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The study focused on the macroscopic, histological, and biometric analysis of European hake females in GSA 17 (Central-North Adriatic Sea). From 2013 to 2015, 976 females were collected and analyzed monthly. Though females in spawning conditions were found during the whole year, the trend of GSI showed a peak of the reproductive season from April to July in 2014 and 2015. HSI and Kn reached the highest values in September, after the spawning peaks. In 2013, the trend of these indices did not highlight an evident peak, probably due to an adverse event that occurred in the previous winter in the Adriatic shelf. The length at first maturity (L50) was estimated by macroscopic and histological approaches, resulting in 30.81 cm for the macroscopical length and 33.73 cm for the histological length; both values are higher than the current catching legal size. For the first time in this area, batch and relative fecundity were estimated. Relative fecundity was similar to the Mediterranean and the Atlantic stocks, whereas batch fecundity values were lower compared to other fishing grounds. Overall, the analysis of reproductive parameters plays a fundamental role in the sustainable management of this resource in an area as overfished as the Central-North Adriatic Sea.
Michela Candelma; Luca Marisaldi; Daniela Bertotto; Giuseppe Radaelli; Giorgia Gioacchini; Alberto Santojanni; Sabrina Colella; Oliana Carnevali. Aspects of Reproductive Biology of the European Hake (Merluccius merluccius) in the Northern and Central Adriatic Sea (GSA 17-Central Mediterranean Sea). Journal of Marine Science and Engineering 2021, 9, 389 .
AMA StyleMichela Candelma, Luca Marisaldi, Daniela Bertotto, Giuseppe Radaelli, Giorgia Gioacchini, Alberto Santojanni, Sabrina Colella, Oliana Carnevali. Aspects of Reproductive Biology of the European Hake (Merluccius merluccius) in the Northern and Central Adriatic Sea (GSA 17-Central Mediterranean Sea). Journal of Marine Science and Engineering. 2021; 9 (4):389.
Chicago/Turabian StyleMichela Candelma; Luca Marisaldi; Daniela Bertotto; Giuseppe Radaelli; Giorgia Gioacchini; Alberto Santojanni; Sabrina Colella; Oliana Carnevali. 2021. "Aspects of Reproductive Biology of the European Hake (Merluccius merluccius) in the Northern and Central Adriatic Sea (GSA 17-Central Mediterranean Sea)." Journal of Marine Science and Engineering 9, no. 4: 389.
Loggerhead sea turtle (C. caretta) is the official European bioindicator of marine litter in the Mediterranean Sea. In 2019, 8 sea turtles, out of 28 specimens loggerhead on the Adriatic coast of Molise, were subjected to necropsy. The intestinal contents were collected and the microplastics until 0.45 μm were extracted. Qualitative and quantitative assessments were performed by stereomicroscope observation and spectroscopic analyses (attenuated total reflection-Fourier transform infrared spectroscopy, ATR-FTIR and Raman microspectroscopy, RMS). Moreover, the analytical quantification of polyethylene terephthalate (PET), polycarbonate (PC), para phthalic acid (PTA) and bisphenol A (BPA) in fat and liver tissues was performed by LC-MS/MS. Microparticles ranging from 0.45 μm to 1 mm were found in all turtles, for a total of 623, while plastic litter greater than 1 mm were found only in 4 specimens (ranging from 0.03 to 0.11 g). Nineteen different polymers and 10 pigments, including polyester (100% of animals), high-density polyethylene (50%) and polypropylene (50%) were identified. BPA, PTA and PET were detected in fat and liver tissues of all animals, while PC was found only in 50%. A major prevalence was registered in the abdominal fat tissue, although only PC compounds were significantly higher in abdominal tissue (p < 0.05), except for free PTA with liver tissue being the most contaminated (p < 0.05). Microplastics and additives surely impact the health status of turtles that showed gastrointestinal impairment and an important level of contamination in tissues. Graphical abstract
L. Di Renzo; G. Mascilongo; M. Berti; T. Bogdanović; E. Listeš; M. Brkljača; V. Notarstefano; G. Gioacchini; E. Giorgini; V. Olivieri; C. Silvestri; M. Matiddi; N. D’Alterio; N. Ferri; F. Di Giacinto. Potential Impact of Microplastics and Additives on the Health Status of Loggerhead Turtles (Caretta caretta) Stranded Along the Central Adriatic Coast. Water, Air, & Soil Pollution 2021, 232, 1 -20.
AMA StyleL. Di Renzo, G. Mascilongo, M. Berti, T. Bogdanović, E. Listeš, M. Brkljača, V. Notarstefano, G. Gioacchini, E. Giorgini, V. Olivieri, C. Silvestri, M. Matiddi, N. D’Alterio, N. Ferri, F. Di Giacinto. Potential Impact of Microplastics and Additives on the Health Status of Loggerhead Turtles (Caretta caretta) Stranded Along the Central Adriatic Coast. Water, Air, & Soil Pollution. 2021; 232 (3):1-20.
Chicago/Turabian StyleL. Di Renzo; G. Mascilongo; M. Berti; T. Bogdanović; E. Listeš; M. Brkljača; V. Notarstefano; G. Gioacchini; E. Giorgini; V. Olivieri; C. Silvestri; M. Matiddi; N. D’Alterio; N. Ferri; F. Di Giacinto. 2021. "Potential Impact of Microplastics and Additives on the Health Status of Loggerhead Turtles (Caretta caretta) Stranded Along the Central Adriatic Coast." Water, Air, & Soil Pollution 232, no. 3: 1-20.
Polydatin is a polyphenol, whose beneficial properties, including anti-inflammatory and antioxidant activity, have been largely demonstrated. At the same time, copper has an important role in the correct organism homeostasis and alteration of its concentration can induce oxidative stress. In this study, the efficacy of polydatin to counteract the stress induced by CuSO4 exposure or by caudal fin amputation was investigated in zebrafish larvae. The study revealed that polydatin can reduced the stress induced by a 2 h exposure to 10 µM CuSO4 by lowering the levels of il1b and cxcl8b.1 and reducing neutrophils migration in the head and along the lateral line. Similarly, polydatin administration reduced the number of neutrophils in the area of fin cut. In addition, polydatin upregulates the expression of sod1 mRNA and CAT activity, both involved in the antioxidant response. Most of the results obtained in this study support the working hypothesis that polydatin administration can modulate stress response and its action is more effective in mitigating the effects rather than in preventing chemical damages.
Andrea Pessina; Mariangela Di Vincenzo; Francesca Maradonna; Francesca Marchegiani; Fabiola Olivieri; Basilio Randazzo; Giorgia Gioacchini; Oliana Carnevali. Polydatin Beneficial Effects in Zebrafish Larvae Undergoing Multiple Stress Types. International Journal of Environmental Research and Public Health 2021, 18, 1116 .
AMA StyleAndrea Pessina, Mariangela Di Vincenzo, Francesca Maradonna, Francesca Marchegiani, Fabiola Olivieri, Basilio Randazzo, Giorgia Gioacchini, Oliana Carnevali. Polydatin Beneficial Effects in Zebrafish Larvae Undergoing Multiple Stress Types. International Journal of Environmental Research and Public Health. 2021; 18 (3):1116.
Chicago/Turabian StyleAndrea Pessina; Mariangela Di Vincenzo; Francesca Maradonna; Francesca Marchegiani; Fabiola Olivieri; Basilio Randazzo; Giorgia Gioacchini; Oliana Carnevali. 2021. "Polydatin Beneficial Effects in Zebrafish Larvae Undergoing Multiple Stress Types." International Journal of Environmental Research and Public Health 18, no. 3: 1116.
In the present work, we assembled and characterized a de novo larval transcriptome of the Atlantic bluefin tuna Thunnus thynnus by taking advantage of publicly available databases with the goal of better understanding its larval development. The assembled transcriptome comprised 37,117 protein-coding transcripts, of which 13,633 full-length (>80% coverage), with an Ex90N50 of 3061 bp and 76% of complete and single-copy core vertebrate genes orthologues. Of these transcripts, 34,980 had a hit against the EggNOG database and 14,983 with the KEGG database. Codon usage bias was identified in processes such as translation and muscle development. By comparing our data with a set of representative fish species, 87.1% of tuna transcripts were included in orthogroups with other species and 5.1% in assembly-specific orthogroups, which were enriched in terms related to muscle and bone development, visual system and ion transport. Following this comparative approach, protein families related to myosin, extracellular matrix and immune system resulted significantly expanded in the Atlantic bluefin tuna. Altogether, these results provide a glimpse of how the Atlantic bluefin tuna might have achieved early physical advantages over competing species in the pelagic environment. The information generated lays the foundation for future research on the more detailed exploration of physiological responses at the molecular level in different larval stages and paves the way to evolutionary studies on the Atlantic bluefin tuna.
Luca Marisaldi; Danilo Basili; Giorgia Gioacchini; Adriana Canapa; Oliana Carnevali. De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage. Marine Genomics 2020, 58, 100834 .
AMA StyleLuca Marisaldi, Danilo Basili, Giorgia Gioacchini, Adriana Canapa, Oliana Carnevali. De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage. Marine Genomics. 2020; 58 ():100834.
Chicago/Turabian StyleLuca Marisaldi; Danilo Basili; Giorgia Gioacchini; Adriana Canapa; Oliana Carnevali. 2020. "De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage." Marine Genomics 58, no. : 100834.
The pleiotropic effects of glucocorticoids in metabolic, developmental, immune and stress response processes have been extensively investigated; conversely, their roles in reproduction are still less documented. It is well known that stress or long-lasting therapies can cause a strong increase in these hormones, negatively affecting reproduction. Moreover, the need of glucocorticoid (GC) homeostatic levels is highlighted by the reduced fertility reported in the zebrafish glucocorticoid receptor mutant (nr3c1ia30/ia30) line (hereafter named gr−/−). Starting from such evidence, in this study, we have investigated the role of glucocorticoid receptor (Gr) in the reproduction of female zebrafish. Key signals orchestrating the reproductive process at the brain, liver, and ovarian levels were analyzed using a multidisciplinary approach. An impairment of the kiss-GnRH system was observed at the central level in (gr−/−) mutants as compared to wild-type (wt) females while, in the liver, vitellogenin (vtg) mRNA transcription was not affected. Changes were instead observed in the ovary, particularly in maturing and fully grown follicles (classes III and IV), as documented by the mRNA levels of signals involved in oocyte maturation and ovulation. Follicles isolated from gr−/− females displayed a decreased level of signals involved in the acquisition of competence and maturation, causing a reduction in ovulation with respect to wt females. Fourier transform infrared imaging (FTIRI) analysis of gr−/− follicle cytoplasm showed major changes in macromolecule abundance and distribution with a clear alteration of oocyte composition. Finally, differences in the molecular structure of the zona radiata layer of gr−/− follicles are likely to contribute to the reduced fertilization rate observed in mutants.
Francesca Maradonna; Giorgia Gioacchini; Valentina Notarstefano; Camilla Maria Fontana; Filippo Citton; Luisa Dalla Valle; Elisabetta Giorgini; Oliana Carnevali. Knockout of the Glucocorticoid Receptor Impairs Reproduction in Female Zebrafish. International Journal of Molecular Sciences 2020, 21, 9073 .
AMA StyleFrancesca Maradonna, Giorgia Gioacchini, Valentina Notarstefano, Camilla Maria Fontana, Filippo Citton, Luisa Dalla Valle, Elisabetta Giorgini, Oliana Carnevali. Knockout of the Glucocorticoid Receptor Impairs Reproduction in Female Zebrafish. International Journal of Molecular Sciences. 2020; 21 (23):9073.
Chicago/Turabian StyleFrancesca Maradonna; Giorgia Gioacchini; Valentina Notarstefano; Camilla Maria Fontana; Filippo Citton; Luisa Dalla Valle; Elisabetta Giorgini; Oliana Carnevali. 2020. "Knockout of the Glucocorticoid Receptor Impairs Reproduction in Female Zebrafish." International Journal of Molecular Sciences 21, no. 23: 9073.
Different Follicle Stimulating Hormone (FSH) formulation and Luteinizing Hormone (LH) are used in Assisted Reproductive Technology (ART) to induce follicles development and oocytes maturation, but it is still under debate which protocol is to be preferred. In the present study, the different effects on cumulus cells (CCs) of three controlled ovarian stimulation (COS) protocols, based on urinary FSH, recombinant FSH, or human Menopausal Gonadotropin (hMG) administration, were assessed. CCs were obtained from 42 normal-responders women undergoing COS, randomly divided into three groups according to the used gonadotropin formulation. Differences were found in the expression of genes belonging to the endocannabinoid system (the receptors CNR1, CNR2 and TRPV1, and the enzymes involved in the metabolisms of anandamide, NAPE-PLD and FAAH, and 2-acylglycerol, DAGL and MAGL); consistently, changes in lipid (PPARα, and FASN) and carbohydrate (GLUT1 and GLUT9) metabolisms, in CCs’ macromolecules composition (highlighted by Fourier Transform Infrared Microspectroscopy, FTIRM), and in the number of retrieved oocytes were found. For the first time, statistically significant evidence on the differences related to each COS protocol on the endocannabinoid system, metabolism and macromolecular composition of CCs was found, representing a proof of concept to be further confirmed in a larger cohort of patients.
Valentina Notarstefano; Giorgia Gioacchini; Elisabetta Giorgini; Nina Montik; Andrea Ciavattini; Anna Rita Polidori; Fulvia Antonia Candela; Lisa Vaccari; Maurizio Cignitti; Oliana Carnevali. The Impact of Controlled Ovarian Stimulation Hormones on the Metabolic State and Endocannabinoid System of Human Cumulus Cells. International Journal of Molecular Sciences 2020, 21, 7124 .
AMA StyleValentina Notarstefano, Giorgia Gioacchini, Elisabetta Giorgini, Nina Montik, Andrea Ciavattini, Anna Rita Polidori, Fulvia Antonia Candela, Lisa Vaccari, Maurizio Cignitti, Oliana Carnevali. The Impact of Controlled Ovarian Stimulation Hormones on the Metabolic State and Endocannabinoid System of Human Cumulus Cells. International Journal of Molecular Sciences. 2020; 21 (19):7124.
Chicago/Turabian StyleValentina Notarstefano; Giorgia Gioacchini; Elisabetta Giorgini; Nina Montik; Andrea Ciavattini; Anna Rita Polidori; Fulvia Antonia Candela; Lisa Vaccari; Maurizio Cignitti; Oliana Carnevali. 2020. "The Impact of Controlled Ovarian Stimulation Hormones on the Metabolic State and Endocannabinoid System of Human Cumulus Cells." International Journal of Molecular Sciences 21, no. 19: 7124.
Over the last two decades, many efforts have been invested in attempting to close the life cycle of the iconic Atlantic bluefin tuna (Thunnus thynnus) and develop a true aquaculture-based market. However, the limited molecular resources nowadays available represent a clear limitation towards the domestication of this species. To fill such a gap of knowledge, we assembled and characterized a de novo larval transcriptome by taking advantage of publicly available databases with the final goal of better understanding the larval development. The assembled transcriptome comprised 37,117 protein-coding transcripts, of which 13,633 full-length (>80% coverage), with an Ex90N50 of 3,061 bp and 76% of complete and single-copy core vertebrate genes orthologues. Of these transcripts, 34,980 had a hit against the EggNOG database and 14,983 with the KAAS annotation server. By comparing our data with a set of representative fish species proteomes, it was found that 78.4% of the tuna transcripts were successfully included in orthologous groups. Codon usage bias was identified for processes such as translation, peptide biosynthesis, muscle development and ion transport, supporting the idea of mechanisms at play in regulating stability and translation efficiency of transcripts belonging to key biological processes during the larval growth. The information generated by this study on the Atlantic bluefin tuna represent a relevant improvement of the transcriptomic resources available to the scientific community and lays the foundation for future works aimed at exploring in greater detail physiological responses at molecular level in different larval stages.
Marisaldi Luca; Basili Danilo; Gioacchini Giorgia; Carnevali Oliana; Luca Marisaldi; Danilo Basili; Giorgia Gioacchini; Oliana Carnevali. De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage. 2020, 1 .
AMA StyleMarisaldi Luca, Basili Danilo, Gioacchini Giorgia, Carnevali Oliana, Luca Marisaldi, Danilo Basili, Giorgia Gioacchini, Oliana Carnevali. De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage. . 2020; ():1.
Chicago/Turabian StyleMarisaldi Luca; Basili Danilo; Gioacchini Giorgia; Carnevali Oliana; Luca Marisaldi; Danilo Basili; Giorgia Gioacchini; Oliana Carnevali. 2020. "De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage." , no. : 1.
Effective stock assessment relies on the evaluation of the reproductive potential of a population, but this is often achieved using fast, inexpensive, and inaccurate macroscopic indicators of gonadal maturity. Indeed, this approach suffers from high error rates as a result of misclassification and operator errors. Although histology is considered the gold standard to assign reproductive stages, it represents a time‐consuming and expensive method. Therefore, cost‐effective approaches are currently needed to assess the species‐specific reproductive potential and define the correct size at first maturity (L50). In the present study a histology‐calibrated method based on the gonadal index (GI) was developed to assign maturity stages to the overfished Mediterranean swordfish (Xiphias gladius), supported by histological classification of gonadal development, an estimation of L50, and an analysis of the reproductive output of this species. The performance of this new method had an overall agreement of 90% with histological data, and the L50 calculated by the GI‐based method compared with histology analysis was not statistically different (p > 0.05). Moreover, in order to apply this method without bias, a thorough validation of the relationship between length and gonad weight was carried out and the suitability of the GI to compare individuals of different size and maturity stages was confirmed. The present findings provide a comprehensive picture of the patterns characterizing Mediterranean swordfish reproduction and a promising method to assign maturity stages based on a macroscopic indicator. This method has the potential to be extensively applied during routine fish sampling activities in the field because of its simple requirements.
Luca Marisaldi; Danilo Basili; Michela Candelma; Vittoria Sesani; Paolo Pignalosa; Giorgia Gioacchini; Oliana Carnevali. Maturity assignment based on histology‐validated macroscopic criteria: Tackling the stock decline of the Mediterranean swordfish ( Xiphias gladius ). Aquatic Conservation: Marine and Freshwater Ecosystems 2019, 30, 303 -314.
AMA StyleLuca Marisaldi, Danilo Basili, Michela Candelma, Vittoria Sesani, Paolo Pignalosa, Giorgia Gioacchini, Oliana Carnevali. Maturity assignment based on histology‐validated macroscopic criteria: Tackling the stock decline of the Mediterranean swordfish ( Xiphias gladius ). Aquatic Conservation: Marine and Freshwater Ecosystems. 2019; 30 (2):303-314.
Chicago/Turabian StyleLuca Marisaldi; Danilo Basili; Michela Candelma; Vittoria Sesani; Paolo Pignalosa; Giorgia Gioacchini; Oliana Carnevali. 2019. "Maturity assignment based on histology‐validated macroscopic criteria: Tackling the stock decline of the Mediterranean swordfish ( Xiphias gladius )." Aquatic Conservation: Marine and Freshwater Ecosystems 30, no. 2: 303-314.
During folliculogenesis, primary oocytes of teleosts grow by several orders of magnitude by-self synthesizing proteins and mRNA, or sequestering from blood specific macromolecular components, such as fatty acids and vitellogenin. All these materials are stored into cortical alveoli, yolk globules or oil droplets during oocyte development. The proper synthesis, storage and displacement of these macromolecular components inside the oocyte play a key role for a successful fertilization process and for the subsequently correct embryo development. In this study, for the first time, the FTIR Imaging (FTIRI) spectroscopy has been applied to characterize the chemical building blocks of several cellular components of swordfish oocytes at different developmental stages. In particular, the spectral features of previtellogenic (PV), vitellogenic (VTG), mature (M) and atretic (A) follicles as well as and of cortical alveoli (CA), yolk vesicles (YV), oil droplets (OD) and Zona Radiata (ZR) have been outlined, providing new insights in terms of composition and topographical distribution of macromolecules of biological interest such as lipids, proteins, carbohydrates and phosphates. The macromolecular characterization of swordfish oocytes at different developmental stages represents a starting point and a useful tool for the assessment of swordfish egg quality caught in different conditions, such as periods of the year or different fishing area.
Oliana Carnevali; Michela Candelma; Andrea Sagrati; Paolo Pignalosa; Elisabetta Giorgini; Giorgia Gioacchini. Macromolecular Characterization of Swordfish Oocytes by FTIR Imaging Spectroscopy. Scientific Reports 2019, 9, 1 -9.
AMA StyleOliana Carnevali, Michela Candelma, Andrea Sagrati, Paolo Pignalosa, Elisabetta Giorgini, Giorgia Gioacchini. Macromolecular Characterization of Swordfish Oocytes by FTIR Imaging Spectroscopy. Scientific Reports. 2019; 9 (1):1-9.
Chicago/Turabian StyleOliana Carnevali; Michela Candelma; Andrea Sagrati; Paolo Pignalosa; Elisabetta Giorgini; Giorgia Gioacchini. 2019. "Macromolecular Characterization of Swordfish Oocytes by FTIR Imaging Spectroscopy." Scientific Reports 9, no. 1: 1-9.
The Mediterranean swordfish (Xiphias gladius) has been recently classified as overfished and in 2016, the International Commission for the Conservation of the Atlantic Tunas (ICCAT) established a multi-annual management plan to recover this stock. To successfully achieve this goal, knowledge about swordfish biology is needed. To date, few studies on swordfish have been performed and none of them has provided useful insights into the reproductive biology at molecular level. Here we set to characterise the molecular dynamics underlying ovarian maturation by employing a de novo transcriptome assembly approach. Differential gene expression analysis in mature and immature ovaries identified a number of differentially expressed genes associated with biological processes driving ovarian maturation. Focusing on ovarian steroidogenesis and vitellogenin uptake, we depict the molecular dynamics characterizing these processes while a phylogenetic analysis let us identify a candidate vitellogenin receptor. This is the first swordfish transcriptome assembly and these findings provide in-depth understanding of molecular processes describing ovarian maturation. Moreover, the establishment of a publicly available database containing information on the swordfish transcriptome aims to boost research on this species with the long-term of developing more comprehensive and successful stock management plans.
Giorgia Gioacchini; Luca Marisaldi; Danilo Basili; Michela Candelma; Paolo Pignalosa; Riccardo Aiese Cigliano; Walter Sanseverino; Gary Hardiman; Oliana Carnevali. A de novo transcriptome assembly approach elucidates the dynamics of ovarian maturation in the swordfish (Xiphias gladius). Scientific Reports 2019, 9, 1 -12.
AMA StyleGiorgia Gioacchini, Luca Marisaldi, Danilo Basili, Michela Candelma, Paolo Pignalosa, Riccardo Aiese Cigliano, Walter Sanseverino, Gary Hardiman, Oliana Carnevali. A de novo transcriptome assembly approach elucidates the dynamics of ovarian maturation in the swordfish (Xiphias gladius). Scientific Reports. 2019; 9 (1):1-12.
Chicago/Turabian StyleGiorgia Gioacchini; Luca Marisaldi; Danilo Basili; Michela Candelma; Paolo Pignalosa; Riccardo Aiese Cigliano; Walter Sanseverino; Gary Hardiman; Oliana Carnevali. 2019. "A de novo transcriptome assembly approach elucidates the dynamics of ovarian maturation in the swordfish (Xiphias gladius)." Scientific Reports 9, no. 1: 1-12.
Endometriosis is a chronic gynaecological disease characterised by the presence of endometrial cells in extra-uterine regions. One of the main factors impacting on the fertility of women affected by endometriosis is the poor oocyte quality. Granulosa Cells (GCs) regulate oocyte development and maintain the appropriate microenvironment for the acquisition of its competence; hence, the dysregulation of these functions in GCs can lead to severe cellular damages also in oocytes. In this study, luteinized GCs samples were separately collected from both ovaries of women affected by Unilateral Ovarian Endometriosis and analysed by infrared and Raman microspectroscopy. The spectral data were compared with those of GCs from women with diagnosis of tubal, idiopathic or male infertility (taken as control group). The coupling of these two spectroscopic techniques sheds new light on the alteration induced by this pathology on GCs metabolism and biochemical composition. In fact, the study revealed similar biochemical modifications in GCs from both ovaries of women affected by unilateral ovarian endometriosis, such as the alteration of the protein pattern, the induction of oxidative stress mechanisms, and the deregulation of lipid and carbohydrate metabolisms. These evidences suggest that unilateral endometriosis impairs the overall ovarian functions, causing alterations not only in the ovary with endometriotic lesions but also in the contralateral “healthy” one.
Valentina Notarstefano; Giorgia Gioacchini; Hugh J. Byrne; Carlotta Zacà; Elena Sereni; Lisa Vaccari; Andrea Borini; Oliana Carnevali; Elisabetta Giorgini. Vibrational characterization of granulosa cells from patients affected by unilateral ovarian endometriosis: New insights from infrared and Raman microspectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 2019, 212, 206 -214.
AMA StyleValentina Notarstefano, Giorgia Gioacchini, Hugh J. Byrne, Carlotta Zacà, Elena Sereni, Lisa Vaccari, Andrea Borini, Oliana Carnevali, Elisabetta Giorgini. Vibrational characterization of granulosa cells from patients affected by unilateral ovarian endometriosis: New insights from infrared and Raman microspectroscopy. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019; 212 ():206-214.
Chicago/Turabian StyleValentina Notarstefano; Giorgia Gioacchini; Hugh J. Byrne; Carlotta Zacà; Elena Sereni; Lisa Vaccari; Andrea Borini; Oliana Carnevali; Elisabetta Giorgini. 2019. "Vibrational characterization of granulosa cells from patients affected by unilateral ovarian endometriosis: New insights from infrared and Raman microspectroscopy." Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 212, no. : 206-214.
Due to minimal environmental impact, compared to most conventional feed commodities, insects deserve a growing attention as candidate ingredients for aquafeeds. This study tested, for the first time during zebrafish larval rearing, the effects of an increasing replacement (0%–25%–50%) of fish meal by black soldier fly (BSF) full-fat prepupae meal. All diets were formulated to be isonitrogenous and isolipidic. A multidisciplinary approach, including biometrics, histology, gas chromatography–mass spectrometry, and molecular analyses, was applied to better understand the biological responses of larval zebrafish to the different partial inclusions of BSF in the feed. Generally, results are promising, but a 50% of BSF meal inclusion in the diet affected both lipid composition and accumulation in the larvae.
Matteo Zarantoniello; Leonardo Bruni; Basilio Randazzo; Arturo Vargas; Giorgia Gioacchini; Cristina Truzzi; Anna Annibaldi; Paola Riolo; Giuliana Parisi; Gloriana Cardinaletti; Francesca Tulli; Ike Olivotto. Partial Dietary Inclusion of Hermetia illucens (Black Soldier Fly) Full-Fat Prepupae in Zebrafish Feed: Biometric, Histological, Biochemical, and Molecular Implications. Zebrafish 2018, 15, 1 .
AMA StyleMatteo Zarantoniello, Leonardo Bruni, Basilio Randazzo, Arturo Vargas, Giorgia Gioacchini, Cristina Truzzi, Anna Annibaldi, Paola Riolo, Giuliana Parisi, Gloriana Cardinaletti, Francesca Tulli, Ike Olivotto. Partial Dietary Inclusion of Hermetia illucens (Black Soldier Fly) Full-Fat Prepupae in Zebrafish Feed: Biometric, Histological, Biochemical, and Molecular Implications. Zebrafish. 2018; 15 (5):1.
Chicago/Turabian StyleMatteo Zarantoniello; Leonardo Bruni; Basilio Randazzo; Arturo Vargas; Giorgia Gioacchini; Cristina Truzzi; Anna Annibaldi; Paola Riolo; Giuliana Parisi; Gloriana Cardinaletti; Francesca Tulli; Ike Olivotto. 2018. "Partial Dietary Inclusion of Hermetia illucens (Black Soldier Fly) Full-Fat Prepupae in Zebrafish Feed: Biometric, Histological, Biochemical, and Molecular Implications." Zebrafish 15, no. 5: 1.
Endocrine disrupting chemicals (EDCs) are known to disrupt normal metabolism and can influence the incidence of obesity in animals and humans. EDCs can exert adverse effects at low concentrations, often in a non-monotonic dose-related fashion. Among EDCs, Bisphenol A (BPA) is extensively used in the production of polycarbonate plastic, and is among the most abundant contaminants in the world. Diethylene glycol dibenzoate (DGB), an approved alternative to phthalates in the production of plastic and latex products, however, is less abundant and its effects are almost completely unknown. The aim of this study is to provide information on the hepatic effects of BPA and DGB on lipid metabolism, and investigate possible links between these contaminants and the increased incidence of obesity. In the present study, we exposed zebrafish to three different BPA doses (5; 10; 20 μg/L) and five different doses of DGB (0.01; 0.1; 1; 10; 100 μg/L) for a period of 21 days, and investigated transcript levels for genes involved in lipid metabolism as well as measuring liver content of phosphates, lipids and proteins. The results demonstrate disruptive effects of BPA and DGB on lipid metabolism in a non-monotonic dose-related fashion. The lowest dose of BPA increased the storage of triglycerides and promoted fatty acid synthesis, while the highest concentration promoted de novo lipogenesis and cholesterologenesis. Exposure to DGB was also found to affect lipid metabolism leading to increased lipid production and mobilization in a non-monotonic dose-related fashion. Analysis of BPA and DGB by FT-IR revealed that exposure to both compounds lead to changes in the biochemical composition of liver. The findings provide a support for the hypothesis that BPA and DGB may be among the environmental contaminants with obesogenic property.
Stefania Santangeli; Valentina Notarstefano; Francesca Maradonna; Elisabetta Giorgini; Giorgia Gioacchini; Isabel Forner-Piquer; Hamid R. Habibi; Oliana Carnevali. Effects of diethylene glycol dibenzoate and Bisphenol A on the lipid metabolism of Danio rerio. Science of The Total Environment 2018, 636, 641 -655.
AMA StyleStefania Santangeli, Valentina Notarstefano, Francesca Maradonna, Elisabetta Giorgini, Giorgia Gioacchini, Isabel Forner-Piquer, Hamid R. Habibi, Oliana Carnevali. Effects of diethylene glycol dibenzoate and Bisphenol A on the lipid metabolism of Danio rerio. Science of The Total Environment. 2018; 636 ():641-655.
Chicago/Turabian StyleStefania Santangeli; Valentina Notarstefano; Francesca Maradonna; Elisabetta Giorgini; Giorgia Gioacchini; Isabel Forner-Piquer; Hamid R. Habibi; Oliana Carnevali. 2018. "Effects of diethylene glycol dibenzoate and Bisphenol A on the lipid metabolism of Danio rerio." Science of The Total Environment 636, no. : 641-655.
STUDY QUESTIONDoes the molecular and metabolic profile of human mural granulosa cells (GCs) correlate with oocyte fate?SUMMARY ANSWERA close relation between the metabolic profile of mural GCs and the fate of the corresponding oocyte was revealed by the analysis of selected biomarkers defined by GC Fourier transform infrared microspectroscopy (FTIRM) analysisWHAT IS KNOWN ALREADYIn ART, oocyte selection is mainly based on the subjective observation of its morphological features; despite recent efforts, the success rate of this practice is still unsatisfactory. FTIRM is a well-established vibrational technique recently applied to evaluate oocytes quality in several experimental models, including human.STUDY DESIGN SIZE, DURATIONGCs retrieved from single-follicle aspirates were obtained with informed consent from 55 women undergoing controlled ovarian stimulation for IVF treatment.GCs were analysed by FTIRM to retrospectively correlate their spectral features with the fate of the companion oocytes. The study has been conducted between March 2016 and September 2017.PARTICIPANTS/MATERIALS, SETTING, METHODSPatients were selected according to the following inclusion criteria: age < 40 years; non-smokers; no ovarian infertility diagnosis (only tubal, idiopathic and male infertility); regular ovulatory menstrual cycles (25-30 days) with FSH < 10 IU/I on day 3 of the menstrual cycle; sperm sample with a total motility count after treatment ≥ 300.000; number of retrieved oocytes ≥ 8. Based on the clinical outcome of the corresponding oocyte, GCs were retrospectively classified into the following experimental groups: clinical pregnancy (CP), fertilization failure (FF), embryo development failure (EDF), and implantation failure (IF). All samples were analysed by the FTIRM technique. The spectral biomarker signature of different oocyte fates was derived by several feature selection procedures (‘Leave-one-out’ method on factorial discriminant analysis (FDA), variable characterization method, and logistic regression method with the multinomial Logit model). ANOVA, permutational multivariate ANOVA, FDA, and canonical analysis of principal co-ordinates statistical tools were also applied to validate the identified spectral biomarkers.MAIN RESULTS AND THE ROLE OF CHANCEIn total, 284 GCs samples were retrieved and retrospectively classified as FF: (N = 92), EDF (N = 113), IF (N = 56), and CP (N = 23). From the spectral profiles of GCs belonging to CP, FF, EDF and IF experimental groups, 17 spectral biomarkers, were identified by several feature selection procedures (p < 0.0001). These biomarkers were then validated by applying multivariate tools, to evaluate their ability to segregate GCs samples into the four experimental groups. FDA showed a clear separation along the F1-axis (62.75% of discrimination) between GCs from oocytes able (CP, IF groups) or not (FF, EDF groups) to develop into embryos; the F2-axis (24.14% of discrimination) segregated the embryos that gave pregnancy (CP) from those that failed implantation (IF). The confusion matrix (total percentage of correctness=80.25%) obtained from this analysis pinpointed that GCs from oocytes unable to develop into embryos (FF, EDF) were better characterized than those from oocytes able to give viable embryos (CP, IF). ANOVA (p < 0.05) analysis pinpointed that: each experimental group showed specific macromolecular traits, ascribable to different biological and metabolic characteristics of GCs; these metabolic features were likely associated with different oocytes fates, but not to patient characteristics, since from the same patient we obtained GCs with different metabolic profiles.LIMITATIONS REASONS FOR CAUTIONThe study is based on a small sample size but provides proof of concept that the GCs’ metabolic profile is associated with the companion oocyte fate. The generated model should be further tested on a larger cohort of patients, classified in a similar manner, to assess the potential predictive value of this approach. Ultimately, validity of the proposed approach should be tested in a RCT.WIDER IMPLICATIONS OF THE FINDINGSFor the first time, the FTIRM analysis of human GCs has demonstrated an approach to better understand the molecular crosstalk between follicular cells and oocytes and has identified potential spectral biomarkers for improving human IVF success rate.STUDY FUNDING/COMPETING INTEREST(S)The study was funded by GFI grant. The authors declare that there is no conflict of interest.
Giorgia Gioacchini; Valentina Notarstefano; Elena Sereni; Carlotta Zacà; Giovanni Coticchio; Elisabetta Giorgini; Lisa Vaccari; Oliana Carnevali; Andrea Borini. Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis. Molecular Human Reproduction 2018, 24, 521 -532.
AMA StyleGiorgia Gioacchini, Valentina Notarstefano, Elena Sereni, Carlotta Zacà, Giovanni Coticchio, Elisabetta Giorgini, Lisa Vaccari, Oliana Carnevali, Andrea Borini. Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis. Molecular Human Reproduction. 2018; 24 (11):521-532.
Chicago/Turabian StyleGiorgia Gioacchini; Valentina Notarstefano; Elena Sereni; Carlotta Zacà; Giovanni Coticchio; Elisabetta Giorgini; Lisa Vaccari; Oliana Carnevali; Andrea Borini. 2018. "Does the molecular and metabolic profile of human granulosa cells correlate with oocyte fate? New insights by Fourier transform infrared microspectroscopy analysis." Molecular Human Reproduction 24, no. 11: 521-532.
A desirable goal of the aquaculture sector is to replace most of fish meal and fish oil with more sustainable, cost-effective, and environmental friendly ingredients ensuring fish health and welfare standards. Due to minimal environmental impact, compared with most conventional feed commodities, insects deserve a growing attention as candidate ingredients for aquafeeds. The present study investigated, for the first time, the possible application of a 100% insect diet in zebrafish larval rearing. Through a multidisciplinary approach, the major biological responses of fish to the new diets were assessed. Results of biometry, fatty acid composition, expression of genes involved in fish growth, stress response, lipid metabolism, chitinolytic activity, gut inflammation, and liver macromolecular composition suggested a possible application of insect larvae for zebrafish larval rearing. However, further studies are necessary to better understand the use of this insect species in the rearing of fish.
Arturo Vargas; Basilio Randazzo; Paola Riolo; Cristina Truzzi; Giorgia Gioacchini; Elisabetta Giorgini; Nino Loreto; Sara Ruschioni; Matteo Zarantoniello; Matteo Antonucci; Sara Polverini; Gloriana Cardinaletti; Simona Sabbatini; Francesca Tulli; Ike Olivotto. Rearing Zebrafish on Black Soldier Fly (Hermetia illucens): Biometric, Histological, Spectroscopic, Biochemical, and Molecular Implications. Zebrafish 2018, 15, 404 -419.
AMA StyleArturo Vargas, Basilio Randazzo, Paola Riolo, Cristina Truzzi, Giorgia Gioacchini, Elisabetta Giorgini, Nino Loreto, Sara Ruschioni, Matteo Zarantoniello, Matteo Antonucci, Sara Polverini, Gloriana Cardinaletti, Simona Sabbatini, Francesca Tulli, Ike Olivotto. Rearing Zebrafish on Black Soldier Fly (Hermetia illucens): Biometric, Histological, Spectroscopic, Biochemical, and Molecular Implications. Zebrafish. 2018; 15 (4):404-419.
Chicago/Turabian StyleArturo Vargas; Basilio Randazzo; Paola Riolo; Cristina Truzzi; Giorgia Gioacchini; Elisabetta Giorgini; Nino Loreto; Sara Ruschioni; Matteo Zarantoniello; Matteo Antonucci; Sara Polverini; Gloriana Cardinaletti; Simona Sabbatini; Francesca Tulli; Ike Olivotto. 2018. "Rearing Zebrafish on Black Soldier Fly (Hermetia illucens): Biometric, Histological, Spectroscopic, Biochemical, and Molecular Implications." Zebrafish 15, no. 4: 404-419.
The original version of this article unfortunately contained mistakes in Authors First name/Family name order. Please refer to this paper as Gioacchini et al. and not as Giorgia et al
Giorgia Gioacchini; Elia Ciani; Andrea Pessina; Cinzia Cecchini; Stefania Silvi; Ana Rodiles; Daniel L. Merrifield; Ike Olivotto; Oliana Carnevali. Correction to: Effects of Lactogen 13, a New Probiotic Preparation, on Gut Microbiota and Endocrine Signals Controlling Growth and Appetite of Oreochromis niloticus Juveniles. Microbial Ecology 2018, 76, 1075 -1075.
AMA StyleGiorgia Gioacchini, Elia Ciani, Andrea Pessina, Cinzia Cecchini, Stefania Silvi, Ana Rodiles, Daniel L. Merrifield, Ike Olivotto, Oliana Carnevali. Correction to: Effects of Lactogen 13, a New Probiotic Preparation, on Gut Microbiota and Endocrine Signals Controlling Growth and Appetite of Oreochromis niloticus Juveniles. Microbial Ecology. 2018; 76 (4):1075-1075.
Chicago/Turabian StyleGiorgia Gioacchini; Elia Ciani; Andrea Pessina; Cinzia Cecchini; Stefania Silvi; Ana Rodiles; Daniel L. Merrifield; Ike Olivotto; Oliana Carnevali. 2018. "Correction to: Effects of Lactogen 13, a New Probiotic Preparation, on Gut Microbiota and Endocrine Signals Controlling Growth and Appetite of Oreochromis niloticus Juveniles." Microbial Ecology 76, no. 4: 1075-1075.
Di-isononyl phthalate (DiNP) is a high molecular weight phthalate commonly used as a plasticizer. It was introduced as a replacement for bis (2-ethylhexyl) phthalate (DEHP) which is used in the production of plasticized polyvinyl chloride (PVC). The purpose of this study was to investigate for the first time the effect of DiNP on female reproductive physiology in Danio rerio. Fish were exposed to five different doses of DiNP plus control (0 μg/L; 0.42 μg/L; 4.2 μg/L; 42 μg/L; 420 μg/L; 4200 μg/L) for a period of 21 days. We evaluated fish fecundity, oocyte growth, autophagic and apoptotic processes, as well as changes in morphological and biochemical composition of oocytes, using, qPCR analysis, histology and Fourier transform infrared imaging. The results demonstrate a non-monotonic dose response to DiNP. Greater differences were observed at the lowest (0.42 μg/L) and higher concentrations (420 μg/L; 4200 μg/L) of DiNP. The findings provide evidence that exposure to DiNP adversely affect oocytes growth and maturation, leading to abnormal gonadal development and reproduction in zebrafish.
Stefania Santangeli; Francesca Maradonna; Maya Zanardini; Valentina Notarstefano; Giorgia Gioacchini; Isabel Forner-Piquer; Hamid Habibi; Oliana Carnevali. Effects of diisononyl phthalate on Danio rerio reproduction. Environmental Pollution 2017, 231, 1051 -1062.
AMA StyleStefania Santangeli, Francesca Maradonna, Maya Zanardini, Valentina Notarstefano, Giorgia Gioacchini, Isabel Forner-Piquer, Hamid Habibi, Oliana Carnevali. Effects of diisononyl phthalate on Danio rerio reproduction. Environmental Pollution. 2017; 231 ():1051-1062.
Chicago/Turabian StyleStefania Santangeli; Francesca Maradonna; Maya Zanardini; Valentina Notarstefano; Giorgia Gioacchini; Isabel Forner-Piquer; Hamid Habibi; Oliana Carnevali. 2017. "Effects of diisononyl phthalate on Danio rerio reproduction." Environmental Pollution 231, no. : 1051-1062.
Phthalates, used as plasticizers, have become a ubiquitous contaminant and have been reported for their potential to induce toxicity in living organisms. Among them, di-isononyl phthalate (DiNP) has been recently used to replace di(2-ethylhexyl) phthalate (DEHP). Nowadays, there is evidence that DiNP is an endocrine-disrupting chemical; however, little is known about its effects on the endocannabinoid system (ECS) and lipid metabolism. Hence, the aim of our study was to investigate the effects of DiNP on the ECS in zebrafish liver and brain and on hepatic lipid storage. To do so, adult female zebrafish were exposed to three concentrations (0.42 µg/L, 4.2 µg/L, and 42 µg/L) of DiNP via water for 3 weeks. Afterwards, we investigated transcript levels for genes involved in the ECS of the brain and liver as well as liver histology and image analysis, Fourier-transform infrared spectroscopy imaging, and measurement of endocannabinoid levels. Our results demonstrate that DiNP upregulates orexigenic signals and causes hepatosteatosis together with deregulation of the peripheral ECS and lipid metabolism. A decrease in the levels of ECS components at the central level was observed after exposure to the highest DiNP concentration tested. These findings suggest that replacement of DEHP with DiNP should be considered with caution because of observed adverse DiNP effects on aquatic organisms.
Isabel Forner-Piquer; Francesca Maradonna; Giorgia Gioacchini; Stefania Santangeli; Marco Allarà; Fabiana Piscitelli; Hamid R Habibi; Vincenzo Di Marzo; Oliana Carnevali. Dose-Specific Effects of Di-Isononyl Phthalate on the Endocannabinoid System and on Liver of Female Zebrafish. Endocrinology 2017, 158, 3462 -3476.
AMA StyleIsabel Forner-Piquer, Francesca Maradonna, Giorgia Gioacchini, Stefania Santangeli, Marco Allarà, Fabiana Piscitelli, Hamid R Habibi, Vincenzo Di Marzo, Oliana Carnevali. Dose-Specific Effects of Di-Isononyl Phthalate on the Endocannabinoid System and on Liver of Female Zebrafish. Endocrinology. 2017; 158 (10):3462-3476.
Chicago/Turabian StyleIsabel Forner-Piquer; Francesca Maradonna; Giorgia Gioacchini; Stefania Santangeli; Marco Allarà; Fabiana Piscitelli; Hamid R Habibi; Vincenzo Di Marzo; Oliana Carnevali. 2017. "Dose-Specific Effects of Di-Isononyl Phthalate on the Endocannabinoid System and on Liver of Female Zebrafish." Endocrinology 158, no. 10: 3462-3476.
Epigenetic modifications are classified as heritable and reversible chemical modifications of chromatin that do not cause changes in DNA sequence. Changes in epigenetic modifications can be caused by exposure to certain environmental factors, such as contaminants like bisphenol A (BPA). Bisphenol A is ubiquitous in the environment and produced in large quantities, and known to have hormone-like activity, whereby disrupting endocrine function. Because of evidence for disruption of sex steroid mediated pathways, there is a concern that BPA could have adverse effects on female reproduction. The purpose of this review is to summarize the effects of BPA on adult female reproduction with focus on epigenetic changes that can be heritable.
Stefania Santangeli; Francesca Maradonna; Ike Olivotto; Chiara Carla Piccinetti; Giorgia Gioacchini; Oliana Carnevali. Effects of BPA on female reproductive function: The involvement of epigenetic mechanism. General and Comparative Endocrinology 2017, 245, 122 -126.
AMA StyleStefania Santangeli, Francesca Maradonna, Ike Olivotto, Chiara Carla Piccinetti, Giorgia Gioacchini, Oliana Carnevali. Effects of BPA on female reproductive function: The involvement of epigenetic mechanism. General and Comparative Endocrinology. 2017; 245 ():122-126.
Chicago/Turabian StyleStefania Santangeli; Francesca Maradonna; Ike Olivotto; Chiara Carla Piccinetti; Giorgia Gioacchini; Oliana Carnevali. 2017. "Effects of BPA on female reproductive function: The involvement of epigenetic mechanism." General and Comparative Endocrinology 245, no. : 122-126.
Ambra1 is a positive regulator of autophagy, a lysosome-mediated degradative process involved both in physiological and pathological conditions. Nowadays, Ambra1 has been characterized only in mammals and zebrafish. Through bioinformatics searches and targeted cloning, we report the identification of the complete Ambra1 transcript in a non-vertebrate chordate, the tunicate Botryllus schlosseri. Tunicata is the sister group of Vertebrata and the only chordate group possessing species that reproduce also by blastogenesis (asexual reproduction). B. schlosseri Ambra1 deduced amino acid sequence is shorter than vertebrate homologues but still contains the typical WD40 domain. qPCR analyses revealed that the level of B. schlosseri Ambra1 transcription is temporally regulated along the colonial blastogenetic cycle. By means of similarity searches we identified Wdr5 and Katnb1 as proteins evolutionarily associated to Ambra1. Phylogenetic analyses on Bilateria indicate that: (i) Wdr5 is the most related to Ambra1, so that they may derive from an ancestral gene, (ii) Ambra1 forms a group of ancient genes evolved before the radiation of the taxon, (iii) these orthologous Ambra1 share the two conserved WD40/YVTN repeat-like-containing domains, and (iv) they are characterized by ancient duplications of WD40 repeats within the N-terminal domain.
Fabio Gasparini; Tatjana Skobo; Francesca Benato; Giorgia Gioacchini; Ayelet Voskoboynik; Oliana Carnevali; Lucia Manni; Luisa Dalla Valle. Characterization of Ambra1 in asexual cycle of a non-vertebrate chordate, the colonial tunicate Botryllus schlosseri, and phylogenetic analysis of the protein group in Bilateria. Molecular Phylogenetics and Evolution 2015, 95, 46 -57.
AMA StyleFabio Gasparini, Tatjana Skobo, Francesca Benato, Giorgia Gioacchini, Ayelet Voskoboynik, Oliana Carnevali, Lucia Manni, Luisa Dalla Valle. Characterization of Ambra1 in asexual cycle of a non-vertebrate chordate, the colonial tunicate Botryllus schlosseri, and phylogenetic analysis of the protein group in Bilateria. Molecular Phylogenetics and Evolution. 2015; 95 ():46-57.
Chicago/Turabian StyleFabio Gasparini; Tatjana Skobo; Francesca Benato; Giorgia Gioacchini; Ayelet Voskoboynik; Oliana Carnevali; Lucia Manni; Luisa Dalla Valle. 2015. "Characterization of Ambra1 in asexual cycle of a non-vertebrate chordate, the colonial tunicate Botryllus schlosseri, and phylogenetic analysis of the protein group in Bilateria." Molecular Phylogenetics and Evolution 95, no. : 46-57.