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Objectives: To evaluate physicians' opinions concerning continuous deep sedation until death (CDSUD) and implementation of Claeys-Leonetti; a law intended to be applicable to all patients, but without a specific framework for children thus giving rise to ethically and legally complex situations. The secondary objective was to identify if physicians' characteristics could influence their opinions. Study Design: This was a national, multicenter, noninterventional cross-sectional survey from January 30, 2020, until March 1, 2020. The target population consisted of French physicians involved in children's end-of-life situations. The validated questionnaire explored respondents' characteristics and their opinions on four hypothetical pediatric clinical cases. Results: Analysis was conducted on 391 respondents. The oncological situation was more easily recognized as end of life compared with the neurological pathology (77% vs. 40.4%). Dependence on mechanical ventilation was another major factor influencing physicians in identifying end-of-life situations. Physicians clearly recognized the difference in intention between CDSUD and euthanasia. They accepted to implement CDSUD more easily in newborns. The withdrawal of artificial nutrition and hydration gave rise to divergent opinions. Respondents were in favor of adolescents' decision-making autonomy and their access to drafting advance directives. The child's best interest prevailed in case of objection by parents, except in situations outside the law's framework or in cases of disagreement within the health care team. Conclusion: Results of our study showed differences in the interpretation of the law concerning the CDSUD application framework and provide elements for reflection, which may ultimately contribute to the development of specific guidelines in CDSUD in children at the end of life.

Circadian rhythms have been described in numerous tissues of living organisms and are necessary for homeostasis. The understanding of their role in normal and pathological pregnancy is only just emerging. It has been established that clock genes are expressed in the placenta of animals and humans, but the rhythmicity of placenta immune cells is not known. Macrophages from healthy placenta of women at term were isolated and the expression of clock genes BMAL1, CLOCK, PER2, CRY2, and NR1D1 was assessed by qRT-PCR every 4 h over 24 h. Raw data were treated with cosinor analysis to evaluate the significance of the oscillations. Placental macrophages exhibited significant circadian expression of clock genes but one third of placental macrophages lost clock gene rhythmicity; the clock gene oscillations were restored by co-culture with trophoblasts. We wondered if melatonin, a key hormone regulating circadian rhythm, was involved in the oscillations of placental cells. We showed that macrophages and trophoblasts produced melatonin and expressed MT2 receptor. In women who developed preeclampsia during pregnancy, circadian oscillations of placental macrophages were lost and could not be rescued by coculture with trophoblasts from healthy women. Moreover, production and oscillations of melatonin were altered in preeclamptic macrophages. For the first time to our knowledge, this study shows circadian rhythms and melatonin production by placental macrophages. It also shows that preeclampsia is associated with a disruption of the circadian rhythm of placental cells. These results represent a new scientific breakthrough that may contribute to the prevention and treatment of obstetrical pathologies.

Background: Antireflux mucosectomy, a new endoscopic treatment for gastroesophageal reflux disease, consists of endoscopic mucosal resection at the esophagogastric junction. This study aim was to evaluate the medium-term efficacy of the antireflux mucosectomy technique for patients with severe gastroesophageal reflux disease symptoms (proton pump inhibitor treatment-dependent or proton pump inhibitor treatment-resistant gastroesophageal reflux disease)., Methods: Between January 2017 and June 2018, 13 patients with severe gastroesophageal reflux disease without hiatal hernia, with positive pH reflux, were included in this monocentric prospective pilot study. The primary outcome was clinical success, defined by improvement evaluated by the Gastroesophageal Reflux Disease Health Related Quality of Life Questionnaire at 24 months. Secondary outcomes were technical success, decreased use of proton pump inhibitors, patient satisfaction, and adverse events., Results: Thirteen patients [females = 8 (62%)], mean age 59 (range, 54-68), were included. The antireflux mucosectomy procedure had technical success in all patients. At 24 months, for 11 patients, gastroesophageal reflux disease symptoms were significantly improved, and mean gastroesophageal reflux disease score decreased from 33 (range, 26-42) to 3 (range, 0-7) (p = 0.001). Ninety-one percent ( n = 10) of patients had a lower proton pump inhibitor intake at 24 months. One patient had 3 endoscopic balloon dilatations for EGJ stenosis, two patients had melena ten days after procedure, and seven patients had thoracic or abdominal pain. Patient's satisfaction at 24 months was 81%., Conclusions: In patients with severe gastroesophageal reflux disease, despite occurrence of several short-term adverse events, antireflux mucosectomy seemed effective in improving gastroesophageal reflux disease symptoms at 24 months. This trial is registered with ClinicalTrials: NCT03357809., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2022 Arthur Laquière et al.)

Background: During endoscopic retrograde cholangiopancreatography (ERCP), access to the common bile duct (CBD) can be problematic after unintentional insertion of the guidewire into the pancreatic duct. We conducted a prospective, randomized study in order to compare biliary cannulation success rates of early double-guidewire (EDG) and repeated single-guidewire (RSG) techniques in patients with inadvertent passage of the guidewire into the pancreatic duct., Methods: Patients with a native papilla were randomly assigned to either the EDG or RSG groups after unintentional insertion of the guidewire into the pancreatic duct. The primary outcome was successful selective CBD cannulation within 10 minutes. The secondary outcomes were successful final selective bile duct cannulation, time to bile duct cannulation, and frequency of post-ERCP pancreatitis (PEP)., Results: 142 patients were randomized and selective bile duct cannulation was achieved in 57/68 patients (84 %) in the EDG group and in 37/74 patients (50 %) in the RSG group within 10 minutes (relative risk 1.34; 95 % confidence interval 1.08-6.18; P  < 0.001). The overall final selective bile duct cannulation rate was 99.3 %. The time to access the CBD was shorter using the EDG technique (6.0 vs. 10.4 minutes; P  = 0.002). Mild PEP was not observed more frequently in the EDG group than in the RSG group., Conclusion: The EDG technique significantly increased the success rate of biliary duct cannulation within 10 minutes compared with an RSG approach., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Preterm birth is the first cause of neonatal mortality and is associated with elevated risks of long-term complications such as neurodevelopmental impairment. Prediction of spontaneous preterm birth, one of the biggest challenges in obstetrics, aims at delaying birth in order to allow corticosteroid therapy and, if necessary, transfer of patient to a higher-level maternity care unit. We aimed to assess the predictive role of phIGFBP-1 (Actim® Partus) diagnostic test on patients at risk of preterm labor, routinely used in our institution. We conducted a retrospective cohort study on 99 patients admitted in the high-risk pregnancy unit of our institution from June 2012 to November 2014. The primary outcome measures were delivery before 34 +0 and 37 +0 weeks. Data analysis allowed measure of Actim® Partus test sensitivity (Se), specificity (Sp), positive and negative predictive values (PPV and NPV), diagnostic efficiency as well as positive and negative likelihood ratios. Actim® Partus test features (Se, Sp, PPV and NPV) were 53.3, 67.9, 23.5 and 88.7% respectively for deliveries occurring ≤ 34 +0 weeks and 54.2, 75.4, 55.8, and 74.2%, respectively, for deliveries occurring ≤ 37 +0 weeks. Diagnostic efficiency of the test was 65.7% (≤ 34 +0 weeks) and 67.7% (≤ 37 +0 weeks). Positive likelihood ratios were 1.6 (≤ 34 +0 weeks) and 2.2 (≤ 37 +0 weeks). Negative likelihood ratios were 0.7 (≤ 34 +0 weeks) and 0.6 (≤ 37 +0 weeks). Results of our study show that phIGFBP-1 diagnostic test is not accurate enough in predicting preterm birth before 34 +0 or 37 +0 weeks, and therefore, there is little clinical interest in its everyday use.

What is the perception of health and well-being of adolescents from an assisted reproductive technology (ART) cohort? We conducted a survey, from September 2015 to June 2016, through self-completion questionnaires, on 487 singleton or twin ART-conceived 11- to 15-year-old adolescents, followed up since 1994, as part of an ART cohort. Collected data concerned perinatal characteristics, health indicators and perception, eating habits, behavior, and living standards. A total of 60.6% of the questionnaires were returned and could therefore be analyzed. This concerned 295 adolescents who were representative of the 788 remaining adolescents of our cohort, in terms of type of ART, maternal and perinatal characteristics, but not gender (sex ratio = 0.77). Overall, 15.3% reported chronic diseases, and only 13.3% of them considered that their chronic disease had an impact on their school life. Moreover, 94.2% of adolescents perceived that their health was "excellent" or "good"; 97.3% adolescents had normal weight or were underweight; onset of menstruation was 12 years old (± 1) for girls, age usually reported for puberty in girls; 51.9% declared having regular physical activity, boys more frequently than girls. Moreover, 70.6% of the boys had a sedentary behavior compared to 44.8% of the girls. A total of 73.5% of the adolescents were stressed at school, but school demand was considered high only in 12.2% of cases. Finally, 90% declared to have high life satisfaction. Overall, ART does not appear to have particular effect on the health indicators and behavior of adolescents who participated in the survey except for higher family affluence scale.

Our objective was to describe and discuss management of recent cases of drug hypersensitivity in children reported in a pharmacovigilance center. Two pediatric allergy units conducted a collaborative retrospective analysis of 101 adverse drug reactions reported to a regional pharmacovigilance center between January 2016 and July 2019. Time lapse between hypersensitivity reaction onset and allergy consultation varied from 1 month to 12 years. Sixty-two patients (61.4%) presented with immediate reactions, 11 (10.9%) with non-immediate reactions, and 28 (27.7%) had reactions impossible to classify through medical interview. Overall, 92 children (91%) were explored for simultaneously administered drugs. All 113 prick tests were negative, and 2 were uncertain. Among 108 intradermal tests, 2 were positive to penicillin and to an iodinated contrast medium, 105 were negative, and 1 was uncertain. Overall, 129 drug provocation tests were proposed. Nine provocation tests among 80 were positive (11.25%): 6 to penicillin, 1 to sulfonamide antibiotics, and 2 to non-steroidal anti-inflammatory drugs; the remaining 71 were negative. No severe reaction was observed during these tests. Finally, drug allergy was only retained in 11 reported cases (10.9%).Conclusion: These pharmacovigilance reports show the difficulty in defining drug allergy in children only by anamnesis, and that explorations, particularly provocation tests, should take place at a reasonable time lapse after drug hypersensitivity reaction onset. What is Known: • True drug allergy is rarely observed in children. • Absence of full workup leads to falsely labeling children as "allergic." What is New: • Short time lapse between hypersensitivity onset and consultation improves classification of pediatric allergy. • Timely allergy consultations are essential, and tests are useful to confirm or exclude pediatric allergy.

Objective: To develop a modified version of Objective Structured Assessment of Technical Skill (OSATS) rating scale for evaluation of surgical skills specific to caesarean and to assess its relevance in documenting the residents' learning curve during their training. Secondarily, to verify the scale's stability to caesarean's level of difficulty and comparing self-assessment to hetero-assessment in order to propose a practical application of this rating scale during residency., Study Design: We conducted a multicentre observational prospective study, from May 2018 to November 2018. All residents at that time could participate and fill in the rating scale after caesarean. Senior surgeons had to fill in the same rating scale. We analysed correlation between self-assessments and hetero-assessments and sensitivity to change of the rating scale. Analysis of feature's relevance was performed by principal component analysis, factor analysis and reliability analysis., Results: In total, 234 rating scales were completed evaluating 18 residents. Our study demonstrated that our rating scale could be used to evaluate surgical skills of residents during caesarean and distinguish their year of residency (p < 0.001) with a high correlation between self and hetero-assessment (Intraclass Correlation coefficient for global score: 0.78; 95% CI 0.68-0.86). The principal component analysis revealed two dimensions corresponding to the two parts of the rating scale and the factorial analysis allowed us to confirm distribution of features according to these two dimensions. Cronbach's alpha allowed us to highlight the percentage of representation of the scale's features in relation to all potential theoretical features (0.93, 95% CI 0.82-0.95)., Conclusion: Our rating scale could be used for self-assessment during residency and as a hetero-assessment tool for validating defined stages of the internship., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Placental macrophages are a heterogenous population of immune cells present throughout pregnancy. They are essential for maintenance of the homeostatic placenta environment and host defense against infections. The characterization of placental macrophages as well as their activation have been limited for a long time by the lack of convenient tools. The emergence of unbiased methods makes it possible to reappraise the study of placental macrophages. In this review, we discuss the diversity and the functions of placental macrophages to better understand their dysfunctions during placental infections., Competing Interests: Conflict of competing interest The authors declare no competing interests. Infection with (a) bacteria, (b) virus or (c) parasites results in the expression of membrane receptors and the secretion of several key proteins by placental macrophages. CC and CXC: chemokines; CD: Cluster of differentiation; HIV: Human Immunodeficiency virus; HLA: Human leukocyte antigen; IL: Interleukin; IFN: Interferon; MHC: Major Histocompatibility complex; TGF: Transforming growth factor; TNF: Tumor necrosis factor., (Copyright © 2020. Published by Elsevier Ltd.)

Objective: To study whether history of cesarean delivery for arrest of descent (failure of fetal head engagement or unsuccessful instrumental delivery), is a factor of unsuccessful vaginal birth after cesarean delivery., Methods: Multicenter prospective study of patients undergoing TOL after previous caesarean delivery between May 2012 and May 2013 in 6 maternities. Univariate statistical analysis was performed depending of previous cesarean delivery indication. Multivariate analysis was used to determine independent association between these factors and TOLAC success., Results: Four hundred and eighty women with previous cesarean delivery were included and separated into two groups: the study group was composed of patients with history of CD for arrest of descent (failure of fetal head engagement or unsuccessful instrumental delivery) (n=31); control group included all other indications for CD (n=449). Overall, of the 480 women included in the study, 71.2 % underwent a TOL for a subsequent delivery (n=342): 68 % in the study group (n=21) vs 71.5 % in the control group (n=321). Vaginal birth after cesarean (VBAC) was obtained in 66.6 % vs 61% in the study and control group respectively (p=0.656). Univariate analysis of factors that may influence the success rate of (VBAC) did not show any difference between the two groups. Multivariate analysis showed that VBAC was only significantly associated with history of vaginal delivery subsequent to prior CD for arrest of descent., Conclusion: This study reassures us in our clinical practice allowing TOL in cases of history of CD for fetal head engagement failure or instrumental delivery failure in the second stage of labor., Competing Interests: Declaration of Competing Interest The other authors report no conflict of interest., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Background: Prostate cancer is a major public health issue, mainly because patients relapse after androgen deprivation therapy. Proteomic strategies, aiming to reflect the functional activity of cells, are nowadays among the leading approaches to tackle the challenges not only of better diagnosis, but also of unraveling mechanistic details related to disease etiology and progression., Methods: We conducted here a large SILAC-based Mass Spectrometry experiment to map the proteomes and phosphoproteomes of four widely used prostate cell lines, namely PNT1A, LNCaP, DU145 and PC3, representative of different cancerous and hormonal status., Results: We identified more than 3000 proteins and phosphosites, from which we quantified more than 1000 proteins and 500 phosphosites after stringent filtering. Extensive exploration of this proteomics and phosphoproteomics dataset allowed characterizing housekeeping as well as cell-line specific proteins, phosphosites and functional features of each cell line. In addition, by comparing the sensitive and resistant cell lines, we identified protein and phosphosites differentially expressed in the resistance context. Further data integration in a molecular network highlighted the differentially expressed pathways, in particular migration and invasion, RNA splicing, DNA damage repair response and transcription regulation., Conclusions: Overall, this study proposes a valuable resource toward the characterization of proteome and phosphoproteome of four widely used prostate cell lines and reveals candidates to be involved in prostate cancer progression for further experimental validation., Competing Interests: This work was supported by the French Plan Cancer 2009-2013 (Systems Biology call). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The funder ProGeLife provided support in the form of salaries for author AV, but had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. This commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. The specific roles of the authors are articulated in the ‘author contributions’ section.

The intracellular bacterium Coxiella burnetii is responsible for Q fever, an infectious disease that increases the risk of abortion, preterm labor, and stillbirth in pregnant women. It has been shown that C. burnetii replicates in BeWo trophoblast cell line and inhibits the activation and maturation of decidual dendritic cells. Although tissue macrophages are known to be targeted by C. burnetii , no studies have investigated the interplay between placental macrophages and C. burnetii . Here, CD14 + macrophages from 46 full-term placentas were isolated by positive selection. They consisted of a mixed population of maternal and fetal origin as shown by genotype analysis. We showed that C. burnetii organisms infected placental macrophages after 4 h. When these infected macrophages were incubated for an additional 9-day culture, they completely eliminated organisms as shown by quantitative PCR. The ability of placental macrophages to form multinucleated giant cells was not affected by C. burnetii infection. The transcriptional immune response of placental macrophages to C. burnetii was investigated using quantitative real-time RT-PCR on 8 inflammatory and 10 immunoregulatory genes. C. burnetii clearly induced an inflammatory profile. Interestingly, the production by placental macrophages of interferon-γ, a cytokine known to be involved in efficient immune responses, was dramatically increased in response to C. burnetii . In addition, a clear correlation between interferon-γ production and C. burnetii elimination was found, suggesting that macrophages from full-term placentas eliminate C. burnetii under the control of an autocrine production of interferon-γ., (Copyright © 2019 Mezouar, Benammar, Boumaza, Diallo, Chartier, Buffat, Boudjarane, Halfon, Katsogiannou and Mege.)

Introduction: Transabdominal chorionic villus sampling (CVS) is an invasive procedure for prenatal diagnosis reported to be associated with anxiety and pain. In this context, the need for analgesia during CVS has been considered useful. Even though several authors have been interested in pain management during amniocentesis, no study has been published on pain reduction during CVS. Our objective was to evaluate pain and anxiety management during transabdominal CVS using nitrous oxide (N 2 O) and local anesthesia., Material and Methods: In a randomized controlled noninferiority trial, self-administered nitrous oxide (N 2 O) inhalation (equimolar premix of oxygen and nitrous oxide) was compared with local anesthesia (1% lidocaine) before CVS. Primary outcome was pain and secondary outcome was anxiety, both measured on a visual analog scale 30-60 minutes before, immediately after (5-10 minutes) and 30-60 minutes after CVS. With a statistical power of 90%, type I error of 5% and two-sided test and potential exclusions, a sample size of 96 patients per group was enrolled and randomized. No patient was enrolled before the trial registration date., Results: From 13 March 2013 through 10 February 2015, 192 patients (96 per group) were screened and randomized. Most characteristics were similar across groups. Pain in the N 2 O group was 2.65 ± 0.22 vs 3.32 ± 0.26 in local anesthesia group [mean ± standard error of mean  (SEM)]. Mean anxiety in the N 2 O group was 3.17 ± 0.27 vs 5.19 ± 0.30 in the local anesthesia group., Conclusion: N 2 O was as efficient and even superior to local anesthesia for both pain and anxiety reduction during CVS, as the 95% confidence intervals were both below the prespecified noninferiority margin of 0.8 and below zero., (© 2018 Nordic Federation of Societies of Obstetrics and Gynecology.)

[This corrects the article DOI: 10.18632/oncotarget.20469.].

The actual strategy to improve current therapies in advanced prostate cancer involves targeting genes activated by androgen withdrawal, either to delay or prevent the emergence of the castration-refractory phenotype. However, these genes are often implicated in several physiological processes, and long-term inhibition of survival proteins might be accompanied with cytotoxic effects. To avoid this problem, an alternative therapeutic strategy relies on the identification and use of compounds that disrupt specific protein-protein interactions involved in androgen withdrawal. Specifically, the interaction of the chaperone protein Hsp27 with the initiation factor eIF4E leads to the protection of protein synthesis initiation process and enhances cell survival during cell stress induced by castration or chemotherapy. Thus, in this work we aimed at i) identifying the interaction site of the Hsp27/eIF4E complex and ii) interfere with the relevant protein/protein association mechanism involved in castration-resistant progression of prostate cancer. By a combination of experimental and modeling techniques, we proved that eIF4E interacts with the C-terminal part of Hsp27, preferentially when Hsp27 is phosphorylated. We also observed that the loss of this interaction increased cell chemo-and hormone-sensitivity. In order to find a potential inhibitor of Hsp27/eIF4E interaction, BRET assays in combination with molecular simulations identified the phenazine derivative 14 as the compound able to efficiently interfere with this protein/protein interaction, thereby inhibiting cell viability and increasing cell death in chemo- and castration-resistant prostate cancer models in vitro and in vivo ., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Prostate cancer has become a real public health issue in industrialized countries, mainly due to patients' relapse by castration-refractory disease after androgen ablation. Castration-resistant prostate cancer is an incurable and highly aggressive terminal stage of prostate cancer, seriously jeopardizing the patient's quality of life and lifespan. The management of castration-resistant prostate cancer is complex and has opened new fields of research during the last decade leading to an improved understanding of the biology of the disease and the development of new therapies. Most advanced tumors resistant to therapy still maintain the androgen receptor-pathway, which plays a central role for survival and growth of most castration-resistant prostate cancers. Many mechanisms induce the emergence of the castration resistant phenotype through this pathway. However some non-related AR pathways like neuroendocrine cells or overexpression of anti-apoptotic proteins like Hsp27 are described to be involved in CRPC progression. More recently, loss of expression of tumor suppressor gene, post-transcriptional modification using miRNA, epigenetic alterations, alternatif splicing and gene fusion became also hallmarks of castration-resistant prostate cancer. This review presents an up-to-date overview of the androgen receptor-related mechanisms as well as the latest evidence of the non-AR-related mechanisms underlying castration-resistant prostate cancer progression., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Previously, we identified the stress-induced chaperone, Hsp27, as highly overexpressed in castration-resistant prostate cancer and developed an Hsp27 inhibitor (OGX-427) currently tested in phase I/II clinical trials as a chemosensitizing agent in different cancers. To better understand the Hsp27 poorly-defined cytoprotective functions in cancers and increase the OGX-427 pharmacological safety, we established the Hsp27-protein interaction network using a yeast two-hybrid approach and identified 226 interaction partners. As an example, we showed that targeting Hsp27 interaction with TCTP, a partner protein identified in our screen increases therapy sensitivity, opening a new promising field of research for therapeutic approaches that could decrease or abolish toxicity for normal cells. Results of an in-depth bioinformatics network analysis allying the Hsp27 interaction map into the human interactome underlined the multifunctional character of this protein. We identified interactions of Hsp27 with proteins involved in eight well known functions previously related to Hsp27 and uncovered 17 potential new ones, such as DNA repair and RNA splicing. Validation of Hsp27 involvement in both processes in human prostate cancer cells supports our system biology-predicted functions and provides new insights into Hsp27 roles in cancer cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Understanding the mechanisms that control stress-induced survival is critical to explain how tumors frequently resist to treatment and to improve current anti-cancer therapies. Cancer cells are able to cope with stress and escape drug toxicity by regulating heat shock proteins (Hsps) expression and function. Hsp27 (HSPB1), a member of the small Hsp family, represents one of the key players of many signaling pathways contributing to tumorigenicity, treatment resistance, and apoptosis inhibition. Hsp27 is overexpressed in many types of cancer and its functions are regulated by post-translational modifications, such as phosphorylation. Protein phosphorylation is the most widespread signaling mechanism in eukaryotic cells, and it is involved in all fundamental cellular processes. Aberrant phosphorylation of Hsp27 has been associated with cancer but the molecular mechanisms by which it is implicated in cancer development and progression remain undefined. This mini-review focuses on the role of phosphorylation in Hsp27 functions in cancer cells and its potential usefulness as therapeutic target in cancer.

It is strongly suspected that potassium (K(+)) channels are involved in various aspects of prostate cancer development, such as cell growth. However, the molecular nature of those K(+) channels implicated in prostate cancer cell proliferation and the mechanisms through which they control proliferation are still unknown. This study uses pharmacological, biophysical and molecular approaches to show that the main voltage-dependent K(+) current in prostate cancer LNCaP cells is carried by large-conductance BK channels. Indeed, most of the voltage-dependent current was inhibited by inhibitors of BK channels (paxillin and iberiotoxin) and by siRNA targeting BK channels. In addition, we reveal that BK channels constitute the main K(+) channel family involved in setting the resting membrane potential in LNCaP cells at around -40 mV. This consequently promotes a constitutive calcium entry through T-type Cav3.2 calcium channels. We demonstrate, using single-channel recording, confocal imaging and co-immunoprecipitation approaches, that both channels form macromolecular complexes. Finally, using flow cytometry cell cycle measurements, cell survival assays and Ki67 immunofluorescent staining, we show that both BK and Cav3.2 channels participate in the proliferation of prostate cancer cells.

The endoplasmic reticulum (ER) is involved in many cellular functions, including protein folding and Ca(2+) homeostasis. The ability of cells to respond to the ER stress is critical for cell survival, and disruption in such regulation can lead to apoptosis. ER stress is accompanied by alterations in Ca(2+) homeostasis, and the ER Ca(2+) store depletion by itself can induce ER stress and apoptosis. Despite that, the ER Ca(2+) leak channels activated in response to the ER stress remain poorly characterized. Here we demonstrate that ER Ca(2+) depletion during the ER stress occurs via translocon, the ER protein complex involved in translation. Numerous ER stress inducers stimulate the ER Ca(2+) leak that can be prevented by translocon inhibitor, anisomycin. Expression of GRP78, an ER stress marker, increased following treatment with puromycin (a translocon opener) and was suppressed by anisomycin, confirming a primary role of translocon in ER stress induction. Inhibition of ER store depletion by anisomycin significantly reduces apoptosis stimulated by the ER stress inducers. We suggest that translocon opening is physiologically modulated by GRP78, particularly during the ER stress. The ability to modulate the ER Ca(2+) permeability and subsequent ER stress can lead to development of a novel therapeutic approach.

Heat shock protein 27 (Hsp27) is highly overexpressed in castration-resistant prostate cancer (CRPC) and an antisense inhibitor (OGX-427) is currently in phase II clinical trials. In order to understand mechanisms of action of Hsp27 and find new therapeutic targets specific of CRPC, we screened for Hsp27 client proteins. Here, we report that translationally controlled tumor protein (TCTP) is a new Hsp27 client protein involved in Hsp27 cytoprotection. We found that TCTP expression is absent or weak in normal prostate cells, moderately expressed in 18.5% of treatment naive PC, and becomes uniformly and strongly expressed in 75% of CRPC. To define TCTP function, we developed and worldwide patented a TCTP antisense oligonucleotide (ASO). Interestingly, we found that CRPC progression correlates with TCTP overexpression and loss of P53. TCTP knockdown restored P53 expression and function, suggesting that castration-sensitivity is directly linked to P53 expression. Collectively, these findings provide a new Hsp27 cytoprotection mechanism in CRPC, and preclinical proof-of-concept that combining ASO-mediated TCTP knockdown with castration and/or docetaxel therapy could serve as a novel strategy to treat CRPC, with no or little toxicity for normal prostate cells.

Hsp27, αB-crystallin and HSP22 are ubiquitous small heat shock proteins (sHsp) whose expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. These sHsp protect cells from otherwise lethal conditions mainly by their involvement in cell death pathways such as necrosis, apoptosis or autophagy. At a molecular level, the mechanisms accounting for sHsp functions in cell death are (1) prevention of denatured proteins aggregation, (2) regulation of caspase activity, (3) regulation of the intracellular redox state, (4) function in actin polymerization and cytoskeleton integrity and (5) proteasome-mediated degradation of selected proteins. In cancer cells, these sHsp are often overexpressed and associated with increased tumorigenicity, cancer cells metastatic potential and resistance to chemotherapy. Altogether, these properties suggest that Hsp27, αB-crystallin and Hsp22 are appropriate targets for modulating cell death pathways. In the present, we briefly review recent reports showing molecular evidence of cell death regulation by these sHsp and co-chaperones. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Orai1, together with STIM1 and STIM2, constitutes the molecular basis for store-operated calcium entry (SOCE) and we have investigated their role in cell proliferation and cell cycle progression in HEK293 cells. 48-h serum deprival, and a 24-h treatment with 1 mM hydroxyurea or with 10 microM RO-3306--a cyclin-dependent kinase 1 inhibitor--induced cell cycle block in G1, S and G2/M, respectively. SOCE amplitude, monitored in whole-cell voltage clamped cells, was markedly reduced (60-70%) in all conditions, with full reversibility within 4h. Silencing of Orai and STIM1 using siRNA resulted in a large inhibition of SOCE (70-80%) whereas siSTIM2 had a smaller but significant effect (30%). However, the cell population doubling time was not affected in siSTIM1 cells (18 h, the same as in control cells) but was increased in both siOrai1 cells (29 h) and in siSTIM2 (23 h) even when combined with siSTIM1. This suggests that STIM1 plays no role in cell proliferation in HEK293 cells while STIM2 is involved in both SOCE and cell proliferation in these cells. Finally, the cell cycle block induced SOCE inhibition was associated with reduced Orai1 expression with full recovery within 4h, whereas the expression of STIM1 and STIM2 remained unaltered. These observations reveal a tight relation between cell proliferation, calcium entry and Orai1 expression in HEK293 cells., (2010 Elsevier Ltd. All rights reserved.)

Background: During androgen ablation prostate cancer cells' growth and survival become independent of normal regulatory mechanisms. These androgen-independent cells acquire the remarkable ability to adapt to the surrounding microenvironment whose factors, such as neurotransmitters, influence their survival. Although findings are becoming evident about the expression of alpha(1A)-adrenoceptors in prostate cancer epithelial cells, their exact functional role in androgen-independent cells has yet to be established. Previous work has demonstrated that membrane lipid rafts associated with key signalling proteins mediate growth and survival signalling pathways in prostate cancer cells., Methodology/principal Findings: In order to analyze the membrane topology of the alpha(1A)-adrenoceptor we explored its presence by a biochemical approach in purified detergent resistant membrane fractions of the androgen-independent prostate cancer cell line DU145. Electron microscopy observations demonstrated the colocalization of the alpha(1A)-adrenoceptor with caveolin-1, the major protein component of caveolae. In addition, we showed that agonist stimulation of the alpha(1A)-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process. Further, immunohistofluorescence revealed the relation between high levels of alpha(1A)-adrenoceptor and caveolin-1 expression with advanced stage prostate cancer. We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK)., Conclusions/significance: In conclusion, we propose that alpha(1A)-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.

Because prostate cancer is, in its early stages, an androgen-dependent pathology, treatments aiming at decreasing testosterone plasma concentration have been developed for many years now. However, a significant proportion of patients suffer a relapse after a few years of hormone therapy. The androgen-independent stage of prostate cancer has been shown to be associated with the development of neuroendocrine differentiation. We previously demonstrated that neuroendocrine prostate cancer cells derived from LNCaP cells overexpress CaV3.2 T-type voltage-dependent calcium channels. We demonstrate here using prostatic acid phosphatase as a marker of prostate secretion and FM1-43 fluorescence imaging of membrane trafficking that neuroendocrine differentiation is associated with an increase in calcium-dependent secretion which critically relies on CaV3.2 T-type calcium channel activity. In addition, we show that these channels are expressed by neuroendocrine cells in prostate cancer tissues obtained from patients after surgery. We propose that CaV3.2 T-type calcium channel up-regulation may account for the alteration of secretion during prostate cancer development and that these channels, by promoting the secretion of potential mitogenic factors, could participate in the progression of the disease toward an androgen-independent stage.