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Cholera toxoid is an established tool for use in cellular tracing in neuroscience and cell biology. We use a sortase labeling approach to generate site-specific N-terminally modified variants of both the A2-B5 heterohexamer and B5 pentamer forms of the toxoid. Both forms of the toxoid are endocytosed by GM1-positive mammalian cells, and while the heterohexameric toxoid was principally localized in the ER, the B5 pentamer showed an unexpectedly specific localization in the medial/trans-Golgi. This study suggests a future role for specifically labeled cholera toxoids in live-cell imaging beyond their current applications in neuronal tracing and labeling of lipid rafts in fixed cells.
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Toxina del Cólera , Cisteína Endopeptidasas , Aparato de Golgi , Humanos , Toxina del Cólera/metabolismo , Cisteína Endopeptidasas/metabolismo , Aparato de Golgi/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/genética , EndocitosisRESUMEN
BACKGROUND: Optic disc edema is a feature of many ophthalmic and neurologic conditions. It remains an underappreciated feature of birdshot chorioretinitis (BSCR), leading to delay in diagnosis and treatment. The purpose of our study was to identify clinical features that are concomitant with optic disc edema and suggest a diagnosis of BSCR. METHODS: Retrospective multicenter case series of 29 patients who were referred to a neuro-ophthalmologist or uveitis specialist for evaluation of disc edema and were ultimately diagnosed with BSCR. RESULTS: Fifty-four eyes of 30 patients, from the practices of 15 uveitis specialists, met the eligibility criteria. In addition to disc edema, concomitant features in all patients included vitritis, chorioretinal lesions, and retinal vasculitis. Visual recovery to 20/40 or better occurred in 26 of 29 patients. Visual acuity remained 20/100 or worse in 2 patients previously diagnosed with idiopathic intracranial hypertension, 1 patient previously diagnosed with optic neuritis, and 1 patient for whom treatment was delayed for years, leading to optic disc atrophy. CONCLUSIONS: Optic disc edema is a presenting feature in some cases of BSCR. A diagnosis of BSCR should be considered when disc edema occurs with vitritis, chorioretinal inflammation, and retinal vasculitis. Patients should be referred to a uveitis specialist for treatment.
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Uveitis is one of the most common manifestations of juvenile idiopathic arthritis (JIA). Currently, JIA is associated with decreased gut microbiota diversity. Studies confirm that perinatal events can cause aberrant microbial colonization. The objective of this study is to determine if JIA is associated with perinatal events with a secondary focus on these variables to the development of JIA-uveitis. 369 patients with strabismus (n = 200) or JIA (n = 196) were included in the study. Completed surveys (JIA 37; strabismus 18) collected data about birth route, pregnancy and labor complications, JIA medications, and the presence of eye disorders. Analysis indicates that there is no relationship between JIA development and the perinatal events investigated. Similarly, no significance was found between JIA-uveitis and birth route or labor complications. Pregnancy complications, namely gestational diabetes (GD), were statistically higher in the JIA group with uveitis compared to JIA without uveitis. The data from this survey study showed that JIA-uveitis was highly associated with pregnancy complications, particularly with GD. However, no statistically significant association was found between JIA and route of delivery, labor complications, or pregnancy complications. Further studies are needed to understand the ways that GD interrelates with the development of uveitis in JIA patients.
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Artritis Juvenil , Complicaciones del Trabajo de Parto , Complicaciones del Embarazo , Estrabismo , Uveítis , Humanos , Femenino , Artritis Juvenil/complicaciones , Artritis Juvenil/epidemiología , Artritis Juvenil/tratamiento farmacológico , Uveítis/etiología , Uveítis/complicaciones , Complicaciones del Embarazo/epidemiologíaRESUMEN
PRCIS: Kahook Dual Blade (KDB) goniotomy can successfully lower intraocular pressure in some patients with uveitis-associated ocular hypertension or glaucoma. PURPOSE: The purpose of this study was to report a case series of patients that underwent KDB goniotomy at a single institution for uveitis-associated ocular hypertension or glaucoma with an open angle. METHODS: We performed a retrospective chart review of all patients with uveitis-associated ocular hypertension or glaucoma who underwent KDB goniotomy with trabecular meshwork excision alone or in combination with phacoemulsification cataract surgery at a single center between August 2017 and February 2020. The case series included 45 eyes of 37 patients. All eyes developed ocular hypertension refractory to maximum-tolerated medical therapy and required surgical intervention. Two eyes were excluded as they were lost to follow-up before 5 months postoperatively. Surgical success was defined as reaching the goal intraocular pressure or lower for each patient, including ongoing medical therapy. RESULTS: At most recent follow-up, 25 (55.6%) of 45 eyes had an intraocular pressure that was at goal. Mean follow-up time was 15.2±12.1 months ranging from 0.5 to 36 months postoperatively, considering that patients were eliminated from the data analysis once they required a second surgery. The mean number of preoperative medications, including oral carbonic anhydrase inhibitors was 3.7±1.2 medications. The mean number of postoperative medications through the last clinic visit was 2.5±1.9 medications for a mean reduction of 1.2±1.6 medications ( P -value <0.0001*). CONCLUSIONS: This larger case series shows that some patients with uveitis-associated ocular hypertension or glaucoma with an open angle may have success with KDB goniotomy.
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Glaucoma de Ángulo Abierto , Glaucoma , Hipertensión Ocular , Trabeculectomía , Uveítis , Humanos , Glaucoma de Ángulo Abierto/complicaciones , Glaucoma de Ángulo Abierto/cirugía , Presión Intraocular , Estudios Retrospectivos , Resultado del Tratamiento , Glaucoma/cirugía , Hipertensión Ocular/etiología , Hipertensión Ocular/cirugía , Uveítis/complicaciones , Uveítis/diagnóstico , Uveítis/cirugíaRESUMEN
PURPOSE: This study aims to report the long-term outcomes of uveitis-associated optic disc and epiretinal neovascularization (NV) treated with immunomodulatory therapy alone. METHODS: This is a retrospective, multi-center chart review conducted at Northwestern University (Chicago, IL) and San Raffaele Scientific Institute (Milan, Italy) from 2014 to 2021 of patients with optic disc and/or retinal neovascularization associated with uveitis. The data collected included age at the time of NV detection, gender, medications, and follow-up period. Imaging was reviewed if available. RESULTS: Eight eyes of six patients were identified. The mean age was 22 years (range 10-52 years); the median follow-up was 3 years (range 6 months to 7 years). All eyes presented with active NV at the time of uveitis onset; 7 eyes were treatment-naïve. None had clinical or angiographic evidence of retinal ischemia. All patients received a variable combination of local steroids, systemic steroids, and systemic immunosuppression. Complete resolution of uveitic NV occurred in all eyes within a median of 8 weeks (ranging 2-20 weeks) from initiating treatment. No NV recurrence was noted. CONCLUSION: Immunomodulatory therapy alone may be successful in achieving long-term control of uveitis-associated NV, without the use of destructive measures.
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Disco Óptico , Neovascularización Retiniana , Uveítis , Adolescente , Adulto , Niño , Estudios de Seguimiento , Humanos , Inmunomodulación , Persona de Mediana Edad , Estudios Retrospectivos , Uveítis/diagnóstico , Uveítis/tratamiento farmacológico , Adulto JovenRESUMEN
PRECIS: In a small case series of patients with uveitis-associated ocular hypertension (OHTN) or glaucoma, goniotomy with Kahook Dual Blade (KDB) was an effective surgical treatment option for lowering intraocular pressure (IOP). PURPOSE: The purpose of this study is to review the success of goniotomy using the KDB with trabecular meshwork (TM) excision in lowering the IOP of patients with uveitis-associated OHTN or glaucoma. MATERIALS AND METHODS: A retrospective chart review was completed for all patients with uveitis-associated OHTN or glaucoma who underwent KDB goniotomy with TM excision alone or in combination with phacoemulsification cataract surgery at a single institution. The study included 12 patients (16 eyes). Patients were followed for a minimum of 5 months postoperatively. The main outcome measures of this case series included postoperative IOPs, percent IOP reduction, and reduction of glaucoma medications. RESULTS: The mean maximum IOP of the patients before maximum-tolerated medical therapy or surgery was 35.6±5.8 mm Hg. The mean preoperative IOP at the clinical office visit before surgery of these patients was 28.1±8.5 mm Hg on maximum-tolerated medical therapy. Ten eyes (62.5%) have maintained an IOP at or below their goal through their most recent follow-up visit (mean follow-up time of 9.6±5.6 mo). The mean number of glaucoma medications was significantly reduced from 3.6±0.9 medications to 2.1±1.7 medications, for a mean reduction of 1.5±1.4 medications (P=0.004). CONCLUSIONS: On the basis of this small case series, KDB goniotomy may be a safe, less invasive, and effective first-line surgical alternative for patients with uveitis-associated OHTN or glaucoma refractory to medical therapy.
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Hipertensión Ocular/complicaciones , Hipertensión Ocular/cirugía , Trabeculectomía , Uveítis/etiología , Uveítis/cirugía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Presión Intraocular , Masculino , Persona de Mediana Edad , Facoemulsificación , Estudios Retrospectivos , Tonometría Ocular , Malla Trabecular/patología , Malla Trabecular/cirugía , Trabeculectomía/efectos adversos , Trabeculectomía/instrumentación , Trabeculectomía/métodos , Resultado del Tratamiento , Adulto JovenRESUMEN
Trisomy 18 is an autosomal trisomy condition characterized by minor to major birth defects, severe disabilities, and high rates of pre- and postnatal mortality. Interventions for these infants have traditionally been withheld with focus instead on palliative support. The issues and attitudes surrounding corrective surgery of congenital heart defects, which is a birth defect that occurs in approximately 90% of infants with trisomy 18, is of our study's interest as recent literature has indicated that cardiac surgery is being performed and may lead to improved survival compared to palliative care. Thus, our study aimed to describe clinician attitudes toward cardiac surgery and trisomy 18. We surveyed 378 clinicians from multiple specialties, including genetic counselors, involved in the pre- and postnatal care of infants with trisomy 18. Descriptive statistics were performed to describe all clinicians' responses, and a secondary analysis with stratifications by clinician type was also performed. Forty-eight percent (n = 378) of clinicians felt it was appropriate to discuss the option of cardiac surgery. Ethical concerns and insufficient outcome data were the most agreed upon reasons for not offering cardiac surgery. Trisomy 18 not being uniformly lethal and expressed parental wishes were the most agreed upon justifications for offering surgery. Clinicians felt the discussion of the option of cardiac surgery is appropriate, however are hesitant due to ethical concerns and insufficient outcome data. Results from this study aim to promote discussion and collaboration among clinicians to improve consistency in patient care.
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Actitud del Personal de Salud , Procedimientos Quirúrgicos Cardíacos/psicología , Cardiopatías Congénitas/cirugía , Síndrome de la Trisomía 18/psicología , Adolescente , Adulto , Femenino , Cardiopatías Congénitas/genética , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Cuidados Paliativos , Adulto JovenRESUMEN
The 'Stakeholder Perspectives on Noninvasive Prenatal Genetic Screening' Symposium was held in conjunction with the 2015 annual meeting of the International Society for Prenatal Diagnosis. During the day-long meeting, a panel of patient advocacy group (PAG) representatives discussed concerns and challenges raised by prenatal cell-free DNA (cfDNA) screening, which has resulted in larger demands upon PAGs from concerned patients receiving prenatal cfDNA screening results. Prominent concerns included confusion about the accuracy of cfDNA screening and a lack of patient education resources about genetic conditions included in cfDNA screens. Some of the challenges faced by PAGs included funding limitations, lack of consistently implemented standards of care and oversight, diverse perspectives among PAGs and questions about neutrality, and lack of access to training and genetic counselors. PAG representatives also put forward suggestions for addressing these challenges, including improving educational and PAG funding and increasing collaboration between PAGs and the medical community. © 2016 John Wiley & Sons, Ltd.
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Trastornos de los Cromosomas/diagnóstico , ADN/sangre , Organizaciones sin Fines de Lucro , Defensa del Paciente , Diagnóstico Prenatal , Femenino , Asesoramiento Genético , Accesibilidad a los Servicios de Salud , Humanos , Defensa del Paciente/economía , Defensa del Paciente/ética , Educación del Paciente como Asunto/economía , Educación del Paciente como Asunto/normas , Valor Predictivo de las Pruebas , Embarazo , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Nivel de Atención , Estados UnidosRESUMEN
Collagen is the most abundant protein in the animal kingdom. It is of fundamental importance during development for cell differentiation and tissue morphogenesis as well as in pathological processes such as fibrosis and cancer cell migration. However, our understanding of the mechanisms of procollagen secretion remains limited. Here, we show that TFG organizes transitional ER (tER) and ER exit sites (ERESs) into larger structures. Depletion of TFG results in dispersion of tER elements that remain associated with individual ER-Golgi intermediate compartments (ERGICs) as largely functional ERESs. We show that TFG is not required for the transport and packaging of small soluble cargoes but is necessary for the export of procollagen from the ER. Our work therefore suggests a key relationship between the structure and function of ERESs and a central role for TFG in optimizing COPII assembly for procollagen export.
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Colágeno/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Manosidasas/metabolismo , Modelos Biológicos , Transporte de Proteínas , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los ResultadosRESUMEN
UNLABELLED: Polychromasia capsulare is a rare condition in which the anterior lens capsule exhibits an extraordinary array of colors during biomicroscopy that change with the incident angle of direct illumination consistent with iridescence. We present the case of a 59-year-old man with bilateral polychromasia capsulare who had successful cataract surgery. Routine light microscopy of the patient's capsulorhexis specimen was normal; however, transmission electron microscopy showed an unusual pattern of polygonal profiles with a periodicity estimated to be approximately 400 to 500 nm. This was not found in a control sample of normal lens capsule, which was relatively uniform in structure and significantly more osmiophilic. The iridescence in this patient's lens capsule is thought to be derived from a complex interplay between light and the abnormal nanoarchitecture of the lens capsule, producing an iridescent appearance. Thus, polychromasia capsulare is evidence of the phenomenon known as structural color in human biology. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.
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Cápsula Anterior del Cristalino , Capsulorrexis , Cápsula del Cristalino , Extracción de Catarata , Humanos , Iridiscencia , Implantación de Lentes Intraoculares , Masculino , Persona de Mediana EdadRESUMEN
The ability to deplete specific proteins from cells has transformed cell biology. Targeting of gene transcripts using RNA interference has allowed for a highly refined approach to the analysis of gene function that has been applied to all aspects of cell biology. Developments of the technology have reached a point where it is now a relatively trivial task to assess the role of an individual protein in a particular cell function. RNAi also allows for genome-wide screening as a discovery step toward the identification of new components of cellular pathways and machines. The technique has been applied extensively to the analysis of Golgi complex function, leading to significant insight into the biology of this complex organelle. Here, we describe the commonly used options for targeting individual genes for both transient and stable knockdown. We consider the alternative methods for introducing these reagents into cells and outline methods that we and others have used widely for validation of specificity and efficacy of gene targeting.
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Técnicas de Silenciamiento del Gen , Aparato de Golgi/fisiología , Animales , Técnicas de Cultivo de Célula , Galactosiltransferasas/genética , Galactosiltransferasas/metabolismo , Proteínas de la Matriz de Golgi , Células HEK293 , Humanos , Lentivirus/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas/genética , Proteínas/metabolismo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transfección , Tubulina (Proteína) , Cultivo de VirusRESUMEN
Protein sorting between eukaryotic compartments requires vesicular transport, wherein tethering provides the first contact between vesicle and target membranes. Here we map and start to functionally analyze the interaction network of the conserved oligomeric Golgi (COG) complex that mediates retrograde tethering at the Golgi. The interactions of COG subunits with members of transport factor families assign the individual subunits as specific interaction hubs. Functional analysis of selected interactions suggests a mechanistic tethering model. We find that the COG complex interacts with two different Rabs in addition to each end of the golgin "TATA element modulatory factor" (TMF). This allows COG to potentially bridge the distance between the distal end of the golgin and the target membrane thereby promoting tighter docking. Concurrently we show that the central portion of TMF can bind to Golgi membranes that are liberated of their COPI cover. This latter interaction could serve to bring vesicle and target membranes into close apposition prior to fusion. A target selection mechanism, in which a hetero-oligomeric tethering factor organizes Rabs and coiled transport factors to enable protein sorting specificity, could be applicable to vesicle targeting throughout eukaryotic cells.
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Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Proteínas de Unión al ADN/metabolismo , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Complejos Multiproteicos/metabolismo , Factores de Transcripción/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteína Coat de Complejo I/genética , Proteína Coat de Complejo I/metabolismo , Proteínas de Unión al ADN/genética , Aparato de Golgi/genética , Células HEK293 , Células HeLa , Humanos , Complejos Multiproteicos/genética , Unión Proteica , Transporte de Proteínas/fisiología , Factores de Transcripción/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The conserved oligomeric Golgi (COG) complex co-ordinates retrograde vesicle transport within the Golgi. These vesicles maintain the distribution of glycosylation enzymes between the Golgi's cisternae, and therefore COG is intimately involved in glycosylation homeostasis. Recent years have greatly enhanced our knowledge of COG's composition, protein interactions, cellular function and most recently also its structure. The emergence of COG-dependent human glycosylation disorders gives particular relevance to these advances. The structural data have firmly placed COG in the family of multi-subunit tethering complexes that it shares with the exocyst, Dsl1 and Golgi-associated retrograde protein (GARP) complexes. Here, we review our knowledge of COG's involvement in vesicle tethering at the Golgi. In particular, we consider what this knowledge may add to our molecular understanding of vesicle tethering and how it impacts on the fine tuning of Golgi function, most notably glycosylation.
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Aparato de Golgi/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Trastornos Congénitos de Glicosilación/etiología , Trastornos Congénitos de Glicosilación/metabolismo , Glicosilación , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
Microtubule nucleation by the γ-tubulin complex occurs primarily at centrosomes, but more diverse types of microtubule organizing centers (MTOCs) also exist, especially in differentiated cells. Mechanisms generating MTOC diversity are poorly understood. Fission yeast Schizosaccharomyces pombe has multiple types of cytoplasmic MTOCs, and these vary through the cell cycle. Cytoplasmic microtubule nucleation in fission yeast depends on a complex of proteins Mto1 and Mto2 (Mto1/2), which localizes to MTOCs and interacts with the γ-tubulin complex. Localization of Mto1 to prospective MTOC sites has been proposed as a key step in γ-tubulin complex recruitment and MTOC formation, but how Mto1 localizes to such sites has not been investigated. Here we identify a short conserved C-terminal sequence in Mto1, termed MASC, important for targeting Mto1 to multiple distinct MTOCs. Different subregions of MASC target Mto1 to different MTOCs, and multimerization of MASC is important for efficient targeting. Mto1 targeting to the cell equator during division depends on direct interaction with unconventional type II myosin Myp2. Targeting to the spindle pole body during mitosis depends on Sid4 and Cdc11, components of the septation initiation network (SIN), but not on other SIN components.
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Proteínas Portadoras/fisiología , Centro Organizador de los Microtúbulos/ultraestructura , Microtúbulos/ultraestructura , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/ultraestructura , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Secuencia Conservada , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Señales de Clasificación de Proteína , Transporte de Proteínas , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Huso Acromático/metabolismoRESUMEN
Cytoplasmic microtubule nucleation in the fission yeast Schizosaccharomyces pombe involves the interacting proteins Mto1 and Mto2, which are thought to recruit the gamma-tubulin complex (gamma-TuC) to prospective microtubule organizing centres. Mto1 contains a short amino-terminal region (CM1) that is conserved in higher eukaryotic proteins implicated in microtubule organization, centrosome function and/or brain development. Here we show that mutations in the Mto1 CM1 region generate mutant proteins that are functionally null for cytoplasmic microtubule nucleation and interaction with the gamma-TuC (phenocopying mto1Delta), even though the Mto1-mutant proteins localize normally in cells and can bind Mto2. Interestingly, the CM1 region is not sufficient for efficient interaction with the gamma-TuC. Mutation within a different region of Mto1, outside CM1, abrogates Mto2 binding and also impairs cytoplasmic microtubule nucleation and Mto1 association with the gamma-TuC. However, this mutation allows limited microtubule nucleation in vivo, phenocopying mto2Delta rather than mto1Delta. Further experiments suggest that Mto1 and Mto2 form a complex (Mto1/2 complex) independent of the gamma-TuC and that Mto1 and Mto2 can each associate with the gamma-TuC in the absence of the other, albeit extremely weakly compared to when both Mto1 and Mto2 are present. We propose that Mto2 acts cooperatively with Mto1 to promote association of the Mto1/2 complex with the gamma-TuC.