High-Throughput Quantification of GFP-LC3+ Dots by Automated Fluorescence Microscopy.

Macroautophagy is a specific variant of autophagy that involves a dedicated double-membraned organelle commonly known as autophagosome. Various methods have been developed to quantify the size of the autophagosomal compartment, which is an indirect indicator of macroautophagic responses, based on the peculiar ability of microtubule-associated protein 1 light chain 3 beta (MAP1LC3B; best known as LC3) to accumulate in forming autophagosomes upon maturation. One particularly convenient method to monitor the accumulation of mature LC3 within autophagosomes relies on a green fluorescent protein (GFP)-tagged variant of this protein and fluorescence microscopy. In physiological conditions, cells transfected temporarily or stably with a GFP-LC3-encoding construct exhibit a diffuse green fluorescence over the cytoplasm and nucleus. Conversely, in response to macroautophagy-promoting stimuli, the GFP-LC3 signal becomes punctate and often (but not always) predominantly cytoplasmic. The accumulation of GFP-LC3 in cytoplasmic dots, however, also ensues the blockage of any of the steps that ensure the degradation of mature autophagosomes, calling for the implementation of strategies that accurately discriminate between an increase in autophagic flux and an arrest in autophagic degradation. Various cell lines have been engineered to stably express GFP-LC3, which-combined with the appropriate controls of flux, high-throughput imaging stations, and automated image analysis-offer a relatively straightforward tool to screen large chemical or biological libraries for inducers or inhibitors of autophagy. Here, we describe a simple and robust method for the high-throughput quantification of GFP-LC3+ dots by automated fluorescence microscopy.

Assessment of Glycolytic Flux and Mitochondrial Respiration in the Course of Autophagic Responses.

Autophagy is an evolutionarily conserved process that mediates prominent homeostatic functions, both at the cellular and organismal level. Indeed, baseline autophagy not only ensures the disposal of cytoplasmic entities that may become cytotoxic upon accumulation, but also contributes to the maintenance of metabolic fitness in physiological conditions. Likewise, autophagy plays a fundamental role in the cellular and organismal adaptation to homeostatic perturbations of metabolic, physical, or chemical nature. Thus, the molecular machinery for autophagy is functionally regulated by a broad panel of sensors that detect indicators of metabolic homeostasis. Moreover, increases in autophagic flux have a direct impact on core metabolic circuitries including (but not limited to) glycolysis and mitochondrial respiration. Here, we detail a simple methodological approach to monitor these two processes in cultured cancer cells that mount a proficient autophagic response to stress.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Spermidine induces autophagy by inhibiting the acetyltransferase EP300.

Several natural compounds found in health-related food items can inhibit acetyltransferases as they induce autophagy. Here we show that this applies to anacardic acid, curcumin, garcinol and spermidine, all of which reduce the acetylation level of cultured human cells as they induce signs of increased autophagic flux (such as the formation of green fluorescent protein-microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta and the depletion of sequestosome-1, p62/SQSTM1) coupled to the inhibition of the mammalian target of rapamycin complex 1 (mTORC1). We performed a screen to identify the acetyltransferases whose depletion would activate autophagy and simultaneously inhibit mTORC1. The knockdown of only two acetyltransferases (among 43 candidates) had such effects: EP300 (E1A-binding protein p300), which is a lysine acetyltranferase, and NAA20 (N(α)-acetyltransferase 20, also known as NAT5), which catalyzes the N-terminal acetylation of methionine residues. Subsequent studies validated the capacity of a pharmacological EP300 inhibitor, C646, to induce autophagy in both normal and enucleated cells (cytoplasts), underscoring the capacity of EP300 to repress autophagy by cytoplasmic (non-nuclear) effects. Notably, anacardic acid, curcumin, garcinol and spermidine all inhibited the acetyltransferase activity of recombinant EP300 protein in vitro. Altogether, these results support the idea that EP300 acts as an endogenous repressor of autophagy and that potent autophagy inducers including spermidine de facto act as EP300 inhibitors.

[Cerebellopontine angle meningeal melanocytoma: a benign tumor?]. / Melanocitoma meníngeo del ángulo pontocerebeloso: un tumor benigno?

We report a case of a rare meningeal melanocytoma in the cerebellopontine angle. One year after tumor gross total removal, the patient suffered a sudden and devastating meningeal melanomatosis. The relevant literature is reviewed looking for the keys to establish preoperative diagnosis and to obtain information about its treatment and postsurgical management.

Melanocitoma meníngeo del ángulo pontocerebeloso, ¿Un tumor benigno? / Cerebellopontine angle meningeal melanocytoma: a benign tumor?

Se presenta un paciente con un raro melanocitoma meníngeo del ángulo pontocerebeloso que, tras su extirpación quirúrgica radical, evolucionó en el plazo de un año hacia una melanomatosis meníngea fulminante. Se realiza una revisión bibliográfica en busca de las claves para hacer una aproximación diagnóstica preoperatoria de este tipo de tumor y obtener información sobre su tratamiento y manejo postoperatorio (AU)

We report a case of a rare meningeal melanocytoma in the cerebellopontine angle. One year after tumor gross total removal, the patient suffered a sudden and devastating meningeal melanomatosis. The relevant literature is reviewed looking for the keys to establish preoperative diagnosis and to obtain information about its treatment and postsurgical management (AU)

Cavernomas in children with brain tumors: a late complication of radiotherapy.

BACKGROUND: Delayed complications of radiation therapy comprise cerebral atrophy, radionecrosis and induction of tumors. Recent reports indicate the possibility of the "de-novo" formation of intracranial cavernomas in patients submitted to radiation therapy to the brain. OBJECTIVES: To report three children, two with medulloblastomas and one with a pineal germinoma, treated with radiotherapy that developed intracerebral cavernous hemangiomas some years after treatment. With this work, we aim to draw attention to this occurrence in the neurosurgical community. RESULTS: The patients were two girls and one boy with ages comprised between 2.5 and 7 years (mean 5.2 years). The average interval from irradiation to the appearance of cavernoma was of 5.3 years (range 5-6 years). The lesions were found during the routine neuroimaging studies performed for the follow-up of their primary neoplasms. No patient showed signs or symptoms related to the cavernomas. However, the three children will need both clinical and neuroimaging surveillance to monitor the evolution of these incidentally discovered lesions. CONCLUSIONS: Intracranial cavernomas can occur years after cerebral radiation therapy. In spite of previous reports that show a high incidence of bleeding lesions, cavernomas may be found incidentally during the neuroimaging surveillance studies that are performed to children with brain tumors previously treated with radiotherapy. In these cases, a conservative attitude seems to be advisable, reserving surgery only for those lesions that grow or bleed.

Cavernomas in children with brain tumors: a late complication of radiotherapy / Cavernomas en niños con tumores cerebrales: una complicación ardía de la radioterapia

Antecedentes. Entre las complicaciones tardías dela radioterapia se encuentran la atrofia cerebral, la radio necrosis y el desarrollo de tumores radio inducidos. Se ha descrito recientemente la formación de cavernomas intracraneales en pacientes tratados con radioterapia cerebral. Objetivo. Presentar los casos de tres niños, dos con meduloblastomas y uno con un germinoma pineal, en los que se encontraron cavernomas cerebrales años después del tratamiento con radioterapia, a fin de contribuir al conocimiento de esta eventualidad en el medio neuroquirúrgico. Resultados. Los pacientes fueron dos niñas y un niño entre 2,5 y 7 años de edad (media 5,2 años). El intervalo medio entre la radioterapia y la aparición del cavernoma fue de 5,3 años (extremos 5 y 6 años). Las lesiones fueron encontradas en los estudios de neuroimagen que se realizan habitualmente a estos pacientes para seguimiento de sus tumores originarios. Durante el período de observación, ningún paciente presentó síntomas o signos relacionados con el cavernoma. No obstante, los tres niños requerirán un seguimiento clínico y de neuroimagen para vigilar la evolución clínica de estas lesiones descubiertas accidentalmente. Conclusiones. La presencia de cavernomas intracraneales puede detectarse años después del tratamiento con radioterapia. Al contrario de casos previos publicados en los que fue frecuente su presentación con hemorragia, los cavernomas también pueden ser detectados de manera casual en los estudios de seguimiento que se realizan habitualmente a niños con tumores cerebrales tratados previamente con radioterapia. En este caso, parece aconsejable mantener una actitud conservadora, reservando la cirugía para las lesiones que crecen o sangran

Background. Delayed complications of radiation therapy comprise cerebral atrophy, radionecrosis and induction of tumors. Recent reports indicate the possibility of the “de-novo” formation of intracranial cavernomas in patients submitted to radiation therapy to the brain. Objectives. To report three children, two with medullo blastomas and one with a pineal germinoma, treated with radiotherapy that developed intracerebral cavernous hemangiomas some years after treatment. With this work, we aim to draw attention to this occurrence in the neurosurgical community. Results. The patients were two girls and one boy with ages comprised between 2.5 and 7 years (mean5.2 years). The average interval from irradiation to the appearance of cavernoma was of 5.3 years (range5-6 years). The lesions were found during the routine neuroimaging studies performed for the follow-up of their primary neoplasms. No patient showed signs or symptoms related to the cavernomas. However, the three children will need both clinical and neuroimaging surveillance to monitor the evolution of these incidentally discovered lesions. Conclusions. Intracranial cavernomas can occur years after cerebral radiation therapy. In spite of previous reports that show a high incidence of bleeding lesions, cavernomas may be found incidentally during the neuroimaging surveillance studies that are performed to children with brain tumors previously treated with radiotherapy. In these cases, a conservative attitude seems to be advisable, reserving surgery only for those lesions that grow or bleed

[Spinal cord tethering in myelomeningocele and lipomeningocele patients: the second operation]. / Reanclaje medular en pacientes con mielomeningocele y lipomeningocele: la segunda operación.

BACKGROUND: Spinal cord rethetering can occur after the primary surgical repair of myelomeningoceles (MMC) and lipomeningoceles (LMC) and produce devastating physical and psychological consequences. The inadvertent introduction of skin elements at the time of the initial surgery can lead to the growth of intraspinal epidermoid or dermoid cysts. OBJECTIVES: To review the incidence of spinal cord tethering following surgery for open and occult spinal dysraphism and to analyze factors that might influence the appearance of this complication. We also aimed to search technical measures at the time of the primary operation that might prevent the occurrence of symptomatic cord retethering. MATERIAL AND METHODS: We reviewed the medical records of patients submitted to surgical repair of MMC (n=162) or occult spinal dysraphism (n= 54) during the period 1975-2005 who developed symptomatic tethered cord syndrome. RESULTS: Eleven of 162 (6.79%) patients with MMC and 2 of 54 (3.7%) with LMC developed clinical symptoms and signs of spinal cord tethering after intervals ranging from 2 to 37 years after the initial surgical repair of their back lesions. Indications for surgical re-exploration were based mainly on clinical grounds. Postoperative fibrosis was a constant finding in all instances. Other surgical findings included inclusion tumors of cutaneous origin (n=3), lumbar canal stenosis (n= 2), foreign body reactions (n= 2), residual lipoma (n= 1), and a tight hyalinized filum (n=1). Interestingly, 3 of 162 (or 1.85%) myelomeningoceles were found to harbor an intradural epidermoid tumor at the time of spinal cord dethetering, accounting for an incidence of cutaneous inclusion tumors of 27% in cases of post- MMC repair tethering. After a mean follow-up time of 5.5 years, eight patients were improved, two were unchanged and one was worsened. CONCLUSIONS: Neurological deterioration is not a necessary consequence of the natural history of patients with MMC or LMC. Early or late clinical deterioration can be due to spinal cord re-tethering and deserves timely investigation and surgical exploration. Results of surgical intervention were rewarding as 92% of the patients showed improvement or stabilization in their otherwise deteriorating condition. We also report two infrequent causes of spinal cord deterioration: lumbar canal stenosis and intense foreign-body reactions to implanted materials.

Reanclaje medular en pacientes con mielomeningocele y lipomeningocele: la segunda operación / Spinal cord tethering in myelomeningocele and lipomeningocele patients: the second operation

Antecedentes. La médula espinal puede fijarse tras la reparación inicial en pacientes operados demielomeningocele (MMC) y lipomeningocele (LMC), produciendo graves lesiones físicas y psicológicas. Asimismo, la introducción accidental de restos cutáneos durante la reparación de estas lesiones puede dar lugar al desarrollo de tumores intraespinales de estirpe cutánea. Objetivos. Averiguar la incidencia del anclaje medular tras la cirugía de MMC y LMC y analizar los factores que puedan explicar su aparición. También, investigamos las maniobras técnicas durante la operación primaria susceptibles de evitar la aparición del síndrome de médula fijada. Pacientes y métodos. Revisión retrospectiva de las historias de los pacientes operados de MMC (n=162) y de espina bífida oculta (n=54) en el período 1975-2005 que desarrollaron cuadros de anclaje medular sintomático. Resultados. Once pacientes con MMC (6,79%) y dos con LMC (3,7%) presentaron manifestaciones de anclaje medular tras intervalos de 2 a 37 años después de la reparación primaria. Las indicaciones de reintervención se basaron fundamentalmente en criterios clínicos. Un hallazgo constante fue la fibrosiscicatricial que estuvo presente en todos los casos. Otros hallazgos operatorios causantes de la fijación medular consistieron en tumores cutáneos de inclusión (n=3),reacciones de cuerpo extraño (n=2), estenosis del canal lumbar (n=2), restos de lipoma (n=1), y filumhialinizado (n=1). En 3 casos de MMC se encontróademás un quiste epidermoide intradural (1,85% delos MMC), lo que supone una tasa de epidermoides en la reintervención de MMC de 27%. El periodo medio de seguimiento fue de 5,5 años y los resultados fueron: mejoría en 8, dos no experimentaron cambios, y uno sufrió empeoramiento. Conclusiones. El deterioro neurológico de los pacientes operados de MMC o LMC no constituye una consecuencia obligada o parte de la historia natural de estos procesos. El deterioro, precoz o tardío, puede estar motivado por el anclaje posquirúrgico de la médula espinal. Ello hace necesario realizar un seguimiento periódico de estos pacientes, acompañado de los oportunos estudios de neuroimagen y, en su caso, de exploración quirúrgica. Los resultados fueron satisfactorios, ya que el 92% de los pacientes reoperados experimentaron mejoría o estabilización de su enfermedad. Se describen además dos causas infrecuentemente descritas de deterioro tardío: la estenosis del canal y las reacciones fibrosas de cuerpo extraño a materiales implantados

Background. Spinal cord rethetering can occur after the primary surgical repair of myelomeningoceles (MMC) and lipomeningoceles (LMC) and produced evastating physical and psychological consequences.The inadvertent introduction of skin elements at the time of the initial surgery can lead to the growth of intraspinal epidermoid or dermoid cysts. Objectives. To review the incidence of spinal cord tethering following surgery for open and occult spinaldys raphism and to analyze factors that might influence the appearance of this complication. We also aimed to search technical measures at the time of the primary operation that might prevent the occurrence of symptomatic cord retethering. Material and methods. We reviewed the medical records of patients submitted to surgical repair of MMC (n=162) or occult spinal dysraphism (n= 54) during the period 1975-2005 who developed symptomatic tethered cord syndrome. Results. Eleven of 162 (6.79%) patients with MMCand 2 of 54 (3.7%) with LMC developed clinical symptoms and signs of spinal cord tethering after intervals ranging from 2 to 37 years after the initial surgical repair of their back lesions. Indications for surgical re-exploration were based mainly on clinical grounds. Postoperative fibrosis was a constant finding in all instances. Other surgical findings included inclusion tumors of cutaneous origin (n=3), lumbar canal stenosis(n= 2), foreign body reactions (n= 2), residual lipoma (n= 1), and a tight hyalinized filum (n=1). Interestingly,3 of 162 (or 1.85%) myelomeningoceles were found to harbor an intradural epidermoid tumor at the time of spinal cord dethetering, accounting for an incidence of cutaneous inclusion tumors of 27% in cases of post-MMC repair tethering. After a mean follow-up time of 5.5 years, eight patients were improved, two were unchanged and one was worsened. Conclusions. Neurological deterioration is not a necessary consequence of the natural history of patients with MMC or LMC. Early or late clinical deterioration can be due to spinal cord re-tethering and deserves timely investigation and surgical exploration. Results of surgical intervention were rewarding as 92% of the patients showed improvement or stabilization in their otherwise deteriorating condition. We also report two infrequent causes of spinal cord deterioration: lumbar canal stenosis and intense foreign-body reactions to implanted materials

Regression of syringomyelia and tonsillar herniation after posterior fossa arachnoid cyst excision. Case report and literature review.

BACKGROUND: Some reports have documented posterior fossa cysts resulting in syringomyelic obstruction of cerebrospinal fluid (CSF) flow caused by cyst displacement within the foramen magnum. Rarely the syringomyelia is caused by acquired Chiari malformation due to a retrocerebellar arachnoid cyst. OBJECTIVE: To report the case of a 38-year-old man with hydrocephalus and syringomyelia, who was found to have a Chiari malformation secondary to a posterior fossa arachnoid cyst. After endoscopic third ventriculostomy, the patient was submitted to foramen magnum decompression and arachnoid cyst removal that were followed by resolution of both the Chiari malformation and the syringomyelia. DISCUSSION: In most published cases the syringomyelia has been attributed to obstruction of CSF flow at the foramen magnum by the arachnoid cyst itself. There is only one previous report of a posterior fossa arachnoid cyst producing tonsillar descent and syringomyelia. CONCLUSIONS: Posterior fossa arachnoid cysts can result in acquired Chiari malformation and syringomyelia. In our view, the management of these patients should be directed at decompressing the foramen magnum and include the removal of the walls of the coexistent arachnoid cyst as it seems to be the crucial factor that accounts for the development of the syringomyelia that these patients present.

Regression of syringomyelia and tonsillar herniation after posterior fossa arachnoid cyst excision. Case report and literature review

Antecedentes. Algunos trabajos han descrito laexistencia de quistes aracnoideos de fosa posterior queproducían siringomielia al obstruir la circulación deliquido cefalorraquídeo (LCR) por bloqueo del agujeromagno ocasionado por el propio quiste. Rara vezla siringomielia asociada a quiste aracnoideo de fosaposterior es producida por malformación de Chiariadquirida.Objetivos. Publicar el caso de un hombre de 38años con hidrocefalia y siringomielia, en el que seencontró descenso de las amígdalas cerebelosas por lapresión ejercida por un quiste aracnoideo retrocerebeloso.Tras tratar primero la hidrocefalia medianteventriculostomía neuroendoscópica, el paciente fueoperado de descompresión del agujero magno yextirpación de las membranas del quiste que condujerona la resolución tanto del Chiari como de lasiringomielia.Discusión. En la mayoría de los casos publicados lasiringomielia se debía a la obstrucción de LCR en el agujeromagno ocasionada por el propio quiste aracnoideo.Hemos encontrado solamente un caso previo en el queel quiste de fosa posterior era causante del descenso delas amígdalas cerebelosas y éste, a su vez, producía lasiringomielia.Conclusiones. Los quistes aracnoideos de fosa posteriorpueden producir descenso de las amígdalas ysiringomielia. En nuestra opinión, el manejo de estospacientes debe de encaminarse no sólo a descomprimirel agujero magno, sino también a comunicar las paredesdel quiste ya que éste parece ser el principal responsabledel complejo cuadro de siringomielia en estos casos

Background. Some reports have documented posteriorfossa cysts resulting in syringomyelic obstruction ofcerebrospinal fluid (CSF) flow caused by cyst displacementwithin the foramen magnum. Rarely the syringomyeliais caused by acquired Chiari malformationdue to a retrocerebellar arachnoid cyst.Objective. To report the case of a 38-year-old manwith hydrocephalus and syringomyelia, who was foundto have a Chiari malformation secondary to a posteriorfossa arachnoid cyst. After endoscopic third ventriculostomy,the patient was submitted to foramen magnumdecompression and arachnoid cyst removal that werefollowed by resolution of both the Chiari malformationand the syringomyelia.Discussion. In most published cases the syringomyeliahas been attributed to obstruction of CSF flow at theforamen magnum by the arachnoid cyst itself. There isonly one previous report of a posterior fossa arachnoidcyst producing tonsillar descent and syringomyelia.Conclusions. Posterior fossa arachnoid cysts canresult in acquired Chiari malformation and syringomyelia.In our view, the management of these patientsshould be directed at decompressing the foramenmagnum and include the removal of the walls of thecoexistent arachnoid cyst as it seems to be the crucialfactor that accounts for the development of the syringomyeliathat these patients present

"Benign" shaken baby syndrome. Case report.

The authors report an infant with clinical and neuroimaging findings of shaken baby syndrome. The pitfalls encountered in the assessment on the cause of the bilateral frontal and interhemispheric subdural hematomas in this child are also briefly discussed. We have called this condition "benign" shaken baby syndrome and emphasize that not always acute subdural hematomas are of non-accidental nature.

"Benign" shaken baby syndrome. Case report

The authors report an infant with clinical and neuroimaging findings of shaken baby syndrome. The pitfalls encountered in the assessment on the cause of the bilateral frontal and interhemispheric subdural hematomas in this child are also briefly discussed. We have called this condition "benign" shaken baby syndrome and emphasize that not always acute subdural hematomas are of non-accidental nature

Los autores describen un lactante con hallazgos clínicos y de neuroimagen sugestivos del síndrome del niño maltratado. Se discuten brevemente las dificultades para la identificación de la causa del hematomasubdural agudo frontal bilateral e interhemisférico que el niño presentaba. Hemos denominado a esta entidad síndrome "benigno" del niño maltratado para destacar que el hallazgo de un hematoma subdural agudo en un niño pequeño no significa necesariamente maltrato infantil

Heterogeneity of vitamin D receptor gene association with celiac disease and type 1 diabetes mellitus.

OBJECTIVE: Vitamin D has been shown to exert multiple immunomodulatory effects and is known to suppress T-cell activation by binding to the vitamin D receptor (VDR). To determine whether VDR gene polymorphisms are related to the susceptibility to celiac disease, we investigated its implication as a candidate gene in the Basque population. Because celiac disease and type 1 diabetes share common susceptibility loci, we also analyzed families with type 1 diabetes mellitus. METHODS: A total of 37 families with celiac disease and 64 type 1 diabetic families of Basque origin with at least one affected offspring were genotyped for four VDR restriction-site polymorphisms (Fok I, Bsm I, Apa I and Taq I). The AFBAC approach was used to test for association. RESULTS: Comparison of VDR genotypes of the patients with those of 88 healthy individuals identified "ff" as a risk genotype for celiac disease [p = 0.01; OR = 3.45 (1.12-10.79)]. On the other hand, a significantly higher frequency of haplotype "fBAt" was observed in the type 1 diabetic group [p(c) = 0.02; OR = 4.4 (1.5-15.3)]. CONCLUSION: Our findings suggest that polymorphisms within the vitamin D receptor gene are markers of susceptibility to or protection from autoimmune diseases, although, at least in the Basque population, association of VDR variants with celiac disease and type 1 diabetes seems to be heterogeneous.