Overall survival and prognostic factors in Stage I lung adenocarcinoma treated with curative intent: A real-life 19-year cohort study.

OBJECTIVES: Important differences in Stage I non-small-cell lung cancer (NSCLC) are related to the delay in the diagnosis to the treatment, hospitals' specialised status, comorbidities, tumour stage and histological type. METHODS: A 19-year retrospective cohort study was conducted, including 681 patients with NSCLC in clinical-stage IA-IB. The variables analysed were gender, age, schooling, type of health care provider, type of treatment, period of 5-year treatment, the time between first attendance to diagnosis and the time between diagnosis and treatment, and hospital's specialised status. RESULTS: Patients who underwent radiotherapy alone had three times more risk of death than those who underwent surgery alone (adjusted hazard ratio [adjHR] = 3.44; 95% confidence interval [CI]: 2.45-4.82; p <0.001). The independent risk of death factors was being treated in nonhigh complexity centres in oncology hospitals and having started the treatment more than 2 months after diagnosis (adjHR = 1.80; 95% CI: 1.26-2.56; p <0.001) and (adjHR = 2.00; 95% CI: 1.33-3.00; p <0.001), respectively. In addition, the patients diagnosed between 2011 and 2015 had a 40% lower risk of death when compared to those diagnosed between 2000 and 2005 (95% CI: 0.38-0.94; p = 0.027). CONCLUSION: The overall survival in curative intent Stage-I lung cancer patients' treatment was associated with the 5-year diagnosis group, the delayed time between diagnosis and treatment and the hospital qualification.

Efficacy and safety of a device used to prevent fogging and residue buildup on surgical optics during video-assisted thoracic surgical procedures (LacrimaSurg): a pilot in vivo study in 30 patients.

INTRODUCTION: Nowadays the majority of the surgical procedures are video surgeries. Despite technological advances, some problems remain. The buildup of residues in front of the lens is an example of this problem. To solve it, Abreu et al. (Surg Technol Int 26:43-47, 2015) developed LacrimaSurg, which works as a lacrimal duct in the optics preventing fogging and residues buildup on it. This had 98.1% of efficiency in an experimental study. OBJECTIVE: Study feasibility and safety of the device under real surgical conditions. MATERIALS AND METHODS: A pilot study included 30 patients undergoing thoracic procedures using LacrimaSurg. Procedures were recorded for further analysis by investigators. The characteristics of patients (age, sex, body mass index, Charlton comorbidity index), data of environment (Temperatures variances), and data of procedures (type of surgery and duration of it) were recorded. The investigators watched the videos and recorded the residue buildup and fogging events that impaired surgeon's vision. The number of times the optics got dirty, need for further jets of crystalloid solution, and need for mechanical cleaning were collected to evaluate efficacy. Comparative serum measurements of electrolytes before and after surgery were studied. Data were organized and analyzed by simple linear correlations and t test for continuous variables and Fisher's test for categorical variables. RESULTS: The use of the device allowed 97% of the procedures to be performed without interruption. No characteristics inherent to the patients or to the procedures that lead to fogging or filth of the optics were identified. There were no statistically significant differences between pre- and postoperative serum ions, and no postoperative complications related to the use of the device were observed. CONCLUSION: The device was effective in preventing interruption of surgical procedures due to dirt or fogging, its use was considered safe, and did not cause postoperative complications.

A new subfamily of ionotropic glutamate receptors unique to the echinoderms with putative sensory role.

Chemosensation is a critical signalling process in animals and especially important in sea cucumbers, a group of ecologically and economically important marine echinoderms (class Holothuroidea), which lack audio and visual organs and rely on chemical sensing for survival, feeding and reproduction. The ionotropic receptors are a recently identified family of chemosensory receptors in insects and other protostomes, related to the ionotropic glutamate receptor family (iGluR), a large family of membrane receptors in metazoan. Here we characterize the echinoderm iGluR subunits and consider their possible role in chemical communication in sea cucumbers. Sequence similarity searches revealed that sea cucumbers have in general a higher number of iGluR subunits when compared to other echinoderms. Phylogenetic analysis and sequence comparisons revealed GluH as a specific iGluR subfamily present in all echinoderms. Homologues of the vertebrate GluA (aka α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA), GluK (aka kainate) and GluD (aka delta) were also identified. The GluN (aka N-methyl-d-aspartate, NMDA) as well as the invertebrate deuterostome subfamily GluF (aka phi) are absent in echinoderms. The echinoderm GluH subfamily shares conserved structural protein organization with vertebrate iGluRs and the ligand binding domain (LBD) is the most conserved region; genome analysis indicates evolution via lineage and species-specific tandem gene duplications. GluH genes (named Grih) are the most highly expressed iGluRs subunit genes in tissues in the sea cucumber Holothuria arguinesis, with Griha1, Griha2 and Griha5 exclusively expressed in tentacles, making them candidates to have a chemosensory role in this species. The multiple GluH subunits may provide alternative receptor assembly combinations, thus expanding the functional possibilities and widening the range of compounds detected during aggregation and spawning in echinoderms.

Sole head transcriptomics reveals a coordinated developmental program during metamorphosis.

Most teleosts undergo a thyroid hormone (TH) regulated larval to juvenile transition known as metamorphosis. In Pleuronectiformes (flatfish), metamorphosis is most dramatic, and one eye of the symmetric pelagic larvae migrates to the opposite side of the head, giving rise to an asymmetric benthic juvenile with both eyes on the same side of the body. Asymmetric development occurs mostly in the head. To understand the genetic mechanisms underlying this developmental change we have generated a Solea senegalensis metamorphosing flatfish head transcriptome. Our results reveal that THs acting as integrative factors direct a stepwise genetic program that initiates a specific organismal level response followed by cell specific responses that lead to the long-term changes that characterise the post-metamorphic identity and physiology of the head. Flatfish head asymmetric development during metamorphosis and its TH dependency is conserved thus we consider the findings in sole most likely representative of all flatfish species.

The Low-Cost, Long-Term Indwelling Pleural Catheter (LunGO): An Inexpensive and Effective Alternative for the Management of Dyspnea Associated with Malignant Pleural Effusions.

BACKGROUND: A malignant pleural effusion (MPE) in patients with cancer is evidence of the underlying disease progression, resulting in many symptoms, and may lead to hospitalization. The treatment options include talc pleurodesis (TP) or the implantation of an indwelling pleural catheter. The costs of the latter approach are often seen to be inhibitive for the Brazilian and other emerging markets' public health system. OBJECTIVES: To assess the feasibility of utilizing a low-cost device (LunGO) through a case-control study. METHODS: Eighteen patients with recurrent neoplastic pleural effusion and contraindications to pleurodesis were recruited between June 2016 and November 2017. The patients were submitted to pleural catheter prototype implantation. Data on the underlying disease and hospital length of stay after the procedure were collected and compared with patients who underwent pleurodesis in the same period (control group, n = 34). RESULTS: In the LunGO group, 7 patients died due to the natural evolution of the underlying disease with the drain, whereas it was removed in 11 patients at a median of 43 days. Recurrence requiring an intervention was seen with the LunGO in 2, compared to 5 (OR = 1.37, p = 1) with TP. Complications were observed in only 1 with the LunGO, compared to 5 with TP. The chances of recurrence in both cohorts do not have a statistically significant difference, with an OR = 1.08 (p = 0.93). There was a tendency towards lower mortality in the LunGO cohort, despite that fact that we did not observe statistical significance (OR = 0.16, p = 0.23). CONCLUSION: LunGO was shown to be a viable and safe device for the treatment of symptomatic MPE.

Prognostic factors in patients with malignant pleural effusion: Is it possible to predict mortality in patients with good performance status?

BACKGROUND AND OBJECTIVES: The aim of this study was to identify predictors of mortality only in patients with malignant pleural effusion (MPE) showing good performance status which required pleural palliative procedures. METHODS: All patients with MPE submitted to pleural palliative procedure were enrolled in a prospective study between 2013 and 2014. Patients with Eastern cooperative oncology group (ECOG) score zero, one, and two were considered with good performance status. The possible prognostic factors were tested for significance using the log-rank test (Kaplan-Meier method) and those with significance on univariate analysis were entered into a multivariable Cox model. RESULTS: A total of 64 patients were included in the analysis. Median follow-up time for surviving patients was 263 days. Median survival for the entire cohort was not reached yet. In the multivariate analysis, gastrointestinal primary site (P = 0.006), low albumin concentration in the pleural fluid (P = 0.017), and high serum NLR (P = 0.007) were associated with mortality. CONCLUSION: In our cohort of ECOG 0-2 patients with MPE submitted to pleural palliative procedures, gastrointestinal malignancy compared to other sites, low pleural fluid albumin and high NLR were significantly associated with mortality. The identification of these prognostic factors may assist the choice of the optimal palliative technique. J. Surg. Oncol. 2016;113:570-574. © 2016 Wiley Periodicals, Inc.

Prognostic Factors of 30-Day Mortality After Palliative Procedures in Patients with Malignant Pleural Effusion.

BACKGROUND: The aim of this study was to identify predictors of 30-day mortality in patients with malignant pleural effusion (MPE) who need pleural palliative procedures. METHODS: Data was prospectively collected from our database, between January 2013 and July 2014 on 86 patients with MPE and complete follow-up 30 days after the procedure. The sample was divided into two groups. The first group (G1) included patients who had died up to 30 days after the palliative procedure, and the second group (G2) included patients who survived for more than 30 days after the palliative procedure. The identification of prognostic factors occurred through univariate analysis using Fisher exact test for analysis of categorical variables and the t test for quantitative variables. Subsequently, all variables were used in the multivariate logistic regression analysis. The cutoff values for any significant continuous variables were determined by receiver operating characteristics analysis. RESULTS: There were 24 patients in G1 and 62 patients in G2. Univariate analysis of factors affecting postprocedural survival disclosed nine factors that were associated with significantly reduced postoperative survival. At the multivariate analysis, high levels of white blood cells, (p = .013), low levels of red blood cells (p < .0001) and protein in pleural fluid (p = .001), and primary lung and gastrointestinal sites (p = .0076) were identified as independent predictors of mortality. CONCLUSIONS: We identified four factors that are easily recognized in daily practice and can help select patients with low life expectancy. Therefore, invasive procedures and hospitalizations for this subgroup of patients can be prevented.

Catheter for Cleaning Surgical Optics During Surgical Procedures: A Possible Solution for Residue Buildup and Fogging in Video Surgery.

Currently, there is a tendency to perform surgical procedures via laparoscopic or thoracoscopic access. However, even with the impressive technological advancement in surgical materials, such as improvement in quality of monitors, light sources, and optical fibers, surgeons have to face simple problems that can greatly hinder surgery by video. One is the formation of "fog" or residue buildup on the lens, causing decreased visibility. Intracavitary techniques for cleaning surgical optics and preventing fog formation have been described; however, some of these techniques employ the use of expensive and complex devices designed solely for this purpose. Moreover, these techniques allow the cleaning of surgical optics when they becomes dirty, which does not prevent the accumulation of residue in the optics. To solve this problem we have designed a device that allows cleaning the optics with no surgical stops and prevents the fogging and residue accumulation. The objective of this study is to evaluate through experimental testing the effectiveness of a simple device that prevents the accumulation of residue and fogging of optics used in surgical procedures performed through thoracoscopic or laparoscopic access. Ex-vivo experiments were performed simulating the conditions of residue presence in surgical optics during a video surgery. The experiment consists in immersing the optics and catheter set connected to the IV line with crystalloid solution in three types of materials: blood, blood plus fat solution, and 200 mL of distilled water and 1 vial of methylene blue. The optics coupled to the device were immersed in 200 mL of each type of residue, repeating each immersion 10 times for each distinct residue for both thirty and zero degrees optics, totaling 420 experiments. A success rate of 98.1% was observed after the experiments, in these cases the device was able to clean and prevent the residue accumulation in the optics.

Heritability of cortisol response to confinement stress in European sea bass dicentrarchus labrax.

BACKGROUND: In fish, the most studied production traits in terms of heritability are body weight or growth, stress or disease resistance, while heritability of cortisol levels, widely used as a measure of response to stress, is less studied. In this study, we have estimated heritabilities of two growth traits (body weight and length) and of cortisol response to confinement stress in the European sea bass. FINDINGS: The F1 progeny analysed (n = 922) belonged to a small effective breeding population with contributions from an unbalanced family structure of just 10 males and 2 females. Heritability values ranged from 0.54 (± 0.21) for body weight to 0.65 (± 0.22) for standard body length and were low for cortisol response i.e. 0.08 (± 0.06). Genetic correlations were positive (0.94) between standard body length and body weight and negative between cortisol and body weight and between cortisol and standard body length (-0.60 and -0.55, respectively). CONCLUSION: This study confirms that in European sea bass, heritability of growth-related traits is high and that selection on such traits has potential. However, heritability of cortisol response to stress is low in European sea bass and since it is known to vary greatly among species, further studies are necessary to understand the reasons for these differences.

Prognostic factors and patients' profile in treated stage I and II lung adenocarcinoma: a Hospital's Cancer Registry-based analysis.

BACKGROUND: It is known that survival from lung cancer can differ between countries and even between different regions of the same country. The variability between hospitals, the age and social profile, and the time when this patient was treated, can influence survival, and these factors are intrinsic to each region. Knowing the profile of patients, hospitals, and other factors associated with the treatment of stage I and II lung cancer in a given region is important to understand outcomes and propose improvements that can be replicated in any region of the world that presents the same profile of patients and care structure. This study evaluates survival and possible predictors in all patients with stage I and II lung cancer adenocarcinoma through the Hospital's Cancer Registry (HCR), responsible for the State of Sao Paulo's cancer registry, a geographical area with 40 million inhabitants. METHODS: Based on the HCR, an observational study was conducted, including 1,278 patients diagnosed with lung adenocarcinoma at clinical stages (CS) I and II. Sex, age at diagnosis, education, neighbourhood, CS at diagnosis, the time between diagnosis and treatment, 5-year periods in which patients were treated, treatment modality and hospitals where patients were treated were analysed. Cox univariate and multiple regression analyses were used to estimate the hazard ratio (HR). RESULTS: A total of 1,278 lung cancer patients with clinical lung cancer adenocarcinoma stages I and II were included. About 40.06% of patients did not receive surgery, and only 55.8% started the treatment within 2 months. The majority of the patients were treated in high complexity hospitals, 69%. Five-year overall survival (OS) was 45.6% in CS I and 27.5% in CS II. Patients treated in high complexity centres have lower mortality rates than those treated in Partial Hospital Complexity Centers in Oncology (PHCCO) (adjHR 1.18; 95% CI: 1.00-1.40; P=0.047). Patients diagnosed between 2010-2014 had a protective factor against the risk of death concerning patients diagnosed between 2000-2004. CONCLUSIONS: The 5-year OS has significantly improved as long as the 5-year group analysed. Also, the 5-year OS of the patients treated in high complexity hospitals is higher than those treated in PHCCO.

Real-Life Long-Term Cohort of Patients With Stage IIIA Non-Small-Cell Lung Cancer: Overall Survival Related to Patients' Characteristics and Multiple Treatment Models.

PURPOSE: This real-life cohort of patients describes the treatment patterns and compares the overall survival (OS) and hazard risk of utilization of multiple therapies. MATERIALS AND METHODS: Electronic medical registries of patients with stage III non-small-cell lung cancer (NSCLC) regularly attended in 72 hospitals were included. Univariate and multivariate analyses were conducted to evaluate the primary patients' characteristics leading to better OS and cancer-specific survival. RESULTS: A total of 3,363 patients with stage III NSCLC followed over 19 years were included in this study. The median age was 66.00 (58.00-72.00) years, 65% male, and 41.2% with squamous cell carcinoma followed by adenocarcinoma (34.6%) and undifferentiated carcinoma (13.1%) in clinical stage T3 (50.3%), T2 (29.3%), and T4 (12.3%). The median survival (in months) was 18.4 (95% CI, 16.9 to 19.5) in patients submitted to radiotherapy plus chemotherapy, 11.2 (95% CI, 10.5 to 12.1) to chemotherapy, 31.5 (95% CI, 25.9 to 37.7) to surgery plus chemotherapy, and 33.8 (95% CI, 28.3 to 47.8) to chemotherapy plus radiotherapy plus surgery. The median cancer-specific survival (in months) was 19.3 (95% CI, 17.9 to 20.9) in patients submitted to radiotherapy plus chemotherapy, 12.1 (95% CI, 11.1 to 12.9) to chemotherapy, 36.9 (95% CI, 29.6 to 43.2) to surgery plus chemotherapy, and 41.3 (95% CI, 32.1 to 61.3) to chemotherapy plus radiotherapy plus surgery. The patients treated with multiple chemotherapy plus radiotherapy followed by surgery had significantly better OS and lower mortality rates than those treated with other treatments (adjusted hazard ratio, 0.55; 95% CI, 0.45 to 0.66; P < .001). At the end of the study, 11.2% and 10.7% of the patients were living with and without cancer, respectively. CONCLUSION: Our real-life 19-year cohort study has shown that only 30.3% of the total patients with stage III NSCLC have been submitted to standard chemotherapy and radiotherapy treatment. This may show a substantial difference between the recruited clinical trials' patients and the real-life patients' characteristics in daily routine treatment.

Holothurians have a reduced GPCR and odorant receptor-like repertoire compared to other echinoderms.

Sea cucumbers lack vision and rely on chemical sensing to reproduce and survive. However, how they recognize and respond to environmental cues remains unknown. Possible candidates are the odorant receptors (ORs), a diverse family of G protein-coupled receptors (GPCRs) involved in olfaction. The present study aimed at characterizing the chemosensory GPCRs in sea cucumbers. At least 246 distinct GPCRs, of which ca. 20% putative ORs, were found in a transcriptome assembly of putative chemosensory (tentacles, oral cavity, calcareous ring, and papillae/tegument) and reproductive (ovary and testis) tissues from Holothuria arguinensis (57 ORs) and in the Apostichopus japonicus genome (79 ORs). The sea cucumber ORs clustered with those of sea urchin and starfish into four main clades of gene expansions sharing a common ancestor and evolving under purifying selection. However, the sea cucumber ORs repertoire was the smallest among the echinoderms and the olfactory receptor signature motif LxxPxYxxxxxLxxxDxxxxxxxxP was better conserved in cluster OR-l1 which also had more members. ORs were expressed in tentacles, oral cavity, calcareous ring, and papillae/tegument, supporting their potential role in chemosensing. This study is the first comprehensive survey of chemosensory GPCRs in sea cucumbers, and provides the molecular basis to understand how they communicate.

A haplotype-resolved draft genome of the European sardine (Sardina pilchardus).

BACKGROUND: The European sardine (Sardina pilchardus Walbaum, 1792) is culturally and economically important throughout its distribution. Monitoring studies of sardine populations report an alarming decrease in stocks due to overfishing and environmental change, which has resulted in historically low captures along the Iberian Atlantic coast. Important biological and ecological features such as population diversity, structure, and migratory patterns can be addressed with the development and use of genomics resources. FINDINGS: The genome of a single female individual was sequenced using Illumina HiSeq X Ten 10x Genomics linked reads, generating 113.8 gigabase pairs of data. Three draft genomes were assembled: 2 haploid genomes with a total size of 935 megabase pairs (N50 103 kilobase pairs) each, and a consensus genome of total size 950 megabase pairs (N50 97 kilobase pairs). The genome completeness assessment captured 84% of Actinopterygii Benchmarking Universal Single-Copy Orthologs. To obtain a more complete analysis, the transcriptomes of 11 tissues were sequenced to aid the functional annotation of the genome, resulting in 40,777 genes predicted. Variant calling on nearly half of the haplotype genome resulted in the identification of >2.3 million phased single-nucleotide polymorphisms with heterozygous loci. CONCLUSIONS: A draft genome was obtained, despite a high level of sequence repeats and heterozygosity, which are expected genome characteristics of a wild sardine. The reference sardine genome and respective variant data will be a cornerstone resource of ongoing population genomics studies to be integrated into future sardine stock assessment modelling to better manage this valuable resource.

A thyroid hormone regulated asymmetric responsive centre is correlated with eye migration during flatfish metamorphosis.

Flatfish metamorphosis is a unique post-embryonic developmental event in which thyroid hormones (THs) drive the development of symmetric pelagic larva into asymmetric benthic juveniles. One of the eyes migrates to join the other eye on the opposite side of the head. Developmental mechanisms at the basis of the acquisition of flatfish anatomical asymmetry remain an open question. Here we demonstrate that an TH responsive asymmetric centre, determined by deiodinase 2 expression, ventrally juxtaposed to the migrating eye in sole (Solea senegalensis) correlates with asymmetric cranial ossification that in turn drives eye migration. Besides skin pigmentation that is asymmetric between dorsal and ventral sides, only the most anterior head region delimited by the eyes becomes asymmetric whereas the remainder of the head and organs therein stay symmetric. Sub-ocular ossification is common to all flatfish analysed to date, so we propose that this newly discovered mechanism is universal and is associated with eye migration in all flatfish.

Wet M1a non-small cell lung cancer: is it possible to predict recurrence of pleural effusion?

BACKGROUND: The propose was to recognize risk factors of malignant pleural effusion (MPE) recurrence in patients with symptomatic M1a non-small cell lung cancer (NSCLC). METHODS: All patients with NSCLC and MPE submitted to pleural palliative procedures were enrolled in a prospective study. Group I contained patients who had pleural recurrence, and Group II with no pleural recurrence. Prognostic factors for pleural recurrence were identified by univariable analysis, using Fisher's exact test for categorical variables and Student's t test for quantitative variables. Afterwards the significant variables were entered into a multivariable logistic regression analysis (with P<0.05 considered significant). Receiver operating characteristics (ROC) analysis determined the cutoff points for continuous variables. RESULTS: A total of 82 patients were included in the analysis. There were 15 patients (18.3%) in Group I and 67 patients (81.7%) in Group II. Univariable analysis regarding factors affecting postoperative recurrence was: adenosine deaminase concentration in pleural fluid <16 mg/dL (P=0.04), albumin concentration in pleural fluid <2.4 mg/dL (P=0.03), administration of second-line palliative chemotherapy (P=0.018) and type of procedure [therapeutic pleural aspiration (TPA)] (P=0.023). At the multivariable analysis, only the type of procedure (TPA) (P=0.031) was identified as independent predictor of recurrence. CONCLUSIONS: The identification of this factor may assist the choice of the optimal palliative technique; at the first episode of MPE in NSCLC patients and definitive procedure as pleurodesis or indwelling pleural catheter are recommended.

Interaction between treatment delivery delay and stage on the mortality from non-small cell lung cancer.

BACKGROUND: The aim of this study is to evaluate the interaction between treatment delay and stage on the mortality from non-small cell lung cancer (NSCLC). METHODS: We performed a survival analysis in a cohort of patients admitted to the reference cancer center. The following data were collected: age, gender, smoking status, tumor staging, type of lung cancer, and time from the date when the patient was diagnosed with cancer to the starting date of effective treatment. Univariable and multivariable Cox proportional hazard models were used to investigate the association between potential confounders identified during the study design. After the final adjusted model was determined, tests for interaction among all predictors were performed. RESULTS: Inclusion criteria were met by 359 patients. In the adjusted analysis, delayed treatment delivery was a protective factor for the risk of death, with a crude hazard ratio (HR) =0.75 (0.59-0.97; P=0.02) and an adjusted HR =0.59 (0.46-0.77; P<0.001). However, a statistically significant interaction with mortality was observed between timely treatment and tumor stage. Patients with stage II disease who received delayed treatment had a higher risk of death [HR =3.08 (1.05-9.0; P=0.04)]. On the other hand, stage IV patients who received delayed treatment had a 52% reduction in mortality [HR =0.48 (0.35-0.66; P<0.001)]. CONCLUSIONS: Stage of disease influenced the association between start of the treatment and mortality, and only the subgroup of stage II patients seemed to benefit from early treatment.

Genomic analysis of Sparus aurata reveals the evolutionary dynamics of sex-biased genes in a sequential hermaphrodite fish.

Sexual dimorphism is a fascinating subject in evolutionary biology and mostly results from sex-biased expression of genes, which have been shown to evolve faster in gonochoristic species. We report here genome and sex-specific transcriptome sequencing of Sparus aurata, a sequential hermaphrodite fish. Evolutionary comparative analysis reveals that sex-biased genes in S. aurata are similar in number and function, but evolved following strikingly divergent patterns compared with gonochoristic species, showing overall slower rates because of stronger functional constraints. Fast evolution is observed only for highly ovary-biased genes due to female-specific patterns of selection that are related to the peculiar reproduction mode of S. aurata, first maturing as male, then as female. To our knowledge, these findings represent the first genome-wide analysis on sex-biased loci in a hermaphrodite vertebrate species, demonstrating how having two sexes in the same individual profoundly affects the fate of a large set of evolutionarily relevant genes.

Persistence of duplicated PAC1 receptors in the teleost, Sparus auratus.

BACKGROUND: : Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC1 receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown. RESULTS: : Duplicate sea bream PACAP receptor genes (sbPAC1A and sbPAC1B), members of family 2 GPCRs, were isolated and share 77% amino acid sequence identity. RT-PCR with specific primers for each gene revealed that they have a differential tissue distribution which overlaps with the distribution of the single mammalian receptor. Furthermore, in common with mammals, the teleost genes undergo alternative splicing and a PAC1Ahop1 isoform has been characterised. Duplicated orthologous receptors have also been identified in other teleost genomes and their distribution profile suggests that function may be species specific. Functional analysis of the paralogue sbPAC1s in Cos7 cells revealed that they are strongly stimulated in the presence of mammalian PACAP27 and PACAP38 and far less with VIP (Vasoactive Intestinal Peptide). The sbPAC1 receptors are equally stimulated (LOGEC50 values for maximal cAMP production) in the presence of PACAP27 (-8.74 +/- 0.29 M and -9.15 +/- 0.21 M, respectively for sbPAC1A and sbPAC1B, P > 0.05) and PACAP38 (-8.54 +/- 0.18 M and -8.92 +/- 0.24 M, respectively for sbPAC1A and sbPAC1B, P > 0.05). Human VIP was found to stimulate sbPAC1A (-7.23 +/- 0.20 M) more strongly than sbPAC1B (-6.57 +/- 0.14 M, P < 0.05) and human secretin (SCT), which has not so far been identified in fish genomes, caused negligible stimulation of both receptors. CONCLUSION: : The existence of functionally divergent duplicate sbPAC1 receptors is in line with previously proposed theories about the origin and maintenance of duplicated genes. Sea bream PAC1 duplicate receptors resemble the typical mammalian PAC1, and PACAP peptides were found to be more effective than VIP in stimulating cAMP production, although sbPAC1A was more responsive for VIP than sbPAC1B. These results together with the highly divergent pattern of tissue distribution suggest that a process involving neofunctionalisation occurred after receptor duplication within the fish lineage and probably accounts for their persistence in the genome. The characterisation of further duplicated receptors and their ligands should provide insights into the evolution and function of novel protein-protein interactions associated with the vertebrate radiation.

A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis.

BACKGROUND: Comparative teleost studies are of great interest since they are important in aquaculture and in evolutionary issues. Comparing genomes of fully sequenced model fish species with those of farmed fish species through comparative mapping offers shortcuts for quantitative trait loci (QTL) detections and for studying genome evolution through the identification of regions of conserved synteny in teleosts. Here a comparative mapping study is presented by radiation hybrid (RH) mapping genes of the gilthead sea bream Sparus aurata, a non-model teleost fish of commercial and evolutionary interest, as it represents the worldwide distributed species-rich family of Sparidae. RESULTS: An additional 74 microsatellite markers and 428 gene-based markers appropriate for comparative mapping studies were mapped on the existing RH map of Sparus aurata. The anchoring of the RH map to the genetic linkage map resulted in 24 groups matching the karyotype of Sparus aurata. Homologous sequences to Tetraodon were identified for 301 of the gene-based markers positioned on the RH map of Sparus aurata. Comparison between Sparus aurata RH groups and Tetraodon chromosomes (karyotype of Tetraodon consists of 21 chromosomes) in this study reveals an unambiguous one-to-one relationship suggesting that three Tetraodon chromosomes correspond to six Sparus aurata radiation hybrid groups. The exploitation of this conserved synteny relationship is furthermore demonstrated by in silico mapping of gilthead sea bream expressed sequence tags (EST) that give a significant similarity hit to Tetraodon. CONCLUSION: The addition of primarily gene-based markers increased substantially the density of the existing RH map and facilitated comparative analysis. The anchoring of this gene-based radiation hybrid map to the genome maps of model species broadened the pool of candidate genes that mainly control growth, disease resistance, sex determination and reversal, reproduction as well as environmental tolerance in this species, all traits of great importance for QTL mapping and marker assisted selection. Furthermore this comparative mapping approach will facilitate to give insights into chromosome evolution and into the genetic make up of the gilthead sea bream.

A genetic linkage map of the hermaphrodite teleost fish Sparus aurata L.

The gilthead sea bream (Sparus aurata L.) is a marine fish of great importance for fisheries and aquaculture. It has also a peculiar sex-determination system, being a protandrous hermaphrodite. Here we report the construction of a first-generation genetic linkage map for S. aurata, based on 204 microsatellite markers. Twenty-six linkage groups (LG) were found. The total map length was 1241.9 cM. The ratio between sex-specific map lengths was 1:1.2 (male:female). Comparison with a preliminary radiation hybrid (RH) map reveals a good concordance, as all markers located in a single LG are located in a single RH group, except for Ad-25 and CId-31. Comparison with the Tetraodon nigroviridis genome revealed a considerable number of evolutionary conserved regions (ECRs) between the two species. The mean size of ECRs was 182 bp (sequence identity 60-90%). Forty-one ECRs have a known chromosomal location in the pufferfish genome. Despite the limited number of anchoring points, significant syntenic relationships were found. The linkage map presented here provides a robust comparative framework for QTL analysis in S. aurata and is a step toward the identification of genetic loci involved both in the determination of economically important traits and in the individual timing of sex reversal.