CRISPR-Barcoding for Intratumor Genetic Heterogeneity Modeling and Functional Analysis of Oncogenic Driver Mutations.

Intratumor genetic heterogeneity underlies the ability of tumors to evolve and adapt to different environmental conditions. Using CRISPR/Cas9 technology and specific DNA barcodes, we devised a strategy to recapitulate and trace the emergence of subpopulations of cancer cells containing a mutation of interest. We used this approach to model different mechanisms of lung cancer cell resistance to EGFR inhibitors and to assess effects of combined drug therapies. By overcoming intrinsic limitations of current approaches, CRISPR-barcoding also enables investigation of most types of genetic modifications, including repair of oncogenic driver mutations. Finally, we used highly complex barcodes inserted at a specific genome location as a means of simultaneously tracing the fates of many thousands of genetically labeled cancer cells. CRISPR-barcoding is a straightforward and highly flexible method that should greatly facilitate the functional investigation of specific mutations, in a context that closely mimics the complexity of cancer.

Arteriovenous fistula after radial catheterization with cardiopulmonary repercussions.

This article describes the case of an 86-year-old coronary disease patient who underwent cardiac catheterization via a left radial access. Around 16 months after the procedure, he presented with dyspnea, unrelated to effort, but associated with nocturnal hypoxia. There was a palpable thrill in the left wrist and he was diagnosed with a radiocephalic arteriovenous fistula in the left wrist. A duplex scan revealed an abnormal wave pattern and increased diastolic velocity compatible with arteriovenous fistula. The fistula was repaired surgically and the patient exhibited improvement in clinical and laboratory parameters after the procedure. Radial access is increasingly being used for cardiac catheterization, primarily because it is associated with fewer and less harmful complications than femoral access. However, complications such as arteriovenous fistula occur and can be particularly harmful in octogenarian patients.

Long-Term Energy Deficit in Mice Causes Long-Lasting Hypothalamic Alterations after Recovery.

Although the short-term effects of fasting or energy deficit on hypothalamic neuropeptide circuitries are now better understood, the effects of long-term energy deficit and refeeding remain to be elucidated. We showed that after a long-term energy deficit, mice exhibited persistent hypoleptinemia following the refeeding period despite restoration of fat mass, ovarian activity, and feeding behavior. We aimed to examine the hypothalamic adaptations after 10 weeks of energy deficit and after 10 further weeks of nutritional recovery. To do so, we assessed the mRNA levels of the leptin receptor and the main orexigenic and anorexigenic peptides, and their receptors regulated by leptin. Markers of hypothalamic inflammation were assessed as leptin can also participate in this phenomenon. Long-term time-restricted feeding and separation induced significant increase in mRNA levels of hypothalamic orexigenic peptides, while both Y1 and Y5 receptor mRNAs were downregulated. No changes occurred in the mRNA levels of orexin (OX), melanin-concentrating hormone, pro-opiomelanocortin, 26RFa (26-amino acid RF-amide peptide), and their receptors despite an increase in the expression of melanocortin receptors (MC3-R and MC4-R) and OXR1 (OX receptor 1). The refeeding period induced an overexpression of leptin receptor mRNA in the hypothalamus. The other assessed mRNA levels were normalized except for Y2, Y5, MC3-R, and MC4-R, which remained upregulated. No convincing changes were observed in neuroinflammatory markers, even if interleukin-1ß mRNA levels were increased in parallel with those of Iba1 (ionized calcium-binding adaptor molecule 1), a marker of microglial activation. Normalization of leptin-regulated functions and hypothalamic gene expressions in refed mice with low plasma leptin levels could be sustained by recalibration of hypothalamic sensitivity to leptin.

Long-term exposure of Allonais inaequalis to a mixture of antibiotics in freshwater and synthetic wastewater matrices: Reproduction, recovery, and swimming responses.

Antibiotics from sulfonamide, fluoroquinolone, and diaminopyrimidine classes are widely used in human and veterinary medicine, and their combined occurrence in the aquatic environment is increasing around the world. In parallel, the understanding of how mixtures of these compounds affect non-target species from tropical freshwaters is scarce. Thus, this work aimed to study the long-term reproductive, recovery, and swimming effects of mixtures of 12 antibiotics from three different classes (up to 10 µg L-1 ) added to freshwater (FWM) and synthetic wastewater (SWM) matrices on freshwater worm Allonais inaequalis. Results revealed that at the reproduction level, the exposure to antibiotics in the SWM matrix does not cause a significant toxic effect on species after 10 days. On the other hand, exposures to initial dose mixtures (10 µg L-1 each) in FWM caused a significant reduction of offspring by 19.2%. In addition, recovery bioassays (10 days in an antibiotic-free environment) suggested that A. inaequalis has reduced offspring production due to previous exposure to antibiotic mixtures in both matrices. Furthermore, despite slight variation in swimming speed over treatments, no significant differences were pointed out. Regarding antibiotics in the water matrices after 10-day exposures, the highest concentrations were up to 2.7, 7.8, and 4.2 µg L-1 for antibiotics from sulfonamide, fluoroquinolone, and diaminopyrimidine classes, respectively. These findings suggest that a species positioned between primary producers and secondary consumers may experience late reproductive damage even in an antibiotic-free zone, after previous 10-day exposure to antibiotic mixtures. PRACTITIONER POINTS: A mixture of sulfonamide, fluoroquinolone, and diaminopyrimidine antibiotics in freshwater affects the offspring production of A. inaequalis after 10 days. After the 10-day antibiotic exposure, the reproduction of A. inaequalis remains affected in an antibiotic-free environment over the recovery period. The swimming speed of the worms does not change after 10 days of exposure to the antibiotic mixture. The concentration of dissolved solids can limit the natural degradation of sulfonamide, fluoroquinolone, and diaminopyrimidine antibiotics in the aquatic environment.

Mechanisms of Acquired Resistance and Tolerance to EGFR Targeted Therapy in Non-Small Cell Lung Cancer.

Non-small cell lung cancers (NSCLC) harboring activating mutations of the epidermal growth factor receptor (EGFR) are treated with specific tyrosine kinase inhibitors (EGFR-TKIs) of this receptor, resulting in clinically responses that can generally last several months. Unfortunately, EGFR-targeted therapy also favors the emergence of drug tolerant or resistant cells, ultimately resulting in tumor relapse. Recently, cellular barcoding strategies have arisen as a powerful tool to investigate the clonal evolution of these subpopulations in response to anti-cancer drugs. In this review, we provide an overview of the currently available treatment options for NSCLC, focusing on EGFR targeted therapy, and discuss the common mechanisms of resistance to EGFR-TKIs. We also review the characteristics of drug-tolerant persister (DTP) cells and the mechanistic basis of drug tolerance in EGFR-mutant NSCLC. Lastly, we address how cellular barcoding can be applied to investigate the response and the behavior of DTP cells upon EGFR-TKI treatment.

The RFamide neuropeptide 26RFa and its role in the control of neuroendocrine functions.

Identification of novel neuropeptides and their cognate G protein-coupled receptors is essential for a better understanding of neuroendocrine regulations. The RFamide peptides represent a family of regulatory peptides that all possess the Arg-Phe-NH2 motif at their C-terminus. In mammals, seven RFamide peptides encoded by five distinct genes have been characterized. The present review focuses on 26RFa (or QRFP) which is the latest member identified in this family. 26RFa is present in all vertebrate phyla and its C-terminal domain (KGGFXFRF-NH2), which is responsible for its biological activity, has been fully conserved during evolution. 26RFa is the cognate ligand of the orphan G protein-coupled receptor GPR103 that is also present from fish to human. In all vertebrate species studied so far, 26RFa-expressing neurons show a discrete localization in the hypothalamus, suggesting important neuroendocrine activities for this RFamide peptide. Indeed, 26RFa plays a crucial role in the control of feeding behavior in mammals, birds and fish. In addition, 26RFa up-regulates the gonadotropic axis in mammals and fish. Finally, evidence that the 26RFa/GPR103 system regulates steroidogenesis, bone formation, nociceptive transmission and arterial blood pressure has also been reported. Thus, 26RFa appears to act as a key neuropeptide in vertebrates controlling vital neuroendocrine functions. The pathophysiological implication of the 26RFa/GPR103 system in human is totally unknown and some fields of investigation are proposed.

Exploring the Mechanisms of Recovery in Anorexia Nervosa through a Translational Approach: From Original Ecological Measurements in Human to Brain Tissue Analyses in Mice.

Anorexia nervosa (AN) is a severe eating disorder where caloric restriction, excessive physical activity and metabolic alterations lead to life-threatening situations. Despite weight restoration after treatment, a significant part of patients experience relapses. In this translational study, we combined clinical and preclinical approaches. We describe preliminary data about the effect of weight gain on the symptomatology of patients suffering from acute AN (n = 225) and partially recovered (n = 41). We measured more precisely physical activity with continuous cardiac monitoring in a sub-group (n = 68). Using a mouse model, we investigated whether a long-term food restriction followed by nutritional recovery associated or not with physical activity may differentially impact peripheral and central homeostatic regulation. We assessed the plasma concentration of acyl ghrelin, desacyl ghrelin and leptin and the mRNA expression of hypothalamic neuropeptides and their receptors. Our data show an effect of undernutrition history on the level of physical activity in AN. The preclinical model supports an important role of physical activity in the recovery process and points out the leptin system as one factor that can drive a reliable restoration of metabolic variables through the hypothalamic regulation of neuropeptides involved in feeding behavior.

On the feasibility of the Rayleigh cycle for dynamic soaring trajectories.

Dynamic soaring is a flight technique used by albatrosses and other birds to cover large distances without the expenditure of energy, which is extracted from the available wind conditions, as brightly perceived five centuries ago by Leonardo da Vinci. Closed dynamic soaring trajectories use spatial variations of wind speed to travel, in principle, indefinitely over a prescribed area. The application of the concept of closed dynamic soaring trajectories to aerial vehicles, such as UAVs, may provide a solution to improve the endurance in certain missions. The main limitation of dynamic soaring is its dependence on the wind characteristics. More than one century ago, Lord Rayleigh proposed a very simple model, based on the repeated crossing of a step wind profile, presently known as Rayleigh cycle, that provides a clear explanation of the physical phenomenon. The present paper studies the feasibility of closed, single-loop, energy-neutral trajectories for a broad set of wind and vehicle conditions. Through the use of trajectory optimization methods, it was possible to see how the shape of the wind profile, the initial flight conditions and the vehicle constraints influence the required wind strength to perform dynamic soaring trajectories and consequently their feasibility. It was possible to conclude that there are optimal values for the initial airspeed and initial height of the vehicle, that minimize the required wind strength. In addition, it was seen how the structural and aerodynamic constraints of the vehicle affect dynamic soaring at high and low airspeeds respectively. Finally, some new trajectories that can be performed in conditions of excess wind are proposed. The purpose is to maximize the time spent aloft and the path length while maintaining the concept of single-loop, energy-neutral trajectories, making them especially useful for aerial vehicles surveillance applications.

Vasotocin and mesotocin stimulate the biosynthesis of neurosteroids in the frog brain.

The neurohypophysial nonapeptides vasopressin (VP) and oxytocin (OT) modulate a broad range of cognitive and social activities. Notably, in amphibians, vasotocin (VT), the ortholog of mammalian VP, plays a crucial role in the control of sexual behaviors. Because several neurosteroids also regulate reproduction-related behaviors, we investigated the possible effect of VT and the OT ortholog mesotocin (MT) in the control of neurosteroid production. Double immunohistochemical labeling of frog brain sections revealed the presence of VT/MT-positive fibers in close proximity of neurons expressing the steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) and cytochrome P450 17alpha-hydroxylase/c17, 20-lyase (P450(C17)). High concentrations of VT and MT receptor mRNAs were observed in diencephalic nuclei containing the 3beta-HSD and P450(C17) neuronal populations. Exposure of frog hypothalamic explants to graded concentrations of VT or MT produced a dose-dependent increase in the formation of progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, and dehydroepiandrosterone. The stimulatory effect of VT and MT on neurosteroid biosynthesis was mimicked by VP and OT, as well as by a selective V1b receptor agonist, whereas V2 and OT receptor agonists had no effect. VT-induced neurosteroid production was completely suppressed by selective V1a receptor antagonists and was not affected by V2 and OT receptor antagonists. Concurrently, the effect of MT on neurosteroidogenesis was markedly attenuated by selective OT and V1a receptor antagonists but not by a V2 antagonist. The present study provides the first evidence for a regulatory effect of VT and MT on neurosteroid biosynthesis. These data suggest that neurosteroids may mediate some of the behavioral actions of VT and MT.

Distribution of 26RFa binding sites and GPR103 mRNA in the central nervous system of the rat.

The novel RFamide peptide 26RFa, the endogenous ligand of the orphan receptor GPR103, affects food intake, locomotion, and activity of the gonadotropic axis. However, little is known regarding the localization of 26RFa receptors. The present report provides the first detailed mapping of 26RFa binding sites and GPR103 mRNA in the rat central nervous system (CNS). 26RFa binding sites were widely distributed in the brain and spinal cord, whereas the expression of GPR103 mRNA was more discrete, notably in the midbrain, the pons, and the medulla oblongata, suggesting that 26RFa can bind to a receptor(s) other than GPR103. Competition experiments confirmed that 26RFa interacts with an RFamide peptide receptor distinct from GPR103 that may be NPFF2. High densities of 26RFa binding sites were observed in olfactory, hypothalamic, and brainstem nuclei involved in the control of feeding behavior, including the piriform cortex, the ventromedial and dorsomedial hypothalamic nuclei, the paraventricular nucleus, the arcuate nucleus, the lateral hypothalamic area, and the nucleus of the solitary tract. The preoptic and anterior hypothalamic areas were also enriched with 26RFa recognition sites, supporting a physiological role of the neuropeptide in the regulation of the gonadotropic axis. A high density of 26RFa binding sites was detected in regions of the CNS involved in the processing of pain, such as the dorsal horn of the spinal cord and the parafascicular thalamic nucleus. The wide distribution of 26RFa binding sites suggests that 26RFa has multiple functions in the CNS that are mediated by at least two distinct receptors.

Expression of PACAP receptors in the frog brain during development.

This study describes the expression of PAC1 and VPAC1 receptor (PAC1-R and VPAC1-R) mRNAs in the brain of frog (Rana esculenta) during development. PAC1-R mRNA was detected in the periventricular and subependymal layers of the thalamus and epithalamus and in the ependymal layer of the mesencephalon and rhombencephalon (stage 20), in the amygdala, in the habenular complex, in the periventricular nucleus of the hypothalamus, and in the ventral cerebellum (stage 30). VPAC1-R mRNA expression was observed only at stage 20, in the floor of the hypothalamus. These results suggest that, in amphibians, pituitary adenylate cyclase-activating polypeptide (PACAP) may play a role in brain development.

The Neuropeptide 26RFa (QRFP) and Its Role in the Regulation of Energy Homeostasis: A Mini-Review.

This mini-review deals with the neuropeptide 26RFa (or QRFP) which is a member of the RFamide peptide family discovered simultaneously by three groups in 2003. 26RFa (or its N-extended form 43RFa) was subsequently shown to be the endogenous ligand of the human orphan receptor GPR103. In the brain, 26RFa and GPR103mRNA are primarily expressed in hypothalamic nuclei involved in the control of feeding behavior, and at the periphery, the neuropeptide and its receptor are present in abundance in the gut and the pancreatic islets, suggesting that 26RFa is involved in the regulation of energy metabolism. Indeed, 26RFa stimulates food intake when injected centrally, and its orexigenic effect is even more pronounced in obese animals. The expression of 26RFa is up-regulated in the hypothalamus of obese animals, supporting that the 26RFa/GPR103 system may play a role in the development and/or maintenance of the obese status. Recent data indicate that 26RFa is also involved in the regulation of glucose homeostasis. 26RFa reduces glucose-induced hyperglycemia, increases insulin sensitivity and insulinemia. Furthermore, an oral ingestion of glucose strongly stimulates 26RFa release by the gut, indicating that 26RFa is a novel incretin. Finally, 26RFa is able to prevent pancreatic ß cell death and apoptosis. This brief overview reveals that 26RFa is a key neuropeptide in the regulation of energy metabolism. Further fields of research are suggested including the pathophysiological implication of the 26RFa/GPR103 system.

The proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1 stimulate neuropeptide gene transcription and secretion in adrenochromaffin cells via activation of extracellularly regulated kinase 1/2 and p38 protein kinases, and activator protein-1 transcription factors.

Immune-autonomic interactions are known to occur at the level of the adrenal medulla, and to be important in immune and stress responses, but the molecular signaling pathways through which cytokines actually affect adrenal chromaffin cell function are unknown. Here, we studied the effects of the proinflammatory cytokines, TNF-alpha and IL-1, on gene transcription and secretion of bioactive neuropeptides, in primary bovine adrenochromaffin cells. TNF-alpha and IL-1 induced a time- and dose-dependent increase in galanin, vasoactive intestinal polypeptide, and secretogranin II mRNA levels. The two cytokines also stimulated the basal as well as depolarization-provoked release of enkephalin and secretoneurin from chromaffin cells. Stimulatory effects of TNF-alpha on neuropeptide gene expression and release appeared to be mediated through the type 2 TNF-alpha receptor, and required activation of ERK 1/2 and p38, but not Janus kinase, MAPKs. In addition, TNF-alpha increased the binding activity of activator protein-1 (AP-1) and stimulated transcription of a reporter gene containing AP-1-responsive elements in chromaffin cells. The AP-1-responsive reporter gene could also be activated through the ERK pathway. These results suggest that neuropeptide biosynthesis in chromaffin cells is regulated by TNF-alpha via an ERK-dependent activation of AP-1-responsive gene elements. Either locally produced or systemic cytokines might regulate biosynthesis and release of neuropeptides in chromaffin cells, integrating the adrenal medulla in the physiological response to inflammation. This study describes, for the first time, a signal transduction pathway activated by TNF-alpha in a major class of neuroendocrine cells that, unlike TNF-alpha signaling in lymphoid cells, employs ERK and p38 rather than Janus kinase and p38 to transmit gene-regulatory signals to the cell nucleus.

Impact of aflatoxin B1 on hypothalamic neuropeptides regulating feeding behavior.

The presence of mycotoxins in food is a major problem of public health as they produce immunosuppressive, hepatotoxic and neurotoxic effects. Mycotoxins also induce mutagenic and carcinogenic effects after long exposure. Among mycotoxins that contaminate food are aflatoxins (AF) such as AFB1, which is the most powerful natural carcinogen. The AF poisoning results in symptoms of depression, anorexia, diarrhea, jaundice or anemia that can lead to death, but very few studies have explored the impact of AF on neuroendocrine regulations. To better understand the neurotoxic effects of AF related to anorexia, we explored in rat the impact of AFB1 on the major hypothalamic neuropeptides regulating feeding behavior, either orexigenic (NPY, Orexin, AgRP, MCH) or anorexigenic (α-MSH, CART, TRH). We also studied the effect of AFB1 on a novel neuropeptide, the secretogranin II (SgII)-derived peptide EM66, which has recently been linked to the control of food intake. For this, adult male rats were orally treated twice a week for 5 weeks with a low dose (150 µg/kg) or a high dose (300 µg/kg) of AFB1 dissolved in corn oil. Repeated exposure to AFB1 resulted in reduced body weight gain, which was highly significant for the high dose of AF. Immunocytochemical and quantitative PCR experiments revealed a dose-related decrease in the expression of all the hypothalamic neuropeptides studied in response to AFB1. Such orexigenic and anorexigenic alterations may underlie appetite disorders as they are correlated to a dose-dependent decrease in body weight gain of treated rats as compared to controls. We also found a decrease in the number of EM66-containing neurons in the arcuate nucleus of AFB1-treated animals, which was associated with a lower expression of its precursor SgII. These findings show for the first time that repeated consumption of AFB1 disrupts the hypothalamic regulation of neuropeptides involved in feeding behavior, which may contribute to the lower body weight gain associated to AF exposure.

Hypothalamic Neuropeptide 26RFa Acts as an Incretin to Regulate Glucose Homeostasis.

26RFa is a hypothalamic neuropeptide that promotes food intake. 26RFa is upregulated in obese animal models, and its orexigenic activity is accentuated in rodents fed a high-fat diet, suggesting that this neuropeptide might play a role in the development and maintenance of the obese status. As obesity is frequently associated with type 2 diabetes, we investigated whether 26RFa may be involved in the regulation of glucose homeostasis. In the current study, we show a moderate positive correlation between plasma 26RFa levels and plasma insulin in patients with diabetes. Plasma 26RFa concentration also increases in response to an oral glucose tolerance test. In addition, we found that 26RFa and its receptor GPR103 are present in human pancreatic ß-cells as well as in the gut. In mice, 26RFa attenuates the hyperglycemia induced by a glucose load, potentiates insulin sensitivity, and increases plasma insulin concentrations. Consistent with these data, 26RFa stimulates insulin production by MIN6 insulinoma cells. Finally, we show, using in vivo and in vitro approaches, that a glucose load induces a massive secretion of 26RFa by the small intestine. Altogether, the present data indicate that 26RFa acts as an incretin to regulate glucose homeostasis.

Novel splice variants of type I pituitary adenylate cyclase-activating polypeptide receptor in frog exhibit altered adenylate cyclase stimulation and differential relative abundance.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts its various effects through activation of two types of G protein-coupled receptors, a receptor with high affinity for PACAP named PAC1-R and two receptors exhibiting similar affinity for both PACAP and vasoactive intestinal polypeptide named VPAC1-R and VPAC2-R. Here, we report the characterization of PAC1-R and novel splice variants in the frog Rana ridibunda. The frog PAC1-R has 78% homology with human PAC1-R and is highly expressed in the central nervous system. Two splice variants of the frog receptor that display additional amino acid cassettes in the third intracellular loop were characterized. PAC1-R25 carries a 25-amino acid insertion that matches the hop cassette of the mammalian receptor, whereas PAC1-R41 carries a cassette with no homology to any mammalian PAC1-R variant. A third splice variant of PAC1-R, exhibiting a completely different intracellular C-terminal domain, named PAC1-Rmc has also been identified. Determination of cAMP formation in cells transfected with the cloned receptors showed that PACAP activated PAC1-R, PAC1-R25, and PAC1-R41 with similar potency. In contrast, PACAP failed to stimulate adenylate cyclase in cells transfected with PAC1-Rmc. Fusion of PAC1-R or PAC1-Rmc with the green fluorescent protein revealed that both receptors are expressed and targeted to the plasma membrane in transfected cells. The different PAC1-R variants are highly expressed in the frog brain and spinal cord and to a lesser extent in peripheral tissues, where only certain isoforms could be detected. The present data indicate that in frog, PACAP may act through different PAC1-R splice variants that differ in their G(s) protein coupling and their abundance in various tissues.

Microarray and suppression subtractive hybridization analyses of gene expression in pheochromocytoma cells reveal pleiotropic effects of pituitary adenylate cyclase-activating polypeptide on cell proliferation, survival, and adhesion.

Pituitary adenylate cyclase-activating polypeptide (PACAP) exerts trophic effects on several neuronal, neuroendocrine, and endocrine cells. To gain insight into the pattern of the transcriptional modifications induced by PACAP during cell differentiation, we studied the effects of this neuropeptide on rat pheochromocytoma PC12 cells. We first analyzed the transcriptome of PC12 cells in comparison to that of terminally differentiated rat adrenomedullary chromaffin cells, using a high-density microarray, to identify genes associated with the proliferative phenotype that are possible targets of PACAP during differentiation of sympathoadrenal normal and tumoral cells. We then studied global gene expression in PC12 cells after 48 h of exposure to PACAP, using both cDNA microarray and suppression subtractive hybridization technologies. These complementary approaches resulted in the identification of 75 up-regulated and 70 down-regulated genes in PACAP-treated PC12 cells. Among the genes whose expression is modified in differentiated cells, a vast majority are involved in cell proliferation, survival, and adhesion/motility. Expression changes of most of these genes have been associated with progression of several neoplasms. A kinetic study of the effects of PACAP on some of the identified genes showed that the neuropeptide likely exerts early as well as late actions to achieve the gene expression program necessary for cell differentiation. In conclusion, the results of the present study underscore the pleiotropic role of PACAP in cell differentiation and provide important information on novel targets that could mediate the effects of this neuropeptide in normal and tumoral neuroendocrine cells.

Molecular characterization of frog chromogranin B reveals conservation of selective sequences encoding potential novel regulatory peptides.

Chromogranin B (CgB) is a member of the granin family of neuroendocrine secretory proteins, which has been proposed to play a role in secretory granule biogenesis and as a precursor to bioactive peptides. The cloning of CgB in a phylogenetically distant vertebrate, the frog Rana ridibunda, reveals a modest overall homology (35-40%) with mammalian CgB. However, the sequences of the N- and C-terminal regions are more highly conserved (57-65% amino acid identity) and may give rise to novel regulatory peptides. In frog, intense expression of CgB mRNA was observed in particular structures of the brain and in the distal lobe of the pituitary.

Fístula arteriovenosa pós-cateterismo radial com repercussões cardiopulmonares / Arteriovenous fistula after radial catheterization with cardiopulmonary repercussions

O presente artigo relata o caso de um paciente coronariopata de 86 anos submetido a cateterismo cardíaco via acesso radial à esquerda. Cerca de 16 meses após o procedimento, manifestou dispneia sem relação com esforço, associada a hipóxia noturna. Apresentava frêmito à palpação do punho esquerdo e foi diagnosticado com fístula arteriovenosa radiocefálica no punho esquerdo. Ao duplex scan apresentava alteração de padrão de onda e aumento da velocidade diastólica compatível com fístula arteriovenosa. Foi submetido a correção cirúrgica da fístula, apresentando melhora clínica e laboratorial após o procedimento. O acesso radial para cateterismo cardíaco tem sido cada vez mais utilizado, principalmente por causar complicações menos frequentes e menos deletérias em comparação ao acesso femoral. Entretanto, complicações como fístula arteriovenosa ocorrem e podem ser especialmente prejudiciais em pacientes octogenários

This article describes the case of an 86-year-old coronary disease patient who underwent cardiac catheterization via a left radial access. Around 16 months after the procedure, he presented with dyspnea, unrelated to effort, but associated with nocturnal hypoxia. There was a palpable thrill in the left wrist and he was diagnosed with a radiocephalic arteriovenous fistula in the left wrist. A duplex scan revealed an abnormal wave pattern and increased diastolic velocity compatible with arteriovenous fistula. The fistula was repaired surgically and the patient exhibited improvement in clinical and laboratory parameters after the procedure. Radial access is increasingly being used for cardiac catheterization, primarily because it is associated with fewer and less harmful complications than femoral access. However, complications such as arteriovenous fistula occur and can be particularly harmful in octogenarian patients

The orexin type 1 receptor is overexpressed in advanced prostate cancer with a neuroendocrine differentiation, and mediates apoptosis.

AIM: In the present study, we have examined the presence of orexins and their receptors in prostate cancer (CaP) and investigated their effects on the apoptosis of prostate cancer cells. METHODS: We have localised the orexin type 1 and 2 receptors (OX1R and OX2R) and orexin A (OxA) in CaP sections of various grades and we have quantified tumour cells containing OX1R. Expression of OX1R was evaluated in the androgeno-dependent (AD) LNCaP and the androgeno-independent (AI) DU145 prostate cancer cells submitted or not to a neuroendocrine differentiation. The effects of orexins on the apoptosis and viability of DU145 cells were also investigated. RESULTS: OX1R is strongly expressed in carcinomatous foci exhibiting a neuroendocrine differentiation, and the number of OX1R-stained cancer cells increases with the grade of the CaP. In contrast, OX2R is only detected in scattered malignant cells in high grade CaP. OX1R is expressed in the AI DU145 cells but is undetectable in the LNCaP cells. Acquisition of a neuroendocrine phenotype by the DU145 cells is associated with an overexpression of OX1R. Orexins induce the apoptosis of DU145 cells submitted to a neuroendocrine differentiation. CONCLUSION: The present data indicate that OX1R-driven apoptosis is overexpressed in AI CaP exhibiting a neuroendocrine differentiation opening a gate for novel therapies for these aggressive cancers which are incurable until now.