V600EBRAF Inhibition Induces Cytoprotective Autophagy through AMPK in Thyroid Cancer Cells.

The dysregulation of autophagy is important in the development of many cancers, including thyroid cancer, where V600EBRAF is a main oncogene. Here, we analyse the effect of V600EBRAF inhibition on autophagy, the mechanisms involved in this regulation and the role of autophagy in cell survival of thyroid cancer cells. We reveal that the inhibition of V600EBRAF activity with its specific inhibitor PLX4720 or the depletion of its expression by siRNA induces autophagy in thyroid tumour cells. We show that V600EBRAF downregulation increases LKB1-AMPK signalling and decreases mTOR activity through a MEK/ERK-dependent mechanism. Moreover, we demonstrate that PLX4720 activates ULK1 and increases autophagy through the activation of the AMPK-ULK1 pathway, but not by the inhibition of mTOR. In addition, we find that autophagy blockade decreases cell viability and sensitize thyroid cancer cells to V600EBRAF inhibition by PLX4720 treatment. Finally, we generate a thyroid xenograft model to demonstrate that autophagy inhibition synergistically enhances the anti-proliferative and pro-apoptotic effects of V600EBRAF inhibition in vivo. Collectively, we uncover a new role of AMPK in mediating the induction of cytoprotective autophagy by V600EBRAF inhibition. In addition, these data establish a rationale for designing an integrated therapy targeting V600EBRAF and the LKB1-AMPK-ULK1-autophagy axis for the treatment of V600EBRAF-positive thyroid tumours.

The ReCaPTa study - a prospective out of hospital cardiac arrest registry including multiple sources of surveillance for the study of sudden cardiac death in the Mediterranean area.

BACKGROUND: Cardiovascular diseases are one of the leading causes of death in the industrialized world. Sudden cardiac death is very often the first manifestation of the disease and it occurs in the prehospital setting. The determination of the sudden cardiac death phenotype is challenging. It requires prospective studies in the community including multiple sources of case ascertainment that help to identify the cause and circumstances of death. The aim of the Clinical and Pathological Registry of Tarragona (ReCaPTa) is to study incidence and etiology of Sudden Cardiac Death in the Tarragona region (Catalonia, Spain). METHODS: ReCaPTa is a population-based registry of OHCA using multiple sources of surveillance. The population base is 511,662. This registry is compiled chronologically in a relational database and it prospectively contains data on all the OHCA attended by the EMS from April 2014 to April 2017. ReCaPTa collects data after each emergency medical assistance using an online application including variables of the onset of symptoms. A quality control is performed and it permits monitoring the percentage of cases included by the emergency crew. Simultaneously, data from the medico-legal autopsies is taken from the Pathology Center of the area. All the examination findings following a specific protocol for the sudden death study are entered into the ReCaPTa database by one trained person. Survivors admitted to hospital are followed up and their clinical variables are collected in each hospital. The primary care researchers analyze the digital clinical records in order to obtain medical background. All the available data will be reviewed after an adjudication process with the aim of identifying all cases of sudden cardiac death. DISCUSSION: There is a lack of population-based registries including multiple source of surveillance in the Mediterranean area. The ReCaPTa study could provide valuable information to prevent sudden cardiac death and develop new strategies to improve its survival.

Somatostatin activates Ras and ERK1/2 via a G protein ßγ-subunit-initiated pathway in thyroid cells.

Somatostatin (SST) is one of the main regulators of thyroid function. It acts by binding to its receptors, which lead to the dissociation of G proteins into Gαi and Gßγ subunits. However, much less is known about the function of Gßγ in thyroid cells. Here, we studied the role of SST and Gßγ dimers released upon SST stimulation on the Ras-ERK1/2 pathway in FTRL-5 thyroid cells. We demonstrate that SST activates Ras through Gi proteins, since SST-induced Ras activation is inhibited by pertussis toxin. Moreover, the specific sequestration of Gßγ dimers decreases Ras-GTP and phosphorylated ERK1/2 levels, and overexpression of Gßγ increases ERK1/2 phosphorylation induced by SST, indicating that Gßγ dimers released after SST treatment mediate activation of Ras and ERK1/2. On the other hand, SST treatment does not modify the expression of the thyroid differentiation marker sodium/iodide symporter (NIS) through ERK1/2 activation. However, SST increases AKT activation and the inhibition of the Src/PI3K/AKT pathway increases NIS levels in SST-treated cells. Thus, we conclude that, in thyroid cells, signalling from SST receptors to ERK1/2 involves a Gßγ-mediated signal acting on a Ras-dependent pathway. Moreover, we demonstrate that SST might regulates NIS expression through a Src/PI3K/AKT-dependent mechanism, but not through ERK1/2 signalling, showing the main role of this hormone in thyroid function.

TGFß induces epithelial-mesenchymal transition of thyroid cancer cells by both the BRAF/MEK/ERK and Src/FAK pathways.

The epithelial-mesenchymal transition (EMT) is a crucial process in tumour progression, by which epithelial cells acquire a mesenchymal phenotype, increasing its motility and the ability to invade distant sites. Here, we describe the molecular mechanisms by which V600E BRAF, TGFß and the Src/FAK complex cooperatively regulate EMT induction and cell motility of anaplastic thyroid cancer cells. Analysis of EMT marker levels reveals a positive correlation between TGFß and Snail expression, with a concomitant downregulation of E-cadherin, accompanied by an increase of cell migration and invasion. Furthermore, we show that V600E BRAF depletion by siRNA or inhibition of its activity by treatment with its inhibitor PLX4720 reverses the TGFß-mediated effects on Snail, E-cadherin, migration and invasion. Moreover, V600E BRAF induces TGFß secretion through a MEK/ERK-dependent mechanism. In addition, TGFß activates the Src/FAK complex, which in turn regulates the expression of Snail and E-cadherin as well as cell migration. The inhibition of Src with the inhibitor SU6656 or abrogation of FAK expression with a specific siRNA reverses the TGFß-induced effects. Interestingly, we demonstrate that activation of the Src/FAK complex by TGFß is independent of V600E BRAF signalling, since inhibition of this oncogene does not affect its phosphorylation. Our data strongly suggest that TGFß induces EMT and aggressiveness of thyroid cancer cells by parallel mechanisms involving both the V600E BRAF/MEK/ERK and Src/FAK pathways independently. Thus, we describe novel functions for Src/FAK in mediating the EMT program and aggressiveness regulated by TGFß, establishing the inhibition of these proteins as a possible effective approach in preventing tumour progression of V600E BRAF-expressing thyroid tumours. © 2015 Wiley Periodicals, Inc.

Passive leg raise (PLR) during cardiopulmonary (CPR) - a method article on a randomised study of survival in out-of-hospital cardiac arrest (OHCA).

BACKGROUND: It is estimated that about 275,000 inhabitants experience an out-of-hospital cardiac arrest (OHCA) every year in Europe. Survival in out-of-hospital cardiac arrest is relatively low, generally between five per cent and 10%. Being able to explore new methods to improve the relatively low survival rate is vital for people with these conditions. Passive leg raise (PLR) during cardiopulmonary resuscitation (CPR) has been found to improve cardiac preload and blood flow during chest compressions. The aim of our study is to evaluate whether early PLR during CPR also has an impact on one-month survival in sudden and unexpected out-of-hospital cardiac arrest (OHCA). METHOD/DESIGN: A prospective, randomized, controlled trial in which all patients (≥18 years) receiving out-of hospital CPR are randomized by envelope to be treated with either PLR or in the flat position. The ambulance crew use a special folding stool which allows the legs to be elevated about 20 degrees. Primary end-point: survival to one month. Secondary end-point: survival to hospital admission to one month and to one year with acceptable cerebral performance classification (CPC) 1-2. DISCUSSION: PLR is a simple and fast maneuver. We believe that the greatest benefit with PLR is when performed early in the process, during the first minutes of CPR and before the first defibrillation. To reach power this study need 3000 patients, we hope that this method article will encourage other sites to contact us and take part in our study. TRIAL REGISTRATION: ClinicalTrials.gov NCT01952197.

(V600E)BRAF promotes invasiveness of thyroid cancer cells by decreasing E-cadherin expression through a Snail-dependent mechanism.

BRAF is a main oncogene in human thyroid cancer. Here, we show that BRAF depletion by siRNA or inhibition of its activity by treatment with BRAF inhibitor PLX4720 decreases migration and invasion in thyroid cancer cells expressing oncogenic (V600E)BRAF through a MEK/ERK-dependent mechanism, since treatment with the MEK inhibitor U0126 exerts the same effect. Moreover, over-expression of (V600E)BRAF increases migration and invasion of wild-type BRAF thyroid cells. Using the same strategies, we demonstrate that these effects are mediated by upregulation of the transcriptional repressor Snail with a concomitant decrease of its target E-cadherin, both hallmarks of EMT. These results reveal a novel (V600E)BRAF-induced mechanism in thyroid tumours progression and provides a rationale for using the PLX4720 inhibitor to target (V600E)BRAF signalling to effectively control progression of thyroid cancer.

[The hospital transfer of a patient with gastrointestinal bleeding from oesophageal varices]. / El traslado interhospitalario de un paciente con hemorragia digestiva por varices esofágicas.

The care of patients with gastrointestinal bleeding from oesophageal varices during inter-hospital transfer is a complex procedure to different causes. Some of these include, the high number of parameters to take into account, the monitoring of the electro-medical equipment, the instability of the patient and their needs or the short time available to know the patient and obtain the maximum information in order to plan a safe and proper transfer. The main role of the nurse in the transfer of critically ill patients focuses on the control and monitoring of all these aspects and the maintenance of the continuity of the caring initiated in the hospital. This requires a personalized care plan to be prepared for the patient who has to be transferred to avoid putting the individual at risk due to their high vulnerability. This should minimise the risk of possible complications or accidental incidents related to the high number of instruments used for monitoring the patients during their transfer. The case presented below describes the transfer of a patient by ambulance from a level 2 hospital to a level 3 hospital of a person who had an urgent condition that reflected all this complexity. It highlights the indications and basic care that has to be taken into account in this type of transfer and the transfer of the patient once in the receiving hospital, especially in this case, where the condition of the patient is highly compromised.

A novel loss-of-function mutation in GPR54/KISS1R leads to hypogonadotropic hypogonadism in a highly consanguineous family.

CONTEXT: The G protein-coupled receptor 54 (GPR54), the kisspeptin receptor, is essential for stimulation of GnRH secretion and induction of puberty. Recently loss-of-function mutations of the GPR54 have been implicated as a cause of isolated idiopathic hypogonadotropic hypogonadism (IHH). OBJECTIVE: The objective of the study was to identify the genetic cause of IHH in a consanguineous pedigree and to characterize the phenotypic features from infancy through early adulthood. DESIGN: In six patients with normosmic IHH belonging to two families of Israeli Muslim-Arab origin highly related to one another, DNA was analyzed for mutations in the GnRHR and GPR54 genes, with functional analysis of the mutation found. The five males underwent comprehensive endocrine evaluation and were under longitudinal follow-up; the one female presented in early adulthood. RESULTS: A new homozygous mutation (c.T815C) in GPR54 leading to a phenylalanine substitution by serine (p.F272S) was detected in all patients. Functional analysis showed an almost complete inhibition of kisspeptin-induced GPR54 signaling and a dramatic decrease of the mutated receptor expression at the cell surface. The males exhibited the same clinical features from infancy to adulthood, characterized by cryptorchidism, a relatively short penis, and no spontaneous pubertal development. The female patient presented at 18 yr with impuberism and primary amenorrhea. Repeated stimulation tests demonstrated complete gonadotropin deficiency throughout follow-up. CONCLUSION: A novel loss-of-function mutation (p.F272S) in the GPR54 gene is associated with familial normosmic IHH. Underdeveloped external genitalia and impuberism point to the major role of GPR54 in the activation of the gonadotropic axis from intrauterine life to adulthood.

Increase of R-/S-methadone enantiomer concentration ratio in serum of patients treated with either nevirapine or efavirenz.

An increased methadone enantiomer ratio (R/S) was associated to both nevirapine (179%, n=5) and efavirenz (36%, n=9) treatments when compared with that of controls (n=52). Additionally, in four follow-up patients, both R- and S-methadone normalized concentrations decreased (19%-93%) while R/S increased (22%-314%) following nevirapine/efavirenz treatment. R/S decreased (42%) after non-compliance with efavirenz treatment. Therefore, the methadone-maintenance-treatment outcome should be evaluated when patients are treated with drugs which are supposed to induce CYP3A4 and CYP2B6 isoforms.

Detection of clinical interactions between methadone and anti-retroviral compounds using an enantioselective capillary electrophoresis for methadone analysis.

A capillary electrophoresis method was developed to detect interactions between methadone and anti-retroviral compounds. Eight subjects, who underwent methadone maintenance treatment in the Province of Alicante (Spain), consented to participate in the present study. Of those, one subject was followed up for 123 days to detect drug-drug interactions. The enantiomers of methadone and those of its main metabolite were conveniently resolved within 4 min using a chiral electrophoresis buffer mixture which consisted of phosphate buffer, pH 5, plus 0.2% highly sulphated-(beta)-cyclodextrin. The effective mobility of the analytes was in the 0.061-0.140 cm(2)/(kV s) range at pH 5. The R-methadone plasma concentration range for seven patients was 91-318 ng/mL, it decreased from 186 to 46 ng/mL in a patient followed-up on commencement of the anti-retroviral therapy, returning to the previous higher levels after progressive dose increases. We conclude that monitoring R-methadone plasma levels can be a useful tool for the dose adjustment of methadone.

Interacción entre metadona y antirretrovirales (estavudina, indinavir, ritonavir, nevirapina) / Interaction between methadone and antirretrovirals (stavudine, indinavir, ritonavir, nevirapine)

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Pilomatrixoma múltiple / Multiple pilomatrixomas

Se comunica el caso de un paciente de sexo masculino, de 12 años de edad, que presenta pilomatrixomas múltiples, ubicados en la región cervical y en las extremidades superiores, con características semiológicas típicas, de varios años de evolución. Se realizó la exéresis quirúrgica de todas ellas y su correspondiente estudio histopatológico. Tanto en microscopía óptica como en la electrónica se comprobaron la presencia de células basofílicas y sombreadas así como depósitos cálcicos, características del epitelioma calcificante de Malherbe. Se realiza una revisión histórica y se consideran además su patogénesis, aspectos clínicos, histopatológicos y los distintos diagnósticos diferenciales, así como también sus posibles asociaciones nosológicas (AU)

Pilomatrixoma múltiple / Multiple pilomatrixomas

Se comunica el caso de un paciente de sexo masculino, de 12 años de edad, que presenta pilomatrixomas múltiples, ubicados en la región cervical y en las extremidades superiores, con características semiológicas típicas, de varios años de evolución. Se realizó la exéresis quirúrgica de todas ellas y su correspondiente estudio histopatológico. Tanto en microscopía óptica como en la electrónica se comprobaron la presencia de células basofílicas y sombreadas así como depósitos cálcicos, características del epitelioma calcificante de Malherbe. Se realiza una revisión histórica y se consideran además su patogénesis, aspectos clínicos, histopatológicos y los distintos diagnósticos diferenciales, así como también sus posibles asociaciones nosológicas