End-of-life care needs in cancer patients: a qualitative study of patient and family experiences.

BACKGROUND: Cancer is a disease that transcends what is purely medical, profoundly affecting the day-to-day life of both patients and family members. Previous research has shown that the consequences of cancer are greatly aggravated in patients at the end of life, at a time when they must also grapple with numerous unmet needs. The main objective of this study was to obtain more in-depth insight into these needs, primarily in patients with end-stage cancer nearing death. METHODS: Semi-structured interviews were conducted in Spain with cancer patients at the end of life (n = 3) and their family members (n = 12). The findings from the interviews were analyzed using qualitative thematic analysis and a grounded theory approach. RESULTS: Four major themes emerged from the interviews that explored the needs and concerns of patients with cancer at the end of life: (1) physical well-being (2) emotional well-being (3) social well-being and (4), needs relating to information and autonomous decision-making. The interviews also shed light on the specific needs of family members during this period, namely the difficulties of managing increased caregiver burden and maintaining a healthy work-life balance. CONCLUSIONS: A lack of support, information and transparency during a period of immense vulnerability makes the end-of-life experience even more difficult for patients with cancer. Our findings highlight the importance of developing a more in-depth understanding of the needs of this population, so that informed efforts can be made to improve palliative healthcare and implement more comprehensive care and support at the end of life.

Use of wood and cork in biofilters for the simultaneous removal of nitrates and pesticides from groundwater.

About 13% and 7% of monitored groundwater stations in Europe exceed the permitted levels of nitrates (50 mg NO3- L-1) or pesticides (0.1 µg L-1), respectively. Although slow sand filtration can remove nitrates via denitrification when oxygen is limited, it requires an organic carbon source. The present study evaluates the performance of the use of wood pellets and granulated cork as carbon sources in bench-scale biofilters operated under water-saturated and water-unsaturated conditions for more than 400 days. The biofilters were monitored for nitrate (200 mg L-1) and pesticide (mecoprop, diuron, atrazine, and bromacil, each at a concentration of 5 µg L-1) attenuation, as well as for the formation of nitrite and pesticide transformation products. Microbiological characterization of each biofilter was also performed. The water-saturated wood biofilter achieved the best nitrate removal (>99%), while the cork biofilters lost all denitrification power over time (from 38% to no removal). The unsaturated biofilter columns were not effective for removing nitrates (20-30% removal). As for pesticides, all the biofilters achieved high removal rates of mecoprop and diuron (>99% and >75%, respectively). Atrazine removal was better in the wood-pellet biofilters than the cork ones (68-96% vs. 31-38%). Bromacil was only removed in the water-unsaturated cork biofilter (67%). However, a bromacil transformation product was formed there. The water-saturated wood biofilter contained the highest number of denitrifying microorganisms, with Methyloversatilis as the characteristic genus. Microbial composition could explain the high removal of pesticides and nitrates achieved in the wood-pellet biofilter. Overall, the results indicate that wood-pellet biofilters operated under water-saturated conditions are a good solution for treating groundwater contaminated with nitrates and pesticides.

Iron-induced cytotoxicity mediated by endolysosomal TRPML1 channels is reverted by TFEB.

Increased brain iron content has been consistently reported in sporadic Parkinson's disease (PD) patients, and an increase in cytosolic free iron is known to cause oxidative stress and cell death. However, whether iron also accumulates in susceptible brain areas in humans or in mouse models of familial PD remains unknown. In addition, whilst the lysosome functions as a critical intracellular iron storage organelle, little is known about the mechanisms underlying lysosomal iron release and how this process is influenced by lysosome biogenesis and/or lysosomal exocytosis. Here, we report an increase in brain iron content also in PD patients due to the common G2019S-LRRK2 mutation as compared to healthy age-matched controls, whilst differences in iron content are not observed in G2019S-LRRK2 knockin as compared to control mice. Chemically triggering iron overload in cultured cells causes cytotoxicity via the endolysosomal release of iron which is mediated by TRPML1. TFEB expression reverts the iron overload-associated cytotoxicity by causing lysosomal exocytosis, which is dependent on a TRPML1-mediated increase in cytosolic calcium levels. Therefore, approaches aimed at increasing TFEB levels, or pharmacological TRPML1 activation in conjunction with iron chelation may prove beneficial against cell death associated with iron overload conditions such as those associated with PD.

Evaluation of Current Methods to Detect Cellular Leucine-Rich Repeat Kinase 2 (LRRK2) Kinase Activity.

BACKGROUND: Coding variation in the Leucine rich repeat kinase 2 gene linked to Parkinson's disease (PD) promotes enhanced activity of the encoded LRRK2 kinase, particularly with respect to autophosphorylation at S1292 and/or phosphorylation of the heterologous substrate RAB10. OBJECTIVE: To determine the inter-laboratory reliability of measurements of cellular LRRK2 kinase activity in the context of wildtype or mutant LRRK2 expression using published protocols. METHODS: Benchmark western blot assessments of phospho-LRRK2 and phospho-RAB10 were performed in parallel with in situ immunological approaches in HEK293T, mouse embryonic fibroblasts, and lymphoblastoid cell lines. Rat brain tissue, with or without adenovirus-mediated LRRK2 expression, and human brain tissues from subjects with or without PD, were also evaluated for LRRK2 kinase activity markers. RESULTS: Western blots were able to detect extracted LRRK2 activity in cells and tissue with pS1292-LRRK2 or pT73-RAB10 antibodies. However, while LRRK2 kinase signal could be detected at the cellular level with over-expressed mutant LRRK2 in cell lines, we were unable to demonstrate specific detection of endogenous cellular LRRK2 activity in cell culture models or tissues that we evaluated. CONCLUSION: Further development of reliable methods that can be deployed in multiple laboratories to measure endogenous LRRK2 activities are likely required, especially at cellular resolution.

Assessment of a novel microalgae-cork based technology for removing antibiotics, pesticides and nitrates from groundwater.

Groundwater pollution has increased in recent years due to the intensification of agricultural and livestock activities. This results in a significant reduction in available freshwater resources. Here, we have studied the long term assessment of a green technology (1-4 L/day) based on a photobioreactor (PBR) containing immobilised microalgae-bacteria in polyurethane foam (PF) followed by a cork filter (CF) for removing nitrates, pesticides (atrazine and bromacil), and antibiotics (sulfamethoxazole and sulfacetamide) from groundwater. The prototype was moderately effective for removing nitrates (58%) at an HRT of 8 days, while its efficiency decreased at a HRT of 4 and 2 days (<20% removal). The combined use of PBR-CF enabled antibiotics and pesticides to be attenuated by up to 95% at an HRT of 8 days, but their attenuation decreased with shorter HRT, with pesticides being the compounds most affected (reducing from 97 to 98% at an HRT of 8 days to 23-45% at an HRT of 2 days). Pesticide transformation products were identified after the CF, supporting biodegradation as the main attenuation process. A gene-based metataxonomic assessment linked the attenuation of micropollutants to the presence of specific pesticide biodegradation species (e.g. genus Phenylobacterium, Sphingomonadaceae, and Caulobacteraceae). Therefore, the results highlighted the potential use of microalgae and cork to treat polluted groundwater.

Anti-cancer and anti-inflammatory activities of a new family of coordination compounds based on divalent transition metal ions and indazole-3-carboxylic acid.

A new family of mononuclear coordination compounds has been synthetized and characterized: [M(3-ind)2(H2O)2] (M = Co (1), Ni (2), Zn (3), Fe (4), Mn (5); 3-ind = indazole-3-carboxylate). These materials are mononuclear coordination compounds that possess strong hydrogen bond interactions. The anti-inflammatory effects of these compounds were assayed in lipopolysaccharide activated RAW 264.7 macrophages by inhibition of NO production. Moreover, the cytotoxicity of the complexes and the ligand in RAW 264.7 cells were determined for the first time. The most significant results were obtained for the compounds 4 and 5 reaching values of NO inhibition close to 80% at 48 h, and above to 90% at 72 h of treatment. The highest inhibitory effects on NO production were showed at the range 7-23 µg/mL for compounds 4 and 5. As a consequence, compounds 4 and 5 could be potential drugs due to the interesting anti-inflammatory properties showed. The anti-cancer potential of these compounds has been also tested against different tumor cell lines. The cytotoxicity of the ligand and of compounds 2 and 3 were assayed in three cell lines: HT29, colon cancer cells, Hep-G2, hepatoma cells and B16-F10 melanoma cells. The best results have been achieved with compound 2 in HepG2 and B16-F10 cell lines, being between 1.5 and 2 times more effective that the ligand in HepG2 cells, and B16-F10 cells. All in all, indazole-3-carboxylic acid is a promising ligand for the formation of coordination compounds with biochemical properties.

Etapas de cambio en alimentos con etiquetado frontal de advertencia / Stages of change in foods with front warning labeling

Los datos en Chile demuestran el efecto que causa la mala alimentación y sus consecuencias. Considerando los problemas alimentarios, el 26 de junio de 2016, la Ley 20.606, entró en vigencia. Determinar el estado de cambio en la intención de compra de alimentos con sello de advertencia en la tercera etapa de la ley. Estudio descriptivo transversal en población chilena, de ambos sexos, entre 18 y 59 años de edad, en usuarios frecuentes de internet. Los datos se obtuvieron a través de una encuesta creada y validada mediante el método de Lawshe, la cual contiene un total de 45 preguntas, en las que se plantea, ¿qué nivel de confianza siente el participante para realizar un cambio? y ¿Cuán importante es para el realizar ese cambio?. Se analizaron 545 encuestas a nivel nacional, el 59,8% corresponde a mujeres, del total de personas el 52,1% es mayor de 30 años de edad. Respecto a la compra de alimentos con sello según sexo, se observa que hay diferencias significativas en la compra de galletas, papas fritas y bebidas azucaradas, en todos los casos los hombres presentan una mayor resistencia al cambio que las mujeres. Pese a que todos los alimentos se encuentran en contemplación, hay diferencias significativas en el puntaje en galletas y bebidas azucaradas y papas fritas en los hombres, respecto a la edad, hay diferencias en todos los alimentos. Al comparar por estado nutricional se observan diferencias significativas en bebidas, galletas, papas fritas y chocolates, en obesidad. Los hombres presentan una mayor resistencia al cambio, por otro lado, se observa que la intención de compra de todos los alimentos se encuentra en contemplación sin embargo hay diferencias en todos los alimentos.

Data in Chile demonstrate the effect caused by poor diet and its consequences. Considering food problems, on June 26, 2016, Law 20,606 came into force. To determine the status of change in the intention to purchase food with a warning labels in the third stage of the law. Cross-sectional descriptive study in a Chilean population, of both sexes, between 18 and 59 years of age, in frequent Internet users. The data were obtained through a survey created and validated using the Lawshe method, which contains a total of 45 questions, in which it is asked, what level of confidence does the participant feel to make a change? And how important is it for making that change?. 545 surveys were analyzed at the national level, 59.8% correspond to women, of the total number of people 52.1% are over 30 years of age. Regarding the purchase of food stamped by sex, it is observed that there are significant differences in the purchase of cookies, French fries and sugary drinks, in all cases men present a greater resistance to change than women. Although all foods are in contemplation, there are significant differences in the score in cookies and sugary drinks and French fries in men, with respect to age, there are differences in all foods. When comparing by nutritional status, significant differences are observed in beverages, cookies, French fries and chocolates, in obesity. Men present greater resistance to change, on the other hand, it is observed that the intention to purchase all foods is in contemplation, however there are differences in all foods.

Antiparasitic, anti-inflammatory and cytotoxic activities of 2D coordination polymers based on 1H-indazole-5-carboxylic acid.

We report on the formation of two novel multifunctional isomorphous (4,4) square-grid 2D coordination polymers based on 1H-indazole-5-carboxylic acid. To the best of our knowledge, these complexes are the first examples of 2D-coordination polymers constructed with this novel ligand. We have analysed in detail the structural, magnetic and anti-parasitic properties of the resulting materials. In addition, the capability of inhibiting nitric oxide production from macrophage cells has been measured and was used as an indirect measure of the anti-inflammatory response. Finally, the photocatalytic activity was measured with a model pollutant, i.e. vanillic acid (phenolic compound), with the aim of further increasing the functionalities and applicability of the compounds.

5-Aminopyridine-2-carboxylic acid as appropriate ligand for constructing coordination polymers with luminescence, slow magnetic relaxation and anti-cancer properties.

Five new coordination polymers (CPs) constructed of aminopyridine-2-carboxylate (ampy) ligand have been synthesized and fully characterized. Three of them correspond to metal-organic chains built from the coordination of ampy to sodium and lanthanides with formulae [MNa(ampy)4]n (M = terbium (2), erbium (1) and ytterbium (3)) resembling a previously reported dysprosium material which shows anticancer activity. On another level, the reaction of Hampy with cobalt and copper ions ({[CoK(ampy)3(H2O)3](H2O)3}n (4) and [Cu(ampy)2]n (5)) lead to CPs with variable dimensionalities, which gives the opportunity of analyzing the structural properties of this new family. Lanthanide materials display solid state intense photoluminescent emissions in both the visible and near-infrared region and exhibit slow relaxation of magnetization with frequency dependence of the out-of-phase susceptibility. More interestingly, in our search for multifunctional materials, we have carried out antitumor measurements of these compounds. These multidisciplinary studies of metal complexes open up the possibility for further exploring the applications in the fields of metal-based drugs.

Centrosomal cohesion deficits as cellular biomarker in lymphoblastoid cell lines from LRRK2 Parkinson's disease patients.

Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson's disease (PD), and orally bioavailable, brain penetrant and highly potent LRRK2 kinase inhibitors are in early stages of clinical testing. Detection of LRRK2 phosphorylation, as well as phosphorylation of Rab10, a LRRK2 kinase substrate, have been proposed as target engagement biomarkers for LRRK2 inhibitor clinical trials. However, these readouts do not seem able to stratify patients based on enhanced LRRK2 kinase activity. Here, we describe a robust cell biological assay based on centrosomal cohesion alterations which were observed in peripheral blood mononuclear cell-derived lymphoblastoid cell lines (LCLs) from patients with G2019S LRRK2 mutations as compared with healthy controls, and could also be detected in a subset of sporadic PD patient samples. We suggest that LCLs may be a valuable resource for LRRK2 research, and that determination of centrosomal cohesion deficits may assist in the stratification of a subset of sporadic PD patients.

Giant cranionasal epithelioid haemangioendothelioma with invasive growth pattern mimicking a skull base chondrosarcoma.

Epithelioid haemangioendothelioma (EHE) is a rare low-grade vascular neoplasm that is composed of mostly epithelioid cells. EHE may arise as a solitary tumour or in the form of multiple body lesions, and commonly occurs in soft tissues, liver, pleura, lung, peritoneum, lymph nodes, breast, and many other sites. EHE in the cranionasal region is extremely rare. There are very few reports of cases of skull-base EHE. We discuss an extremely rare presentation of an aggressive EHE that originated from the sellar region. Based on literature review, our patient is the first reported case of a giant solitary EHE with prepontine cistern invasion and abducens nerve encroachment mimicking a chondrosarcoma. We treated this rare tumour by near subtotal surgical excision with subsequent radiotherapy, considering that complete tumour resection with free margins in both cavernous sinus and clival region avoiding neural and vascular structure encroachment becomes technically difficult.

Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation.

BACKGROUND: Mutations in LRRK2 are a common genetic cause of Parkinson's disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive. METHODS: Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2. RESULTS: Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a. CONCLUSIONS: Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.

Impact on dose and image quality of a software-based scatter correction in mammography.

Background In 2014, Siemens developed a new software-based scatter correction (Progressive Reconstruction Intelligently Minimizing Exposure [PRIME]), enabling grid-less digital mammography. Purpose To compare doses and image quality between PRIME (grid-less) and standard (with anti-scatter grid) modes. Material and Methods Contrast-to-noise ratio (CNR) was measured for various polymethylmethacrylate (PMMA) thicknesses and dose values provided by the mammograph were recorded. CDMAM phantom images were acquired for various PMMA thicknesses and inverse Image Quality Figure (IQFinv) was calculated. Values of incident entrance surface air kerma (ESAK) and average glandular dose (AGD) were obtained from the DICOM header for a total of 1088 pairs of clinical cases. Two experienced radiologists compared subjectively the image quality of a total of 149 pairs of clinical cases. Results CNR values were higher and doses were lower in PRIME mode for all thicknesses. IQFinv values in PRIME mode were lower for all thicknesses except for 40 mm of PMMA equivalent, in which IQFinv was slightly greater in PRIME mode. A mean reduction of 10% in ESAK and 12% in AGD in PRIME mode with respect to standard mode was obtained. The clinical image quality in PRIME and standard acquisitions resulted to be similar in most of the cases (84% for the first radiologist and 67% for the second one). Conclusion The use of PRIME software reduces, in average, the dose of radiation to the breast without affecting image quality. This reduction is greater for thinner and denser breasts.

SDHC Promoter Methylation, a Novel Pathogenic Mechanism in Parasympathetic Paragangliomas.

Context: Germline mutations in the succinate dehydrogenase A, B, C, and D genes (collectively, SDHx) predispose to the development of paragangliomas (PGLs) arising at the parasympathetic or sympathetic neuroendocrine systems. SDHx mutations cause absence of tumoral immunostaining for SDHB. However, negative SDHB immunostaining has also been found in a subset of PGLs that lack SDHx mutations. Settings: Here, we report the comprehensive molecular characterization of one such a tumor of parasympathetic origin compared with healthy paraganglia and other PGLs with or without SDHx mutations. Results: Integration of multiplatform data revealed somatic SDHC methylation and loss of the 1q23.3 region containing the SDHC gene. This correlated with decreased SDHC messenger RNA (mRNA) and protein levels. Furthermore, another genetic event found affected the VHL gene, which showed a decreased DNA copy number, associated with low VHL mRNA levels, and an absence of VHL protein detected by immunohistochemistry. In addition, the tumor displayed a pseudohypoxic phenotype consisting in overexpression of the hypoxia-inducible factor (HIF)-1α and miR-210, as well as downregulation of the iron-sulfur cluster assembly enzyme (ISCU) involved in SDHB maturation. This profile resembles that of SDHx- or VHL-mutated PGLs but not of PGLs with decreased VHL copy number, pointing to SDHC rather than VHL as the pathogenic driver. Conclusions: Collectively, these findings demonstrate the potential importance of both the SDHC epigenomic event and the activation of the HIF-1α/miR-210/ISCU axis in the pathogenesis of SDHx wild-type/SDHB-negative PGLs. To our knowledge, this is the first case of a sporadic parasympathetic PGL that carries silencing of SDHC, fulfilling the two-hit Knudson's model for tumorigenesis.

Biomonitorization of iron accumulation in the substantia nigra from Lewy body disease patients.

Iron levels in the healthy human brain are known to be high in certain areas such as the substantia nigra (SN), and increase further with age. In addition, there is some evidence for a further increase in iron load in the SN of Parkinson's disease (PD) patients as compared to controls, which correlates with motor disability. Here, we have analyzed total iron levels in cells as well as mouse and human brain samples by atomic absorption spectroscopy (AAS). Our data indicate that iron load is more pronounced in cells with dopaminergic features. Moreover, region-specific differences in iron load reflecting those in the human brain were detected in rodent brains as well. Whilst altered iron load was not observed in other regions also affected in PD patients, we report a significant increase in iron load in the SN of Lewy body disease patients as compared to Alzheimer's disease (AD) patients or controls, which correlates with neurodegeneration in this brain area.

Oral anticoagulation in patients with atrial fibrillation and medical non-neoplastic disease in a terminal stage.

Many patients with non-neoplastic disease develop atrial fibrillation in advanced stages of their disease. The aim of this study is to determine the factors associated with the use of oral anticoagulants in patients with atrial fibrillation and non-neoplastic medical disease in a terminal stage, and whether their use is associated with a longer survival. Design is prospective, observational, multicentre study. Patients with atrial fibrillation and non-neoplastic disease (severe not reversible organ insufficiency) in a terminal stage were included between February 2009 and September 2010. A 6-month follow-up was carried out. We included 314 patients with a mean (SD) age of 82.6 (7.0) years. Their mean (SD) scores in CHADS2 and ATRIA scales were 3.4 (1.2) and 4.7 (2.0), respectively. Anticoagulants were prescribed to 112 (37.5 %) patients. The use of anticoagulants was associated with age (OR 0.96 95 % CI 0.93-0.99, p = 0.046) and to the Barthel index (OR 1.01 95 % CI 1.00-1.02; p = 0.034). After performing a propensity score matching analysis, 262 patients were included in the survival analysis. After 6 months, 133 (50.8 %) patients were dead. The mortality is higher among patients who are not treated with oral anticoagulants (57.1 vs. 39.4 %; p = 0.006), but it is independently associated only with the Barthel index score (HR 0.99 95 % CI 0.98-1.00; p = 0.039), delirium (HR 1.60, 95 % CI 1.08-2.36; p = 0.018), anorexia (HR 1.58 95 % CI 1.05-2.38; p = 0.027), and with the use of calcium channel blockers (HR 0.50 95 % CI 0.30-0.84; p = 0.009). In patients with atrial fibrillation and non-neoplastic disease in a terminal stage, the use of oral anticoagulants is not independently associated with a higher probability of survival.

Una experiencia preclínica de enseñanza y aprendizaje en una carrera de especialización en endodoncia / A pre-clinical teaching and learning experiencein a specialization program in endodontics

Objetivo: desarrollar una estrategia para la enseñanza yel aprendizaje, que pueda derivar en una nueva metodología deevaluación individual y grupal de los procedimientos pre clínicosen la Carrera de Especialización en Endodoncia de la Universidad del Salvador / Asociación Odontológica Argentina. Materiales y métodos: Se llevaron a cabo dos experiencias pre clínicas donde cada alumno realizó un tratamiento endodóntico en dientes humanos unir radiculares extraídos, alinicio del curso preclínico –con los saberes y las destrezas previos–y al finalizar. Se consignaron y evaluaron 19 errores procedimentales.Los datos fueron analizados mediante la pruebano paramétrica del signo de Wilcoxon, con un valor de p≤0,05. Resultados: En la primera experiencia se constataron 63errores procedimentales, y en la segunda, 14. Entre la valoracióninicial y la final se hallaron diferencias significativasen las variables procesamiento radiográfico (α=0,014), apertura cameral exagerada (α=0,0013), alteración dela anatomía (α=0,05), pérdida de la longitud de trabajo(α=0,007), falta de adaptación del cono principal(α=0,008), y falta de compactación lateral (α=0,008).En el resto de las variables analizadas no se encontraron diferencias significativas. El índice de la mejoríaindividual fue del 50 al 100%, y el grupal, del 78,7%. Conclusiones: De acuerdo con los resultados obtenidosen esta experiencia, podría considerarse que la propuestapedagógica resultó válida para los objetivos planteados.

Aim: to develop a strategy for teaching and learning, which may lead to a new methodology for individual and group evaluation of pre clinical procedures in the Specialization Course in Endodontics of the Universidad del Salvador /Asociación Odontológica Argentina. Materials and methods: Two preclinical experiments were carried out where each student performed an endodontic treatment in single-rooted extracted human teeth, at the beginningand after finishing the preclinical course with his/herprevious knowledge and skills. Nineteen different proceduralerrors were recorded and evaluated. Wilcoxon’s sign test wasused to compare before and after results setting significanceat p≤0,05. Results: In the initial experiment 63 procedural errors were found and in the second, 14. Between the initial and final evaluations, significant differences were found in thevariables radiographic processing (α=0.014), exaggerated chamber opening (α=0.0013), anatomic form modification(α=0.05), loss of working length (α=0.007), lack of adaptation of the main cone (α=0.008), and lack of lateralcompaction (α=0.008). In the other variables that were evaluated, no significant differences were found. The rateof individual improvement was 50 to 100%, and the group of 78.7%. Conclusions: According to the results obtained in this experience, the pedagogical proposal can be considered to bevalid to reach educational goals.

Iron overload causes endolysosomal deficits modulated by NAADP-regulated 2-pore channels and RAB7A.

Various neurodegenerative disorders are associated with increased brain iron content. Iron is known to cause oxidative stress, which concomitantly promotes cell death. Whereas endolysosomes are known to serve as intracellular iron storage organelles, the consequences of increased iron on endolysosomal functioning, and effects on cell viability upon modulation of endolysosomal iron release remain largely unknown. Here, we show that increasing intracellular iron causes endolysosomal alterations associated with impaired autophagic clearance of intracellular protein aggregates, increased cytosolic oxidative stress and increased cell death. These effects are subject to regulation by NAADP, a potent second messenger reported to target endolysosomal TPCNs (2-pore channels). Consistent with endolysosomal iron storage, cytosolic iron levels are modulated by NAADP, and increased cytosolic iron is detected when overexpressing active, but not inactive TPCNs, indicating that these channels can modulate endolysosomal iron release. Cell death triggered by altered intralysosomal iron handling is abrogated in the presence of an NAADP antagonist or when inhibiting RAB7A activity. Taken together, our results suggest that increased endolysosomal iron causes cell death associated with increased cytosolic oxidative stress as well as autophagic impairments, and these effects are subject to modulation by endolysosomal ion channel activity in a RAB7A-dependent manner. These data highlight alternative therapeutic strategies for neurodegenerative disorders associated with increased intracellular iron load.

Noncovalent Mutant Selective Epidermal Growth Factor Receptor Inhibitors: A Lead Optimization Case Study.

Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model.