Habilidades prácticas en estudiantes de Enfermería Técnica de tercer año, Facultad Finlay-Albarrán, 2021 / Practical skills in third-year Technical Nursing students, Finlay-Albarrán Faculty, 2021 / Competências práticas em estudantes de enfermagem técnica do terceiro ano, Finlay-Albarrán Faculty, 2021

Introducción: El proceso de formación de habilidades es superior a la simple asimilación del contenido, ya que las habilidades resultan indispensables en el aprendizaje, requiriéndose que la enseñanza sea activa a través de diferentes etapas interrelacionadas, pero con cierta flexibilidad en su aplicación, según su complejidad. Objetivo: Diagnosticar el estado de desarrollo de las habilidades prácticas en estudiantes de 3° año (9° grado) de la carrera de Enfermería Técnica Facultad Finlay-Albarrán marzo-mayo 2021. Metodología: El estudio corresponde a un diseño descriptivo de corte transversal en la Facultad Finlay-Albarrán con estudiantes de 3° año de la carrera de Enfermería Técnica durante el período marzo-mayo 2021.El universo estuvo representado por los estudiantes que iniciaron en la rotación de Enfermería Ginecobstétrica (N=28). Para el estudio se combinaron métodos teóricos, empíricos y estadísticos. Resultados: Los alumnos del 3° año de Enfermería, reconocen como habilidades a desarrollar la medición de signos vitales para un 57.14%, el examen físico general, para un 67.85%, la cura de herida quirúrgica para un 53.57% y la evolución de Enfermería para un 82.1%, no consideran el Proceso de Atención de Enfermería, o sea la aplicación de este que es el método científico de la profesión, como un saber esencial en la práctica de Enfermería. Conclusiones: Se diagnosticó el estado de las habilidades prácticas en estudiantes de 3° año de Enfermería.

Introduction: The skills training process is superior to the simple assimilation of the content, since the skills are essential in learning, requiring that the teaching be active through different interrelated stages, but with some flexibility in its application, according to its complexity. Objective: To diagnose the state of development of practical skills in third year students (ninth grade) of the Finlay-Albarrán Faculty of Technical Nursing career, March-May 2021. Methodology: The study corresponds to a descriptive cross-sectional design in the Finlay-Albarrán Faculty with third year students of the Technical Nursing career during the period March-May 2021.The universe was represented by the students who started in the Gyneco-Obstetric Nursing rotation (N = 28) .For the study, theoretical, empirical, and statistical methods were combined. Results: The 3rd year Nursing students recognize as skills to develop the measurement of vital signs for 57.14%, the general physical examination, for a 67.85%, surgical wound healing for 53.57% and Nursing evolution for 82.1%, do not consider the Nursing Care Process, that is, the application of this, which is the scientific method of the profession, as essential knowledge in Nursing practice. Conclusions: The state of practical skills in third year Nursing students was diagnosed.

Introdução: O processo de formação de competências é superior à simples assimilação do conteúdo, uma vez que as competências são essenciais na aprendizagem, exigindo que o ensino seja ativo em diferentes etapas inter-relacionadas, mas com alguma flexibilidade na sua aplicação, de acordo com a sua complexidade. Objetivo: Diagnosticar o estado de desenvolvimento de habilidades práticas em alunos do 3º ano (9º ano) da carreira da Faculdade de Enfermagem Técnica Finlay-Albarrán, março-maio de 2021. Metodologia: O estudo corresponde a um desenho transversal descritivo no Finlay-Albarrán Docente com alunos do 3º ano da carreira de Técnico de Enfermagem durante o período março-maio de 2021. O universo foi representado pelos alunos que iniciaram no rodízio de Enfermagem Gineco-Obstétrica (N = 28). Para o estudo foram combinados métodos teóricos, empíricos e estatísticos. Resultados: Os alunos do 3º ano de Enfermagem reconhecem como habilidades para desenvolver a mensuração dos sinais vitais para 57,14%, o exame físico geral, para 67,85%, a cicatrização de feridas cirúrgicas para 53,57% e a evolução da Enfermagem para 82,1%, não consideram o Processo de Assistência de Enfermagem, ou seja, a aplicação deste, que é o método científico da profissão, como conhecimento essencial na prática da Enfermagem. Conclusões: Foi diagnosticado o estado das habilidades práticas em alunos do 3º ano de Enfermagem.

Identificación de competencias específicas de enfermería para el cuidado del recién nacido en recuperación nutricional, Hospital "Eusebio Hernández Pérez": 2020 / Identification of specific competences for nursing for the care of the newborn in nutritional recovery, hospital 'Eusebio Hernández Pérez': 2020 / Identificação de competências específicas para a enfermagem para o cuidado do recém-nascido na recuperação nutricional, hospital "Eusbio Hernández Pérez": 2020

Introducción: En la investigación desarrollada en el servicio de neonatología del hospital Docente Ginecobstétrico "Eusebio Hernández", se pudo constatar que para el personal de enfermería el cuidado del recién nacido que se encuentra en recuperación nutricional es un elemento importante para el progreso de la atención del neonato en las salas de cuidados intensivos neonatales. Objetivo: Identificar las competencias específicas de enfermería para el cuidado del recién nacido en recuperación nutricional. Métodos: Investigación de desarrollo tecnológico en el Hospital Ginecobstétrico Eusebio Hernández en La Habana durante 2020. El universo lo constituyeron 50 profesionales de enfermería que laboran en el servicio de neonatología. Para el desarrollo de la investigación se utilizó el método DACUM (desarrollo del currículum laboral), se confeccionó las funciones y tareas, con la participación de expertos, se construyó el mapa DACUM. Resultados: Se identificaron 6 competencias específicas en relación con 6 funciones y sus tareas en lo que la totalidad de los expertos estuvieron de acuerdo por tener un alto nivel científico. Conclusiones: Se identificaron las competencias específicas de enfermería para el cuidado de neo-natos en recuperación nutricional, lo que permitió mejorar la calidad de la atención a estos recién nacidos y el perfeccionamiento de los profesionales.

Introduction: In the research carried out in the neonatology service of the Eusebio Hernández Gyneco-Obstetric Teaching Hospital, it was found that for the nursing staff the care of the newborn who is in nutritional recovery is an important element for the progress of care of the neonate in neonatal intensive care wards. Objective: To identify the specific nursing competencies for the care of the newborn in nutritional recovery. Methods: Technological development research at the Eusebio Hernández Gyneco-Obstetric Hospital in Havana during 2020. The universe was made up of 50 nursing professionals working in the neonatology service. For the development of the research, the DACUM method (development of the work curriculum) was used, the functions and tasks were prepared, with the participation of experts, the DACUM map was constructed. Results: 6 specific competences were identified in relation to 6 functions and their tasks, in which all the experts agreed due to having a high scientific level. Conclusions: Specific nursing competencies for the care of neonates in nutritional recovery were identified, which allowed improving the quality of care for these newborns and the improvement of professionals.

Introdução: Na pesquisa realizada no serviço de neonatologia do Hospital Universitário Gineco-Obstétrico "Eusebio Hernández", constatou-se que para a equipe de enfermagem o cuidado ao recém-nascido em recuperação nutricional é um elemento importante para o andamento da assistência do recém-nascido em enfermarias de terapia intensiva neonatal. Objetivo: Identificar as competências específicas de enfermagem para o cuidado ao recém-nascido em recuperação nutricional. Métodos: Pesquisa de desenvolvimento tecnológico no Hospital "Eusebio Hernández" Gyneco-Obstétrico de Havana em 2020. O universo era formado por 50 profissionais de enfermagem que atuavam no serviço de neonatologia. Para o desenvolvimento da pesquisa, foi utilizado o método DACUM (elaboração do currículo do trabalho), foram elaboradas as funções e tarefas, com a participação de especialistas, foi construído o mapa DACUM. Resultados: identificaram-se 6 competências específicas em relação a 6 funções e respetivas tarefas, em que todos os peritos concordaram por possuírem um elevado nível científico. Conclusões: Foram identificadas competências específicas de enfermagem para o cuidado ao recém-nascido em recuperação nutricional, o que permitiu melhorar a qualidade da assistência a esses recém-nascidos e o aprimoramento dos profissionais.

Competencias específicas del enfermero(a) que labora en salón de parto. Hospital Ginecobstétrico ̈Eusebio Hernández Pérez ̈. 2018 / Specific Competences of the Nurse Who Works in the Delivery Room. Gynecobstetric Hospital ̈Eusebio Hernández Pérez ̈. 2018 / Competências específicas do enfermeiro que trabalha na sala de parto.Hospital Ginecobstétrico ̈Eusebio Hernández Pérez ̈. 2018

En la investigación desarrollada en el salón de Partos del hospital Docente "Eusebio Hernández", se pudo constatar que para el personal de enfermería el cuidado de la mujer en trabajo de parto es un elemento importante para el progreso de la perinatología. El presente trabajo es una investiga-ción de desarrollo tecnológico con el objetivo de identifi car las competencias específi cas del enfer-mero que labora en el salón de partos. Se trabajó con: análisis documental, encuestas a expertos y entrevistas a profesionales de enfermería que laboran en el salón de partos. Se elaboró, a partir de la identifi cación de competencias específi cas para el personal de enfermería que labora en el salón de parto, un mapa para el desarrollo del currículo laboral (mapa DACUM) donde se incluyen las tareas y funciones que debe realizar el personal de enfermería

In the investigation developed in the living room of Childbirths of the Educational hospital Eu-sebio Hernández Pérez, could ascertain that for the personnel of infi rmary the care of the woman in work of childbirth. " e present work is an investigation of technological development with the aim to identify thespecifi c competitions of infi rmary. We worked with: documentary analysis, expert surveys and interviews with nursing professionals who work in the delivery room. It was elaborated, from the identifi cation of specifi c competencies for the nursing staff that works in the delivery room, a map for the development of the labor curriculum (DACUM map) which includes the tasks and func-tions that the nursing staff must perform

Na pesquisa realizada na sala de parto do Hospital Universitário "Eusebio Hernández", consta-tou-se que para a equipe de enfermagem o cuidado da mulher em trabalho de parto é um elemento importante para o progresso da perinatologia. O presente trabalho é uma pesquisa de desenvol-vimento tecnológico com o objetivo de identifi car as competências específi cas do enfermeiro que trabalha na sala de parto. Trabalhamos com: análise documental, pesquisas de especialistas e en-trevistas com profi ssionais de enfermagem que atuam na sala de parto. Foi elaborado, a partir da identifi cação de competências específi cas para a equipe de enfermagem que trabalha na sala de parto, um mapa para o desenvolvimento do currículo de trabalho (mapa DACUM) que inclui as tarefas e funções que a equipe de enfermagem deve realizar.

Caracterización clínica, microbiológica y epidemiológica en neonatos con infecciones relacionadas con la atención sanitaria / Clinical, microbiological and epidemiological characterization of neonates with health care-related infections

INTRODUCCIÓN: las infecciones relacionadas con la atención sanitaria representan un problema importante en la etapa neonatal por el compromiso con la mortalidad. OBJETIVO: caracterizar clínica, microbiológica y epidemiológicamente a los neonatos con infecciones relacionadas con la atención sanitaria. MÉTODOS: estudio descriptivo, transversal, retrospectivo, realizado en el Hospital "Eusebio Hernández Pérez" (2007-2014), en 151 neonatos, con análisis de variables epidemiológicas, clínicas y microbiológicas. Se estimaron: tasa de infección, de mortalidad e índice de letalidad, y porcentaje de resistencia antimicrobiana. RESULTADOS: la tasa de infección fue de 3,76 × 100 egresados, la de mortalidad de 1,96 × 100 egresados, y el índice de letalidad de 6,9 × 100 pacientes infectados. Como factores intrínsecos predisponentes se citan la prematuridad (83,1 %) y el bajo peso al nacer (85,6 %); y como extrínsecos, el catéter percutáneo (86,1 %) y la infección sistémica en 50 % de los pacientes. Los microorganismos mayormente aislados fueron: Estafilococo coagulasa negativo (25,3 %), Klebsiella spp. (16,9 %), Candida spp. (13,3 %) y Enterococcus faecalis (10,9 %), que se distribuyen diferentes significativamente (p= 0,000). La resistencia del Estafilococo coagulasa negativo se manifestó en las penicilinas (78,6 %), la eritromicina (63,0 %) y el cefoxitín (35,7 %). CONCLUSIONES: los factores de riesgo conocidos predominantes son la prematuridad, el bajo peso al nacer y el cateterismo percutáneo. Las principales características clínicas y microbiológicas son: la infección sistémica, la identificación de Estafilococo coagulasa negativo resistente a penicilinas, eritromicina y cefoxitìn; y bacilos gramnegativos resistentes a las cefalosporinas y la gentamicina.

INTRODUCTION: health care-related infections represent an important problem in the neonatal phase because of its association with mortality. OBJECTIVE: to characterize neonates with health-care related infections from the clinical, microbiological and epidemiological viewpoints. METHODS: retrospective, cross-sectional and descriptive study of 151 newborns conducted in ¨Eusebio Hernández Pérez¨ hospital from 2007 to 2014, with the analysis of epidemiological, clinical and microbiological variables. The infection rate, the mortality rate and the fatality index, and the antimicrobial resistance percentages were all estimated. RESULTS: the infection rate was 3.76 x 100 discharges; the mortality rate was 1.96 x 100 discharges and the fatality index reached 6.9 x 100 infected patients. Predisposing intrinsic factors were prematurity (83.1 %) and low birthweight (85.6 %) and the extrinsic ones were percutaneous catheter (86.1 %) and systemic infection (50 %) of the patients. The most isolated microorganisms included negative staphylococcus coagulasa (25.3 %), Klebsiella spp. (16.9), Candida spp. (13.3 %) and Enterococcus faecalis (10.9 %), with a significantly different distribution. It was observed that negative Staphylococcus coagulasa was resistant to penicillin (78.6 %), erythromycin (63 %) and cefoxitin (35.7 %). CONCLUSIONS: the predominant risk factors are prematurity, low birth weight and percutaneous catheterism. The main clinical and microbiological characteristics include systemic infections, detection of penicillin, erythromycin and cefoxitin-resistant negative Staphylococcus coagulase and cephalosporin-and gentamycin-resistant Gram-negative bacilli.

p38(MAPK)-regulated induction of p62 and NBR1 after photodynamic therapy promotes autophagic clearance of ubiquitin aggregates and reduces reactive oxygen species levels by supporting Nrf2-antioxidant signaling.

Emerging evidence indicates that oxidative stress instigates the formation of ubiquitin (Ub) aggregates, substrates of autophagy, through a process requiring the ubiquitin binding adaptors p62/SQSTM1 and NBR1. Here, we have investigated the role of p62 and NBR1 in cell survival after hypericin-mediated photodynamic therapy (Hyp-PDT), a procedure known to incite robust reactive oxygen species (ROS)-based endoplasmic reticulum stress and autophagy pathways. We found that Hyp-PDT stimulated the formation of p62- and NBR1-associated Ub aggregates in normal and cancer cells, which were ultimately removed by autophagy, through a mechanism partially regulated by p38(MAPK). In line with this, genetic or pharmacological p38(MAPK) inhibition reduced p62 and NBR1 levels and aggregate formation and impaired Nrf2 activation, thus increasing photo-oxidative stress and cell death. p62-deficient cells, or cells lacking p62 and with reduced levels of NBR1 (through siRNA knockdown), also displayed reduced aggregate formation but exhibited attenuated ROS levels, reduced caspase activation, and improved survival after Hyp-PDT. The increased resistance to photo-oxidative stress exhibited by cells lacking p62 and/or NBR1 was overruled by the inhibition of p38(MAPK), which restored cytotoxic ROS levels, thus indicating the relevance of this signal in the control of cell viability. Taken together these findings provide evidence that in photodynamically treated cells a p38(MAPK)-regulated pathway coordinates the p62/NBR1-mediated clearance of cytosolic aggregates and mitigates PDT-induced proteotoxicity. They also reveal that a functional p38(MAPK)-Nrf2 signal is required to keep ROS levels in check and protect against PDT-induced proteotoxicity, independent of aggregate formation.

Pro-apoptotic signaling induced by photo-oxidative ER stress is amplified by Noxa, not Bim.

Pro-apoptotic signaling instigated by endoplasmic reticulum (ER) stress is tightly governed by the BH3-only proteins like Noxa and Bim, which help trigger apoptosis, in part by inactivating mitochondria protecting proteins like Mcl-1. Bim/Noxa-based pro-apoptotic signaling has been implicated for various ER stressors but not yet for those causing "ER-focused" production of severe oxidative stress. In the present study we found that photo-oxidative (phox)-ER stress induced by hypericin-based photodynamic therapy is associated with activation of PERK (an ER sessile, stress sensor), robust induction of CHOP (a pro-apoptotic transcription factor) and induction of Bim and Noxa (accompanied by an eventual drop in Mcl-1 levels). Interestingly Noxa, but not Bim, contributed toward phox-ER stress induced apoptosis, regulated by PERK in a CHOP-independent, temporally-defined manner. These observations shed further light on complex signaling pathways elicited byphox-ER stress and vouch for directing more investigation toward the role of PERK in cell death governance.

Mitochondria are targets for peroxisome-derived oxidative stress in cultured mammalian cells.

Many cellular processes are driven by spatially and temporally regulated redox-dependent signaling events. Although mounting evidence indicates that organelles such as the endoplasmic reticulum and mitochondria can function as signaling platforms for oxidative stress-regulated pathways, little is known about the role of peroxisomes in these processes. In this study, we employ targeted variants of the genetically encoded photosensitizer KillerRed to gain a better insight into the interplay between peroxisomes and cellular oxidative stress. We show that the phototoxic effects of peroxisomal KillerRed induce mitochondria-mediated cell death and that this process can be counteracted by targeted overexpression of a select set of antioxidant enzymes, including peroxisomal glutathione S-transferase kappa 1, superoxide dismutase 1, and mitochondrial catalase. We also present evidence that peroxisomal disease cell lines deficient in plasmalogen biosynthesis or peroxisome assembly are more sensitive to KillerRed-induced oxidative stress than control cells. Collectively, these findings confirm and extend previous observations suggesting that disturbances in peroxisomal redox control and metabolism can sensitize cells to oxidative stress. In addition, they lend strong support to the ideas that peroxisomes and mitochondria share a redox-sensitive relationship and that the redox communication between these organelles is not only mediated by diffusion of reactive oxygen species from one compartment to the other. Finally, these findings indicate that mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress, and this may have profound implications for our views on cellular aging and age-related diseases.

Autophagy: shaping the tumor microenvironment and therapeutic response.

Autophagy, the major lysosomal pathway for recycling intracellular components including whole organelles, is emerging as a key process modulating tumorigenesis, tumor-stroma interactions, and cancer therapy. Research over the past decade has highlighted a context-dependent and dynamic role for autophagy in cancer: it is tumor suppressive in the early stages of cancer development, but fuels the growth of established tumors. Likewise, the stimulation of autophagy in response to therapeutics can contextually favor or weaken chemoresistance and antitumor immunity. From a therapeutic perspective, understanding whether, when, and how autophagy can be harnessed to kill cancer cells remains challenging. In this review, we discuss new connections that reveal the role of autophagy in shaping tumor-stroma interaction during carcinogenesis and in the context of anticancer treatments.

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species.

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy. Consistent with a primary ER photodamage, kinetics and colocalization studies revealed that photogenerated ROS induced an initial reticulophagy, followed by morphological changes in the mitochondrial network that preceded clearance of mitochondria by mitophagy. Overexpression of cytosolic and mitochondria-associated GPX4 retained the tubular mitochondrial network in response to PD stress and concomitantly blocked the progression toward mitophagy. Preventing the formation of phospholipid hydroperoxides and H(2)O(2) in the cytosol as well as in the mitochondria significantly reduced cardiolipin peroxidation and apoptosis. All together, these results show that in response to apical ER photodamage ROS propagate to mitochondria, which in turn amplify ROS production, thereby contributing to two antagonizing processes, mitophagy and apoptosis.

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death.

Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80(high), CD83(high), CD86(high), MHC-II(high)) and functional stimulation (NO(high), IL-10(absent), IL-1ß(high)) of dendritic cells as well as induction of a protective antitumour immune response. Intriguingly, early after PDT the cancer cells displayed ecto-CRT and secreted ATP before exhibiting biochemical signatures of apoptosis, through overlapping PERK-orchestrated pathways that require a functional secretory pathway and phosphoinositide 3-kinase (PI3K)-mediated plasma membrane/extracellular trafficking. Interestingly, eIF2α phosphorylation and caspase-8 signalling are dispensable for this ecto-CRT exposure. We also identified LRP1/CD91 as the surface docking site for ecto-CRT and found that depletion of PERK, PI3K p110α and LRP1 but not caspase-8 reduced the immunogenicity of the cancer cells. These results unravel a novel PERK-dependent subroutine for the early and simultaneous emission of two critical DAMPs following ROS-mediated ER stress.

5-ALA-PDT induces RIP3-dependent necrosis in glioblastoma.

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are resistant to all current therapies and are associated with a high rate of recurrence. Glioblastoma were previously shown to respond to treatments by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) mainly by activating a necrotic type of cell death. The receptor-interacting protein 3 (RIP3) has recently been outlined as a key mediator of this caspase-independent form of programmed cell death. In the present study, we analyzed the necrotic mechanism induced by 5-ALA-PDT in human glioblastoma cells and explored the role of RIP3 in this context. Our results show that PDT-induced necrosis is dependent on RIP3, which forms aggregates and colocalizes with RIP1 following photosensitization. We demonstrate that PDT-mediated singlet oxygen production is the cause of RIP3-dependent necrotic pathway activation. We also prove that PDT induces the formation of a pro-necrotic complex containing RIP3 and RIP1 but lacking caspase-8 and FADD, two proteins usually part of the necrosome when TNF-α is used as a stimulus. Thus, we hypothesize that PDT might lead to the formation of a different necrosome whose components, besides RIP1 and RIP3, are still unknown. In most cases, glioblastoma are characterized by a constitutive activation of NF-κB. This factor is a key regulator of various processes, such as inflammation, immune response, cell growth or apoptosis. Its inhibition was shown to further sensitize glioblastoma cells to PDT-induced necrosis, however, no difference in RIP3 upshift or aggregation could be observed when NF-κB was inhibited.

NF-kappaB inhibition improves the sensitivity of human glioblastoma cells to 5-aminolevulinic acid-based photodynamic therapy.

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-κB). This factor is known to be a key regulator of various physiological processes such as inflammation, immune response, cell growth or apoptosis. In the present study, we explored the role of NF-κB activation in the sensitivity of human glioblastoma cells to a treatment by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT). 5-ALA is a physiological compound widely used in PDT as well as in tumor photodetection (PDD). Our results show that inhibition of NF-κB improves glioblastoma cell death in response to 5-ALA-PDT. We then studied the molecular mechanisms underlying the cell death induced by PDT combined or not with NF-κB inhibition. We found that apoptosis was induced by PDT but in an incomplete manner and that, unexpectedly, NF-κB inhibition reduced its level. Oppositely PDT mainly induces necrosis in glioblastoma cells and NF-κB is found to have anti-necrotic functions in this context. The autophagic flux was also enhanced as a result of 5-ALA-PDT and we demonstrate that stimulation of autophagy acts as a pro-survival mechanism confering protection against PDT-mediated necrosis. These data point out that 5-ALA-PDT has an interesting potential as a mean to treat glioblastoma and that inhibition of NF-κB renders glioblastoma cells more sensitive to the treatment.

Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.

Reactive oxygen species (ROS) concurrently instigate apoptosis and autophagy pathways, but the link between these processes remains unclear. Because cytotoxic ROS formation is exploited in anticancer therapy, such as in photodynamic therapy (PDT), a better understanding of the complex interplay between autophagy and apoptosis is urgently required. Previously, we reported that ROS generated by PDT with an endoplasmic reticulum (ER)-associated sensitizer leads to loss of ER-Ca(2+) homeostasis, ER stress and apoptosis. Here we show that PDT prompted Akt-mTOR (mammalian target of rapamycin) pathway down-regulation and stimulated macroautophagy (MA) in cancer and normal cells. Overexpression of the antioxidant enzyme glutathione peroxidase-4 reversed mTOR down-regulation and blocked MA progression and apoptosis. Attenuating MA using Atg5 knockdown or 3-methyladenine, reduced clearance of oxidatively damaged proteins and increased apoptosis, thus revealing a cytoprotective role of MA in PDT. Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling. We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg(-/-) cells compensated for MA loss and increased cellular resistance to PDT. CMA-deficient cells were significantly sensitized to photokilling but were protected against the ER stressor thapsigargin. These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.

Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy.

Dyes that bind to DNA, such as Hoechst 33342, are commonly used to visualize chromatin in live cells by fluorescence microscopy. A caveat is that the probes themselves should not perturb cellular responses and under normal conditions the dyes are generally non-toxic. However, researchers are increasingly using computerized time-lapse microscopy (CTLM), where cells stained with fluorescent dyes are often imaged frequently over a period of several days, to follow cellular responses in real time. Little is currently known about possible toxicity of fluorescent DNA dyes under CTLM conditions. In this study we demonstrate that the common live-cell DNA stain Hoechst 33342 can cause apoptosis under CTLM conditions. Although toxicity is evident at long times in the absence of imaging at high dye concentrations, phototoxicity from repeated excitation of the dye in the imaging process is dominant. We show that phototoxicity is a function of the product of light fluence and dye concentration, irrespective of irradiance, frequency and total number of scans. Thus, phototoxicity can be prevented by a combination of dye concentration and imaging procedure that is below this threshold. These quantitative data can be used as a guide to others performing time-lapse microscopy studies with this common live-cell DNA stain and serves as a caution for researchers when using other fluorescent stains under CTLM conditions.

A p38(MAPK)/HIF-1 pathway initiated by UVB irradiation is required to induce Noxa and apoptosis of human keratinocytes.

The signal transduction pathways leading to apoptosis of human keratinocytes responding to UVB irradiation are complex and not completely understood. Previously, we reported that in UVB-irradiated keratinocytes, p38(MAPK) instigates Bcl-2-associated X protein (Bax) activation and mitochondrial apoptosis. However, the molecular mechanism underlying the pro-apoptotic function of p38(MAPK) remained unclear. Here, we show that in UVB-treated human primary keratinocytes the activation of p38(MAPK) is necessary to upregulate Noxa, a BH3-only pro-apoptotic dominantly induced by UVB and required for apoptosis. Whereas p53-silencing was marginally cytoprotective and poorly affected Noxa expression, p38(MAPK) inhibition in p53-silenced keratinocytes or in p53(-/-) cells could still efficiently prevent Noxa induction and intrinsic apoptosis after UVB, indicating that p38(MAPK) signals mainly through p53-independent mechanisms. Furthermore, p38(MAPK) was required for the induction and activation of hypoxia-inducible factor 1 (HIF-1) in response to UVB, and HIF-1 knockdown reduced Noxa expression and apoptosis. In UVB-irradiated keratinocytes, Noxa targeted the anti-apoptotic myeloid cell leukemia sequence 1 (Mcl-1) for degradation, and small-interfering RNA (siRNA)-mediated knockdown of Noxa or p38(MAPK) inhibition restored levels of Mcl-1 and abolished apoptosis. Thus, the pro-apoptotic mechanisms orchestrated by p38(MAPK) in human keratinocytes in response to UVB involve an HIF-1/Noxa axis, which prompts the downregulation of anti-apoptotic Mcl-1, thereby favoring Bax-mediated mitochondrial apoptosis of UVB-damaged keratinocytes.

Real-time imaging of novel spatial and temporal responses to photodynamic stress.

Cells subjected to various forms of stress have been shown to induce bystander responses in nontargeted cells, thus extending the stress response to a larger population. However, the mechanism(s) of bystander responses remains to be clearly identified, particularly for photodynamic stress. Oxidative stress and cell viability were studied on the spatial and temporal levels after photodynamic targeting of a subpopulation of EMT6 murine mammary cancer cells in a multiwell plate by computerized time-lapse fluorescence microscopy. In the targeted population a dose-dependent loss of cell viability was observed in accordance with increased oxidative stress. This was accompanied by increased oxidative stress in bystander populations but on different time scales, reaching a maximum more rapidly in targeted cells. Treatment with extracellular catalase, or the NADPH oxidase inhibitor diphenyleneiodinium, decreased production of reactive oxygen species (ROS) in both populations. These effects are ascribed to photodynamic activation of NADPH-oxidase in the targeted cells, resulting in a rapid burst of ROS formation with hydrogen peroxide acting as the signaling molecule responsible for initiation of these photodynamic bystander responses. The consequences of increased oxidative stress in bystander cells should be considered in the overall framework of photodynamic stress.

Spatial and temporal dynamics of in vitro photodynamic cell killing: extracellular hydrogen peroxide mediates neighbouring cell death.

Photodynamic killing of a cell population is generally considered to result from direct effects that occur in each cell. In some scenarios this may be an over-simplification and the potential for cell-cell signaling processes to contribute to the response of a population to photodynamic stress is addressed in this paper. Photodynamic killing of EMT6 cells in culture was studied in time and space using computerized time-lapse microscopy. The rate of cell killing was dependent on the fluence with both rapid and slower processes evident, the proportion of the former increasing with fluence. The spatial distribution of cell death was non-random and for the slow cell killing process was found to occur preferentially in the vicinity of dead or dying cells, suggesting a local signaling process. An inhibitory effect of extracellular catalase indicated the involvement of hydrogen peroxide in the spread of cell death and NADPH oxidase was determined as the principal source of hydrogen peroxide. This cell signaling pathway was observed for membrane-bound and mitochondrial photosensitizers but not for a nuclear photosensitizer. These secondary cell signalling pathways extend the oxidative damage to cells in space and time.

Photochemical production and characterisation of the radical ions of tetraphenylporphycenes.

Both the radical anion and radical cation of 2,7,12,17-tetraphenylporphycene and of its palladium(II) complex have been produced by photochemical methods. Their electronic absorption spectrum and decay kinetics have been characterised by global analysis of the time-absorbance-wavelength matrix. The results provide deeper insight on the role of these radical ion species in the photodynamic activity of tetraphenylporphycenes.

Effect of aza substitution on the photophysical and electrochemical properties of porphycenes: Characterization of the near-IR-absorbing photosensitizers 2,7,12,17-tetrakis(p-substituted phenyl)-3,6,13,16-tetraazaporphycenes.

The need for new photodynamic-therapy photosensitizers has stimulated the search of new families of compounds absorbing strongly in the 700-900 nm range, the region where tissue is most transparent to radiation capable to induce the photodynamic effect. Using computational chemistry techniques, 3,6,13,16-tetraazaporphycenes were previously identified as interesting target candidates. This work reports on the photophysical and electrochemical properties of selected members of this new family of macrocycles. Compared to porphycenes, the tetra-aza counterparts show stronger absorption in the near-infrared, lower-lying singlet and triplet excited states, and substantially larger internal conversion quantum yield (Phi(IC) = 0.93). Energy transfer to oxygen is observed, which results in the formation of the cytotoxic species singlet oxygen. The process is found to be reversible, consistent with a triplet-energy value close to that of singlet oxygen.