class 3 Arfs, are crucial for lamellipodia. Lamellipodium formation in Arf79F-silenced cells was restored by expressing mammalian Arf1, but not by constitutively active Rac1, showing that Arf79F does not act via Rac1. Abolition of lamellipodium formation in Arf79F-silenced cells was not due to Golgi disruption. Blocking Arf79F activation with guanine nucleotide exchange factor inhibitors impaired WRC localisation to the plasma membrane and concomitant generation of lamellipodia. Our data indicate that the Class I Arf GTPase is a central component in WRC-driven lamellipodium formation.
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Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Seudópodos/metabolismo , Factores de Ribosilacion-ADP/genética , Animales , Línea Celular , Células Cultivadas , Drosophila , Técnica del Anticuerpo FluorescenteRESUMEN
BACKGROUND: Deep brain stimulation (DBS) has proven to be an effective treatment for Parkinson's disease (PD) and other movement disorders, but its usefulness is limited by complications related to the hardware. METHODS: We reviewed the records of all our patients treated with DBS from January 1996 to August 2010 and analyzed those with hardware complications and reasons for surgical revision. RESULTS: A total of 512 patients underwent 856 electrode implantations during the study period. A total of 297 (58%) patients had PD, 127 (24.8%) had essential tremor (ET), 40 (7.8%) had dystonia, and 48 (9.37%) had another movement disorder. The mean age at the first electrode implantation was 57.6 ± 14 years and patients were followed for a mean of 3.9 ± 2.8 years. A total of 44 patients (8.6%) had a hardware complication or system revision. Lead fracture was the most common complication and occurred in 13 (2.5%) patients, followed by infections (n = 10, 1.9%), electrode misplacement (n = 10, 1.9%), electrode migration (n = 9, 1.75%), and other complications (n = 2 , 0.39%). Patients with ET had a higher risk of hardware complications compared to those with PD, 13 vs. 7% (OR 2.03; p = 0.042). CONCLUSIONS: DBS is a safe intervention with a relatively low rate of hardware complications.
Asunto(s)
Estimulación Encefálica Profunda/instrumentación , Estimulación Encefálica Profunda/métodos , Falla de Equipo , Trastornos del Movimiento/terapia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Electrodos Implantados/normas , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Trastornos del Movimiento/fisiopatología , Estudios Retrospectivos , Factores de Riesgo , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Impairment in computerized dynamic posturography scores has been documented in Huntington disease patients. Tetrabenazine is approved to treat chorea in Huntington disease, but its effect on posturography scores, and balance in general, is unknown. MATERIALS AND METHODS: We designed a study to test computerized dynamic posturography performance while taking tetrabenazine and after stopping tetrabenazine for at least three days. RESULTS: 10 Huntington disease patients were studied both ON and OFF tetrabenazine. The composite score was statistically different between ON and OFF conditions and both conditions were significantly worse than reference scores. There was no significant difference between ON and OFF trials in the number of falls. A significant improvement on sensory orientation test conditions 3 (sway-referenced vision) and 5 (sway-referenced motion of the support surface and eyes closed) was seen while ON tetrabenazine. Strategy scores 1-3 were also significantly different while ON tetrabenazine. CONCLUSION: These findings suggest that tetrabenazine aided patients in gating out of abnormal visual cues when other sensory modalities were available, as well as in gating out abnormal kinesthetic cues when visual cues were not available. It could not help with gating out of simultaneous abnormal visual and somatosensory cues. Thus, tetrabenazine can improve postural stability when one sensory modality is irrelevant, but this effect is not sustained when multiple abnormal sensory modalities are present. This is the first study supporting the use of any medicine to treat balance problems in Huntington disease.
Asunto(s)
Enfermedad de Huntington/tratamiento farmacológico , Equilibrio Postural/efectos de los fármacos , Tetrabenazina/uso terapéutico , Adulto , Anciano , Señales (Psicología) , Femenino , Humanos , Masculino , Persona de Mediana Edad , Visión Ocular/efectos de los fármacosRESUMEN
OBJECTIVE: To assess temporal amplitude variability in patients with essential tremor (ET). METHODS: Patients who satisfied the diagnostic criteria for probable or definite ET were enrolled in the study. Each enrolled patient was first rated using the essential tremor rating assessment scale (TETRAS). Postural and kinetic tremors of the arms were then measured using a quantitative motor assessment system (QMAS) starting at 8:00 AM (T0-baseline) every 2 h for 6 h. Subjects were videotaped performing the tasks. Single subjects consecutively performed each assessment twice during every time-interval. At the end of the study, videos were randomized and blindly rated using TETRAS. RESULTS: Twelve ET subjects were enrolled. QMAS and video scores were directly correlated with high test-retest reliability for each time-interval. Furthermore, the QMAS scores at T0 significantly correlated with in-person rated TETRAS scores as well as with subsequent time-intervals instrumental scores. No significant differences were detected between time-intervals QMAS average measurements using ANOVA. There was a maximal 23% absolute variation in tremor amplitude from baseline as determined by the QMAS. Test for equality of variance showed high measurement variability for subjects with high QMAS scores at T0 and throughout the 6 h of assessment. CONCLUSIONS: Baseline measures are predictive of tremor amplitude at subsequent assessments during the day. High amplitude tremor is associated with high intra-assessment variability.