Stochasticity and bistability in horizontal transfer control of a genomic island in Pseudomonas.

Genomic islands (GEI) comprise a recently recognized large family of potentially mobile DNA elements and play an important role in the rapid differentiation and adaptation of bacteria. Most importantly, GEIs have been implicated in the acquisition of virulence factors, antibiotic resistances or toxic compound metabolism. Despite detailed information on coding capacities of GEIs, little is known about the regulatory decisions in individual cells controlling GEI transfer. Here, we show how self-transfer of ICEclc, a GEI in Pseudomonas knackmussii B13 is controlled by a series of stochastic processes, the result of which is that only a few percent of cells in a population will excise ICEclc and launch transfer. Stochastic processes have been implicated before in producing bistable phenotypic transitions, such as sporulation and competence development, but never before in horizontal gene transfer (HGT). Bistability is instigated during stationary phase at the level of expression of an activator protein InrR that lays encoded on ICEclc, and then faithfully propagated to a bistable expression of the IntB13 integrase, the enzyme responsible for excision and integration of the ICEclc. Our results demonstrate how GEI of a very widespread family are likely to control their transfer rates. Furthermore, they help to explain why HGT is typically confined to few members within a population of cells. The finding that, despite apparent stochasticity, HGT rates can be modulated by external environmental conditions provides an explanation as to why selective conditions can promote DNA exchange.

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.

Buccal mucosa graft for laryngotracheal reconstruction in severe laryngeal stenosis.

OBJECTIVE: An operative technique is described as a salvage treatment for severe subglottic and supraglottic laryngeal stenosis. In addition to expansion of the laryngeal framework with an anterior cartilage graft, as used in a classical laryngotracheal reconstruction, the scar tissue obliterating the airway lumen is excised and a mucosal graft is placed to reconstruct the inner lining of the airway. The graft is harvested from buccal mucosa. METHODS: The operative technique is outlined. Three cases, 2 paediatric and one adult, with complete or near complete laryngeal stenosis are presented where this operative technique was employed. In all patients several surgeries had been performed previously which were unsuccessful. RESULTS: In all 3 patients a patent airway was achieved with decannulation of the tracheostomy in the 2 paediatric patients. CONCLUSIONS: In patients with severe subglottic or supraglottic airway stenosis where other surgeries have failed, excision of endoluminal scar tissue and placement of a buccal mucosal graft, in addition to conventional laryngotracheal reconstruction, is a promising technique. In revision cases of subglottic stenosis cricotracheal resection might not be an option because of scarring from previous surgeries. This operation is an alternative, which allows an increase in the airway lumen by excising the scar tissue then re-lining the exposed internal lumen. The buccal mucosa reduces granulation formation and re-stenosis.

Implication of DNA methylation, CTCF and BORIS factors in the transcriptional regulation of the human telomerase catalytic subunit hTERT

RESUME La télomérase confère une durée de vie illimitée et est réactivée dans la plupart des cellules tumorales. Sa sous-unité catalytique hTERT est définie comme le facteur limitant pour son activation. De l'identification de facteurs liant la région régulatrice d'hTERT, au rôle de la méthylation de l'ADN et de la modification des histones, de nombreux modèles de régulation ont été suggérés. Cependant, aucun de ces modèles n'a pu expliquer l'inactivation de la télomérase dans la plupart des cellules somatiques et sa réactivation dans la majorité des cellules tumorales. De plus, les observations contradictoires entre le faible niveau d'expression d'ARN messager d'hTERT dans les cellules télomérase-positives et la très forte activité transcriptionnelle du promoteur d'hTERT en transfection restent incomprises. Dans cette étude, nous avons montré que la région proximale du gène hTERT (exon 1 et 2) était impliquée dans la répression de l'activité de son promoteur. Nous avons identifié le facteur CTCF comme étant un inhibiteur du promoteur d'hTERT, en se liant au niveau de son premier exon. La méthylation de l'exon 1 du gène hTERT, couramment observée dans les tumeurs mais pas dans les cellules normales, empêcherait la liaison de CTCF. L'étude du profil de méthylation du promoteur d'hTERT indique qu'une partie du promoteur reste déméthylée et qu'elle semble suffisante pour permettre une faible activité transcriptionnelle du gène hTERT. Ainsi, la méthylation particulière des régions régulatrices d'hTERT inhibe la liaison de CTCF tout en permettant une faible transcription du gène. Cependant, dans certaines cellules tumorales, le promoteur et la région proximale du gène hTERT ne sont pas méthylés. Dans les lignées cellulaires tumorales de tesitcules et d'ovaires, l'inhibition de CTCF est contrée par son paralogue BORIS, qui se lie aussi au niveau de l'exon 1 d'hTERT, mais permet ainsi l'activation du promoteur. L'étude de l'expression du gène BORIS montre qu'il est exclusivement exprimé dans les tissus normaux de testicules et d'ovaires jeunes, ainsi qu'à différents niveaux dans la plupart des tumeurs. Sa transcription est sous le contrôle de deux promoteurs. Le promoteur proximal est régulé par méthylation et un transcrit alternatif majoritaire, délété de l'exon 6, est trouvé lorsque ce promoteur est actif. Tous ces résultats conduisent à un modèle de régulation du gène hTERT qui tient compte du profil épigénétique du gène et qui permet d'expliquer le faible taux de transcription observé in vivo. De plus, l'expression de BORIS dans les cancers et son implication dans l'activation du gène hTERT pourrait permettre de comprendre les phénomènes de dérégulation épigénétique et d'immortalisation qui ont lieu durant la tumorigenèse. SUMMARY Telomerase confers an unlimited lifespan, and is reactivated in most tumor cells. The catalytic subunit of telomerase, hTERT, is defined as the limiting factor for telomerase activity. Between activators and repressors that bind to the hTERT 5' regulatory region, and the role of CpG methylation and histone acetylation, an abundance of regulatory models have been suggested. None of these models can explain the silence of telomerase in most somatic cells and its reactivation in tumor cells. Moreover, the contradictory observations of the low level of hTERT mRNA in telomerase-positive cells and the high transcriptional activity of the hTERT promoter in transfection experiments remain unresolved. In this study, we demonstrated that the proximal exonic region of the hTERT gene (exon 1 and 2) is involved in the inhibition of its promoter. We identified the protein CTCF as the inhibitor of the hTERT promoter, through its binding to the first exon. The methylation of the first exon region, which is often observed in cancer cells but not in noimal cells, represses CTCF binding. Study of hTERT promoter methylation shows a partial demethylation sufficient to activate the transcription of the hTERT gene. Therefore, we demonstrated that the particular methylation profile of the hTERT regulatory sequences inhibits the binding of CTCF, while it allows a low transcription of the gene. Nevertheless, in some tumor cells, the promoter and the proximal exonic region of hTERT are unmethylated. In testicular and ovarian cancer cell lines, CTCF inhibition is counteracted by its BORIS paralogue that also binds the hTERT first exon but allows the promoter activation. The study of BORIS gene regulation showed that this factor is exclusively expressed in normal tissue of testis and ovary of young woman, as well as in almost all tumors with different levels. Two promoters were found to induce its transcription. The proximal promoter was regulated by methylation. Moreover, a major alternative transcript, deleted of the exon 6, is detected when this promoter is active. All these results lead to a model for hTERT regulation that takes into account the epigenetic profile of the gene and provides an explanation for the low transcriptional level observed in vivo. BORIS expression in cancers and its implication in hTERT activation might also permit the understanding of epigenetic deregulation and immortalization phenomena that occur during tumorigenesis.

Research resource: the dynamic transcriptional profile of sertoli cells during the progression of spermatogenesis.

Sertoli cells (SCs), the only somatic cells within seminiferous tubules, associate intimately with developing germ cells. They not only provide physical and nutritional support but also secrete factors essential to the complex developmental processes of germ cell proliferation and differentiation. The SC transcriptome must therefore adapt rapidly during the different stages of spermatogenesis. We report comprehensive genome-wide expression profiles of pure populations of SCs isolated at 5 distinct stages of the first wave of mouse spermatogenesis, using RNA sequencing technology. We were able to reconstruct about 13 901 high-confidence, nonredundant coding and noncoding transcripts, characterized by complex alternative splicing patterns with more than 45% comprising novel isoforms of known genes. Interestingly, roughly one-fifth (2939) of these genes exhibited a dynamic expression profile reflecting the evolving role of SCs during the progression of spermatogenesis, with stage-specific expression of genes involved in biological processes such as cell cycle regulation, metabolism and energy production, retinoic acid synthesis, and blood-testis barrier biogenesis. Finally, regulatory network analysis identified the transcription factors endothelial PAS domain-containing protein 1 (EPAS1/Hif2α), aryl hydrocarbon receptor nuclear translocator (ARNT/Hif1β), and signal transducer and activator of transcription 1 (STAT1) as potential master regulators driving the SC transcriptional program. Our results highlight the plastic transcriptional landscape of SCs during the progression of spermatogenesis and provide valuable resources to better understand SC function and spermatogenesis and its related disorders, such as male infertility.

Léiomyosarcome de la veine cave inférieure. A propos de trois observations [Leiomyosarcoma of the inferior vena cava. Three cases].

We report three new cases of leiomyosarcoma of the inferior vena cava observed between 1985 and 1990. Two of them developed in women and one in a man. The age of the patients were 56, 29 and 57 years. The three tumors have been removed completely. One patient received postoperative radio- and chemotherapy and is well 68 months after excision. One patient developed liver metastasis 18 months after resection despite chemotherapy and died after 3 years. The last one refused postoperative radio- and chemotherapy, developed massive local recurrence and pulmonary metastasis three months postoperatively, and is still alive with disease after 8 months. The diagnostic modalities of these rare tumors as well as the problems created by their surgical resection and the need for a complementary treatment are discussed.

Mid-gut ACTH-secreting neuroendocrine tumor unmasked with (18)F-dihydroxyphenylalanine-positron emission tomography.

Ectopic ACTH Cushing's syndrome (EAS) is often caused by neuroendocrine tumors (NETs) of lungs, pancreas, thymus, and other less frequent locations. Localizing the source of ACTH can be challenging. A 64-year-old man presented with rapidly progressing fatigue, muscular weakness, and dyspnea. He was in poor condition and showed facial redness, proximal amyotrophy, and bruises. Laboratory disclosed hypokalemia, metabolic alkalosis, and markedly elevated ACTH and cortisol levels. Pituitary was normal on magnetic resonance imaging (MRI), and bilateral inferior petrosal sinus blood sampling with corticotropin-releasing hormone stimulation showed no significant central-to-periphery gradient of ACTH. Head and neck, thoracic and abdominal computerized tomography (CT), MRI, somatostatin receptor scintigraphy (SSRS), and (18)F-deoxyglucose-positron emission tomography (FDG-PET) failed to identify the primary tumor. (18)F-dihydroxyphenylalanine (F-DOPA)-PET/CT unveiled a 20-mm nodule in the jejunum and a metastatic lymph node. Segmental jejunum resection showed two adjacent NETs, measuring 2.0 and 0.5 cm with a peritoneal metastasis. The largest tumor expressed ACTH in 30% of cells. Following surgery, after a transient adrenal insufficiency, ACTH and cortisol levels returned to normal values and remain normal over a follow-up of 26 months. Small mid-gut NETs are difficult to localize on CT or MRI, and require metabolic imaging. Owing to low mitotic activity, NETs are generally poor candidates for FDG-PET, whereas SSRS shows poor sensitivity in EAS due to intrinsically low tumor concentration of type-2 somatostatin receptors (SST2) or to receptor down regulation by excess cortisol. However, F-DOPA-PET, which is related to amine precursor uptake by NETs, has been reported to have high positive predictive value for occult EAS despite low sensitivity, and constitutes a useful alternative to more conventional methods of tumor localization. LEARNING POINTS: Uncontrolled high cortisol levels in EAS can be lethal if untreated.Surgical excision is the keystone of NETs treatment, thus tumor localization is crucial.Most cases of EAS are caused by NETs, which are located mainly in the lungs. However, small gut NETs are elusive to conventional imaging and require metabolic imaging for detection.FDG-PET, based on tumor high metabolic rate, may not detect NETs that have low mitotic activity. SSRS may also fail, due to absent or low concentration of SST2, which may be down regulated by excess cortisol.F-DOPA-PET, based on amine-precursor uptake, can be a useful method to localize the occult source of ACTH in EAS when other methods have failed.

Potential health-care service utilization substitution effects induced by case management for emergency department frequent users

BACKGROUND: In most of the emergency departments (ED) in developed countries, a subset of patients visits the ED frequently. Despite their small numbers, these patients are the source of a disproportionally high number of all ED visits, and use a significant proportion of healthcare resources. They place a heavy economic burden on hospital and healthcare systems budgets overall. Several interventions have been carried out to improve the management of these ED frequent users. Case management has been shown in some North American studies to reduce ED utilization and costs. In these studies, cost analyses have been carried out from the hospital perspective without examining the costs induced by healthcare consumed in the community. However, case management might reduce ED visits and costs from the hospital's perspective, but induce substitution effects, and increase health service utilization outside the hospital. This study examined if an interdisciplinary case-management intervention-compared to standard ED care -reduced costs generated by frequent ED users not only from the hospital perspective, but also from the healthcare system perspective-that is, from a broader perspective taking into account the costs of healthcare services used outside the hospital. METHODS: In this randomized controlled trial, 250 adult frequent emergency department users (5 or more visits during the previous 12 months) who visited the ED of the University Hospital of Lausanne, Switzerland, between May 2012 and July 2013 were allocated to one of two groups: case management intervention (CM) or standard ED care (SC), and followed up for 12 months. Depending on the perspective of the analysis, costs were evaluated differently. For the analysis from the hospital's perspective, the true value of resources used to provide services was used as a cost estimate. These data were obtained from the hospital's analytical accounting system. For the analysis from the health-care system perspective, all health-care services consumed by users and charged were used as an estimate of costs. These data were obtained from health insurance providers for a subsample of participants. To allow comparisons in a same time period, individual monthly average costs were calculated. Multivariate linear models including a fixed effect "group" were run using socio-demographic characteristics and health-related variables as controlling variables (age, gender, educational level, citizenship, marital status, somatic and mental health problems, and risk behaviors).

Frequent users of the emergency department in a universal health coverage system : a randomized controlled trial of a case-management intervention

BACKGROUND: Frequent emergency department users represent a small number of patients but account for a large number of emergency department visits. They should be a focus because they are often vulnerable patients with many risk factors affecting their quality of life (QoL). Case management interventions have resulted in a significant decrease in emergency department visits, but association with QoL has not been assessed. One aim of our study was to examine to what extent an interdisciplinary case management intervention, compared to standard emergency care, improved frequent emergency department users' QoL. METHODS: Data are part of a randomized, controlled trial designed to improve frequent emergency department users' QoL and use of health-care resources at the Lausanne University Hospital, Switzerland. In total, 250 frequent emergency department users (≥5 attendances during the previous 12 months; ≥ 18 years of age) were interviewed between May 2012 and July 2013. Following an assessment focused on social characteristics; social, mental, and somatic determinants of health; risk behaviors; health care use; and QoL, participants were randomly assigned to the control or the intervention group (n=125 in each group). The final sample included 194 participants (20 deaths, 36 dropouts, n=96 in the intervention group, n=99 in the control group). Participants in the intervention group received a case management intervention by an interdisciplinary, mobile team in addition to standard emergency care. The case management intervention involved four nurses and a physician who provided counseling and assistance concerning social determinants of health, substance-use disorders, and access to the health-care system. The participants' QoL was evaluated by a study nurse using the WHOQOL-BREF five times during the study (at baseline, and at 2, 5.5, 9, and 12 months). Four of the six WHOQOL dimensions of QoL were retained here: physical health, psychological health, social relationship, and environment, with scores ranging from 0 (low QoL) to 100 (high QoL). A linear, mixed-effects model with participants as a random effect was run to analyze the change in QoL over time. The effects of time, participants' group, and the interaction between time and group were tested. These effects were controlled for sociodemographic characteristics and health-related variables (i.e., age, gender, education, citizenship, marital status, type of financial resources, proficiency in French, somatic and mental health problems, and behaviors at risk).

Impact of case management on frequent users quality of life : a randomized controlled trial

BACKGROUND: Frequent emergency department users represent a small number of patients but account for a large number of emergency department visits. They should be a focus because they are often vulnerable patients with many risk factors affecting their quality of life (QoL). Case management interventions have resulted in a significant decrease in emergency department visits, but association with QoL has not been assessed. One aim of our study was to examine to what extent an interdisciplinary case management intervention, compared to standard emergency care, improved frequent emergency department users' QoL. METHODS: Data are part of a randomized, controlled trial designed to improve frequent emergency department users' QoL and use of health-care resources at the Lausanne University Hospital, Switzerland. In total, 250 frequent emergency department users (≥5 attendances during the previous 12 months; ≥ 18 years of age) were interviewed between May 2012 and July 2013. Following an assessment focused on social characteristics; social, mental, and somatic determinants of health; risk behaviors; health care use; and QoL, participants were randomly assigned to the control or the intervention group (n=125 in each group). The final sample included 194 participants (20 deaths, 36 dropouts, n=96 in the intervention group, n=99 in the control group). Participants in the intervention group received a case management intervention by an interdisciplinary, mobile team in addition to standard emergency care. The case management intervention involved four nurses and a physician who provided counseling and assistance concerning social determinants of health, substance-use disorders, and access to the health-care system.

Lymphoepithelial carcinoma in the maxillary sinus: a case report.

INTRODUCTION: Lymphoepithelial carcinoma of the maxillary sinus is a very rare malignancy and it can be difficult to make a pre-operative diagnosis. CASE PRESENTATION: A 72-year-old Caucasian woman presented to our facility with an isolated right-side epistaxis that had been present for three months, with the results of a computed tomography scan showing a soft tissue mass in the right maxillary sinus with an impacted tooth. The results of a transnasal endoscopic biopsy were compatible with a lymphoepithelial carcinoma, following which our patient underwent a radical excision of the mass. The final histology results revealed lymphoepithelial carcinoma of the maxillary sinus with negative assays for Epstein-Barr virus. Our patient was given post-operative external radiotherapy and has remained disease-free at three-year follow-up. CONCLUSIONS: This report details the diagnosis and management of a case of lymphoepithelial carcinoma of the maxillary sinus, which is a very rare malignant tumor with very little mention in the literature. Only a strong suspicion with systematic use of various patho-immunological tests helps to arrive at a definitive diagnosis by excluding other better-known tumors.

Embodied memory: unconscious smiling modulates emotional evaluation of episodic memories.

Since Damasio introduced the somatic markers hypothesis in Damasio (1994), it has spread through the psychological community, where it is now commonly acknowledged that somatic states are a factor in producing the qualitative dimension of our experiences. Present actions are emotionally guided by those somatic states that were previously activated in similar experiences. In this model, somatic markers serve as a kind of embodied memory. Here, we test whether the manipulation of somatic markers can modulate the emotional evaluation of negative memories. Because facial feedback has been shown to be a powerful means of modifying emotional judgements, we used it to manipulate somatic markers. Participants first read a sad story in order to induce a negative emotional memory and then were asked to rate their emotions and memory about the text. Twenty-four hours later, the same participants were asked to assume a predetermined facial feedback (smiling) while reactivating their memory of the sad story. The participants were once again asked to fill in emotional and memory questionnaires about the text. Our results showed that participants who had smiled during memory reactivation later rated the text less negatively than control participants. However, the contraction of the zygomaticus muscles during memory reactivation did not have any impact on episodic memory scores. This suggests that manipulating somatic states modified emotional memory without affecting episodic memory. Thus, modulating memories through bodily states might pave the way to studying memory as an embodied function and help shape new kinds of psychotherapeutic interventions.

Western blotting as a tool for the serodiagnosis of farmer's lung disease: validation with Lichtheimia corymbifera protein extracts.

Electrosyneresis and double diffusion are immunoprecipitation techniques commonly used in the serological diagnosis of Farmer's lung disease (FLD). These techniques are reliable but lack standardization. The aim of this study was to evaluate Western blotting for the serodiagnosis of FLD. We carried out Western blotting with an antigenic extract of Lichtheimia corymbifera, an important aetiological agent of the disease. The membranes were probed with sera from 21 patients with FLD and 21 healthy exposed controls to examine the IgG antibody responses against purified somatic antigens. Given the low prevalence of the disease, 21 patients could be considered as a relevant series. Four bands were significantly more frequently represented in membranes probed with FLD sera (bands at 27.7, 40.5, 44.0 and 50.5 kDa) than those probed with control sera. We assessed the diagnostic value of different criteria alone or in combination. The diagnostic accuracy of the test was highest with the inclusion of at least two of the following criteria: at least five bands on the strip and the presence of one band at 40.5 or 44.0 kDa. Sensitivity, specificity and positive and negative predictive values were all 81%, and the odds ratio was 18.06. Inclusion of bands of high intensity diminished rather than improved the diagnostic value of the test. We concluded that Western blotting is a valuable technique for the serodiagnosis of FLD. The industrial production of ready-to-use membranes would enable the routine use of this technique in laboratories, and provide reliable and standardized diagnostic results within a few hours.

Different locations of dermoid cysts in the orbital region.

BACKGROUND: Dermoid cysts are frequent unilateral congenital benign tumors that can be found at different locations throughout the body. In the orbital region, dermoid cysts occur predominantly in the supero-temporal quadrant. However, different orbital locations are possible, making this entity an important differential diagnosis for orbital tumors. PATIENTS AND METHODS: We retrospectively reviewed the charts of 23 consecutive patients operated in our institution between 2005 and 2014 for orbital tumors that were diagnosed as congenital dermoid or epidermoid cysts. RESULTS: There were 21 dermoid and 2 epidermoid cysts. The median age at surgery time was 7 years (range 1-41). There were 15 females and 8 males. Eleven cysts were located supero-temporally (47.8%), seven supero-medially (30.4%), two temporally (8.7%), one at the frontal bone (4.4%) and two in the fossa of the lacrimal gland (8.7%). The mean cyst diameter was 13.1 mm±5.0 (SD). None had signs of malignant disease. No recurrence was observed after complete excision. CONCLUSION: Dermoid cysts of orbital region are mostly located in the supero-temporal quadrant. However, other orbital locations are possible and dermoid cysts must thus be considered in the differential diagnosis of any mass in the orbital region.

Comparison of different methods to forecast hospital bed needs

Objectives : This study compares three methods to forecast the number of acute somatic hospital beds needed in a Swiss academic hospital over the period 2010-2030. Design : Information about inpatient stays is provided through a yearly mandatory reporting of Swiss hospitals, containing anonymized data. Forecast of the numbers of beds needed compares a basic scenario relying on population projections with two other methods in use in our country that integrate additional hypotheses on future trends in admission rates and length of stay (LOS).