Letteratura scientifica selezionata sul tema "Fish cell line"
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Articoli di riviste sul tema "Fish cell line":
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Kolarova, J., J. Velisek e Z. Svobodova. "Comparison of in vitro (fish cell line) and in vivo (fish and crustacean) acute toxicity tests in aquatic toxicology". Veterinární Medicína 66, No. 8 (5 luglio 2021): 350–55. http://dx.doi.org/10.17221/161/2020-vetmed.
Abstract (sommario):
The use of in vitro (fish cell lines) is a cost-effective, very rapid, and informative tool for toxicological assessments. Using the neutral red (NR) assay, we compared the in vitro acute toxicity (20hEC50) of twenty-six chemical substances on a rainbow trout gonad cell line (RTG-2) with their in vivo acute toxicity to Barbados Millions Poecilia reticulata (48hLC50, OECD 203) and crustacean Daphnia magna (48hEC50, OECD 202). The 20hEC50 values obtained by the NR assay were higher in nearly all the cases when compared to the 48hLC50 in P. reticulata and the 48hEC50 in D. magna, indicating that the sensitivity of the RTG-2 cell line was lower compared to P. reticulata and D. magna. A high (r = 0.89) and significant (P < 0.001) correlation was recorded between the 20hEC50 values of the RTG-2 and the 48hEC50 values of D. magna. The correlation between the 20hEC50 values of the RTG-2 and the 48hLC50 values of P. reticulata was lower (r = 0.65; P < 0.001), but also significant. The authors recommend use of the NR assay on the RTG-2 cell lines as a screening protocol to evaluate the toxicity of xenobiotics in aquatic environments to narrow the spectrum of the concentrations for the fish toxicity test.
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Kan, Yuting, Ying Zhong, Muhammad Jawad, Xiao Chen, Dong Liu, Mingchun Ren, Gangchun Xu, Lang Gui e Mingyou Li. "Establishment of a Coilia nasus Gonadal Somatic Cell Line Capable of Sperm Induction In Vitro". Biology 11, n. 7 (13 luglio 2022): 1049. http://dx.doi.org/10.3390/biology11071049.
Abstract (sommario):
Coilia nasus is an important economic anadromous migratory fish of the Yangtze River in China. In recent years, overfishing and the deterioration of the ecological environment almost led to the extinction of the wild resources of C.nasus. Thus, there is an urgent need to protect this endangered fish. Recently, cell lines derived from fish have proven a promising tool for studying important aspects of aquaculture. In this study, a stable C. nasus gonadal somatic cell line (CnCSC) was established and characterized. After over one year of cell culture (>80 passages), this cell line kept stable growth. RT-PCR results revealed that the CnGSC expressed some somatic cell markers such as clu, fshr, hsd3β, and sox9b instead of germ cell markers like dazl, piwi, and vasa. The strong phagocytic activity of CnGSC suggested that it contained a large number of Sertoli cells. Interestingly, CnGSC could induce medaka spermatogonial cells (SG3) to differentiate into elongated spermatids while co-cultured together. In conclusion, we established a C. nasus gonadal somatic cell line capable of sperm induction in vitro. This research provides scientific evidence for the long-term culture of a gonadal cell line from farmed fish, which would lay the foundation for exploring the regulatory mechanisms between germ cells and somatic cells in fish.
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Lee, K. W., S. C. Chi e T. M. Cheng. "Interference of the life cycle of fish nodavirus with fish retrovirus". Journal of General Virology 83, n. 10 (1 ottobre 2002): 2469–74. http://dx.doi.org/10.1099/0022-1317-83-10-2469.
Abstract (sommario):
Interference of the life cycle of grouper nervous necrosis virus (GNNV), a member of the Nodaviridae, genus Betanodavirus, by snakehead retrovirus (SnRV) has been studied in vitro. SGF-1, a new fish cell line that is persistently infected with SnRV, was induced by inoculating SnRV into the grouper fin cell line GF-1. Culture supernatants and cell pellets from both GNNV-infected SGF-1 and GF-1 cells were collected and employed for virus productivity analysis. The yields of GNNV RNA and capsid protein in GNNV-infected SGF-1 cells were similar to those in GNNV-infected GF-1 cells. However, when GF-1 cells were used for titration, the titre of the culture supernatant from GNNV-infected SGF-1 cells was much higher than that from GNNV-infected GF-1 cells. The titration result suggested that SnRV enhanced the infection or cytopathic effect (CPE) of GNNV during GNNV and SnRV coinfection of the GF-1 cell titration system, although SnRV cannot induce any CPE in GF-1 cells alone, nor can it increase the yield of GNNV after GNNV superinfection of SGF-1 cells. Moreover, GNNV cDNA was detected in both the pellet and the supernatant from GNNV-infected SGF-1 cells. This result indicated that SnRV reverse-transcribed the GNNV single-stranded genomic RNA into cDNA during GNNV superinfection of SGF-1 cells and created a new cDNA stage in the life cycle of the fish nodavirus.
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Tanneberger, Katrin, Melanie Knöbel, Frans J. M. Busser, Theo L. Sinnige, Joop L. M. Hermens e Kristin Schirmer. "Predicting Fish Acute Toxicity Using a Fish Gill Cell Line-Based Toxicity Assay". Environmental Science & Technology 47, n. 2 (27 dicembre 2012): 1110–19. http://dx.doi.org/10.1021/es303505z.
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Tung, Li-Chu, Yung-Reui Chen, Shiu-Nan Chen e Guang-Hsiung Kuo. "Fish kidney cell line in response to heat shock". Proceedings, annual meeting, Electron Microscopy Society of America 50, n. 1 (agosto 1992): 704–5. http://dx.doi.org/10.1017/s0424820100123921.
Abstract (sommario):
In the present study, the ultrastructural changes of BPK cells, a fibroblast-like cell line, derived from the kidney of juvenile black porgy Acanthopagrus schlegeli, under heat shock treatment are described.The BPK cells were maintained in L-15 medium supplemented with 10% fetal calf serum and 0.15 M NaCl at 28|C2. The heating was carried out in precalibrated water baths. Monolayers of cells, grown on coverslips in parafilm-sealed petri dishes were submerged under water for 30 min at 40|C treatments. Cells were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer supplemented with 6.6% sucrose, postfixed in 1% OsO4 and flat embedded in Spurr’s resin. Silver section were cut parallel to the substratum, stained with uranyl acetate and Reynold’s lead citrate, and examined in a Hitachi H-600 electron microscope at 75 KV.
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Gagné, F., e C. Blaise. "Evaluation of environmental estrogens with a fish cell line". Bulletin of Environmental Contamination and Toxicology 65, n. 4 (ottobre 2000): 494–500. http://dx.doi.org/10.1007/s001280000151.
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Chaumont, L., L. Jouneau, M. Peruzzi, P. Boudinot e B. Collet. "Functional characterisation of a Viperin knockout fish cell line". Developmental & Comparative Immunology 148 (novembre 2023): 104932. http://dx.doi.org/10.1016/j.dci.2023.104932.
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Zhang, Hetong, Junjian Dong, Yunyun Yan, Shanshan Liu, Xing Ye, Fengying Gao e Chengfei Sun. "Development of a Highly Permissive Mandarin Fish (Siniperca chuatsi) Kidney Cell Line for Mandarin Fish Ranavirus Using a Single-Cell Cloning Method". Cells 13, n. 1 (20 dicembre 2023): 18. http://dx.doi.org/10.3390/cells13010018.
Abstract (sommario):
Mandarin fish ranavirus (MRV) infection poses a substantial challenge to the mandarin fish culture industry as no effective preventive or therapeutic measures currently exist. The creation of a highly permissive cell line from a natural host is crucial for developing a vaccine for MRV and understanding its pathogenic mechanisms. In this research, the mandarin fish (Siniperca chuatsi) kidney cell line (SCK) was isolated from mandarin fish kidneys. Subsequently, SCK-a to SCK-g monoclonal cell lines were derived from the SCK cell population, distinguished by morphological variations. Notably, MRV infection induced an advanced cytopathic effect (CPE) in almost all cells of the SCK-f clone. Further tests showed that MRV achieved a peak viral titer of 1010.7 50% tissue culture infectious dose (TCID50)/mL and consistently exceeded 1010 TCID50/mL across nine passages in SCK-f cells. Electron microscopy verified the MRV virion integrity within SCK-f. In vivo experiments revealed that MRV infections led to cumulative mortality rates of 86.9% in mandarin fish and 88.9% in largemouth bass (Micropterus salmoides). Such results suggest that SCK-f is highly permissive to MRV. This study underscores the importance of cellular diversity in developing viral permissive cell lines. The SCK monoclonal cell line pool may offer potential for generating highly permissive cell lines for other mandarin fish viruses.
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Luo, Xia, Xiaozhe Fu, Min Zhang, Hongru Liang, Yinjie Niu, Qiang Lin, Baofu Ma, Lihui Liu e Ningqiu Li. "Development of a New Marine Fish Continuous Cell Line Derived from Brain of Red Sea Bream (Pagrosomus major) and Its Application to Fish Virology and Heavy Metal Toxicology". Animals 13, n. 22 (15 novembre 2023): 3524. http://dx.doi.org/10.3390/ani13223524.
Abstract (sommario):
Red sea bream (Pagrosomus major) is one of the most popular farmed marine teleost fish species. Fish cell lines are becoming important research tool in the aquaculture field, and they are suitable models to study fish virology, immunology and toxicology. To obtain a Pagrosomus major cell line for biological studies, a continuous cell line from brain of red sea bream (designated as RSBB cell line) was established and has been successfully subcultured over 100 passages. The RSBB cell line predominantly consisted of fibroblast-like cells and multiplied well in M199 medium supplemented with 10% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome numbers of RSBB cells was 48. After transfection with pEGFP-N1, RSBB cells showed bright green fluorescence with a transfection efficiency approaching 8%. For toxicology study, it was demonstrated that metal Cd could induce cytotoxic effects of RSBB cells, accompanied with a dose-dependent MTT conversion capacity. Morphologically, cells treated with metal Cd produced rounding, shrinking and detaching and induced both cell apoptosis and necrosis. For virology study, the RSBB cells were highly susceptible to Nervous necrosis virus (NNV) and Singapore grouper iridovirus (SGIV) with steady titers (i.e., 108.0~8.3 TCID50 mL−1 and 107.0~7.2 TCID50 mL−1 respectively). Furthermore, an obvious cytopathic effect (CPE) could be observed in RSBB cells infected with Infectious spleen and kidney necrosis virus (ISKNV) and Siniperca chuatsi rhabdoviruses (SCRV). Meanwhile, all the infections were confirmed by polymerase chain reaction. The new brain cell line developed and characterized from red sea bream in this study could be used as an in vitro model for fish studies in the fields of toxicology and virology.
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TAKARADA, Yutaka, Yoshio OJIMA e Akinori TAKAI. "Establishment of a fish cell line transformed by MNNG, RFM." Proceedings of the Japan Academy. Ser. B: Physical and Biological Sciences 65, n. 5 (1989): 108–11. http://dx.doi.org/10.2183/pjab.65.108.
Tesi sul tema "Fish cell line":
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Dumont, Sarah. "Caractéristiques cliniques, moléculaires et prise en charge des Rhabdomyosarcomes de l'adulte et identification d'une polythérapie ciblée in vitro". Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10311/document.
Abstract (sommario):
Le rhabdomyosarcome de l'adulte est une tumeur rare au pronostic. Le présent travail propose d'étudier les caractéristiques cliniques et moléculaires et la prise en charge des adolescents et adultes atteints de rhabdomyosarcome ainsi que la possibilité de combinaison de thérapie ciblées sur lignées cellulaires in vitro. Nous avons anamysé rétrospectivement 239 patients âgés de 10 ans ou plus, atteints de rhabdomyosarcome au MD Anderson Cancer Center entre 1957 et 2003 et leur statut fusionnel pour PAX-FOXO1 par hybridation in situ en fluorescence. Trois lignées cellulaire de sarcome à petites cellules ont été soumises à des combinaisons de thérapies ciblées avec analyse de la viabilité. Les patients de plus de 50 ans avaient une survie globale à 5 ans de 13 % (médiane de survi à 1.7 ans) en dépit d'une maladie localisée. Approximativement 13 % des patients métastasiques de moins de 50 ans ont eu une survie prolongée de plus de 15 ans. L'utilisation d'une stratégie thérapeutique triple, intégrant chirurgie, chimiothérapie et radiothérapie était signifcativement associée à une survie prolongée. Auniveau molécualire, la présence du transcrit de fusio PAX3/7-FOXO1 était significativement liée à un risque accru de maladie métastatique. L'étude in vitro de thérapies ciblées a permis d'identifier la combinaison du vorinostat plus le 17DMAG associée à la doxorubicine comme ayant une meilleure efficacité. La prise en charge du rhabdomyosarcome de l'adolescent et de l'adulte semble souffrir d'une approche moins agressive comparée au rhabdomyosarcome pédiatrique. De plus, des combianaisons de thérapies ciblées peuvent être intégrées aux protocoles de chimiothérapies standardsRhabdomyosarcoma is a rare entity adult patient with unfavourable outcome. This work describes the clinical and molecular specificities of adolescent and adult type of rhabdomyosarcoma and investigates the optimal integration of targetd therapy combinations on small cell sarcoma cell lines in vitro. We retrospectively analyzed 239 patients, 10 years of age and greater, diagonsed withrhabdomyosarcoma at MD Anderson Cancer Center from 1957 trough 2003 and their PAX-FOXO1 fusion gene status by fluorescence in situ hybridization on tissues microarray. Three samll cell sarcoma cell lines were exposed to targetd agent combinations. PAtient with metastatic rhabdomyosarcoma were found to have a 18 % survival rate at 5 years from diagnosis with an 12 %survival past 15 years. This outcome was even poorer for patients over 50 of age, even with localized disease. Younger patients were more likely to receive multidisciplinary therapy than their older counterparts. The presence of PAX-FOXO1 tranlocation was significantly associated with a higher frequency of metastatic disease. The four agents with the exception of abacavir synergized two by two with each other in vitro but the triple combinations did not perform beter than the bitherapies. The dual therapies vorinostat 5HDAC inhibitor) plus 17-DMAG (Hsp90 inhibitor) added with doxorubicin achvied better results than dual or triple therapies. Adult patient with rhabdomyosarcoma present similar molecular and clinical characteristics compared pediatric patients but outcome decrease with age partly du to a less multimodal management. Moreover targeted combinations should be integrated to chemotherapy backbone
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Nehls, Sebastian. "Anwendung des Comet Assay (Einzelzell-Gelelektrophorese) an Zellen von Fischen zum Nachweis gentoxischer Wirkungen im aquatischen Biomonitoring". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2013. http://dx.doi.org/10.18452/16830.
Abstract (sommario):
Gewässer sind Lebensgrundlage, jedoch gleichzeitig Schadstoffsenken für eine Vielzahl von Kontaminanten. Biologische Wirkungstests und das Biomonitoring aquatischer Proben sind daher besonders wichtig, um Umwelt-Gefahrenpotenziale erkennen zu können. Der "Comet Assay" (Einzelzell-Gelelektrophorese) ist ein Indikator von DNA-Strangbrüchen und wurde hier als Test auf gentoxische Wirkungen erprobt und angewandt. Mit bekannten, gentoxischen Substanzen wurden Nachweisgrenzen und Dosis-Wirkungs-Beziehungen für die Zelllinien RTG-2 und RTL-W1 (aus der Regenbogenforelle, Oncorhynchus mykiss) in vitro ermittelt und methodische Parameter an die Zellen angepasst. Der Test reagierte sehr sensitiv auf 4-Nitrochinolin-1-oxid. Die Substanz war daher geeignet, um in weiteren Versuchen als Positivkontrolle zu dienen. Zur Bewertung der Messdaten wurde ein geeignetes statistisches Verfahren gefunden, das auch historische Kontrollen mit einbezog. Der zeitliche Verlauf der DNA-Schädigung des Testsystems mit RTG-2-Zellen wurde ermittelt, und durch Inhibition der DNA-Reparatur mit Aphidicolin wurden Zusammenhänge zwischen der Entstehung von DNA-Strangbrüchen, der DNA-Reparaturkapazität sowie der Metabolisierungskapazität untersucht. In einer zweiten Phase wurden unbehandelte Wasserproben aus Rhein, Elbe sowie weitere Oberflächenwasserproben mit dem Comet Assay an RTG-2-Zellen getestet. Bei 15 von 49 Proben zeigten sich gentoxische Effekte. In einer dritten Phase wurden Erythrozyten von freilebenden Döbeln, Leuciscus cephalus, aus der Mosel mit dem Comet Assay untersucht. Die Fische von drei Messstellen zeigten erhöhte Werte von DNA-Schädigungen, gegenüber einer vierten, stromabwärts gelegenen Messstation. Korrelationen mit den Ergebnissen zusätzlicher Biomarker ergaben sich nur teilweise. Chemische Analysen von Wasser- oder Gewebeproben ließen keine Rückschlüsse auf verursachende Kontaminanten zu - gerade dies unterstreicht jedoch die Wichtigkeit biologischer Tests bei komplexen Proben.Bodies of Water are both vital resources and pollutant sinks for a multitude of contaminants. Therefore, biological effect tests and biomonitoring of aquatic samples are of particular importance to detect potential environmental hazards. The "comet assay" (single cell gel electrophoresis) is an indicator for DNA strand breaks and was explored and applied as a genotoxicity test in the present study. Known genotoxic substances were used to determine the detection limits and dose-response relationships for the cell lines RTG-2 and RTL-W1 (from rainbow trout, Oncorhynchus mykiss) in vitro, and to adapt methodological parameters to the cells. The test was very sensitive to 4-Nitroquinoline-1-oxide. This substance was therefore well-suited to serve as positive control in further experiments. In order to evaluate the measurement data, an appropriate statistical procedure was developed, which also took "historical" controls into account. The time course of DNA damage in the test system using RTG-2 cells was determined, and relationships between the origin of DNA strand breaks, DNA repair capacity and the metabolizing capacity of the cells was investigated by means of inhibition of DNA repair with Aphidicoline. In the second stage, native water samples from the rivers Rhine and Elbe and further surface waters were tested with the comet assay, using RTG-2 cells. 15 out of 49 samples showed genotoxic effects. In a third stage, erythrocytes of feral chub, Leuciscus cephalus, from the Moselle river were examined with the comet assay. The fish from three measuring stations showed elevated values of DNA damage compared to fish sampled from a downstream station. There were only partly correlations with the results from additional biomarkers. Chemical analyses of water and tissue samples did not permit conclusions on effect-causing substances.However, this emphasizes the importance of biological tests in dealing with complex environmental samples.
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DeWitte-Orr, Stephanie. "A study of innate antiviral mechanisms using fish cell lines". Thesis, University of Waterloo, 2006. http://hdl.handle.net/10012/1272.
Abstract (sommario):
Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, Anguilla rostrata. RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders.
HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues.
Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked Mx induction, which is the first demonstration of PKR mediating Mx expression.
The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication.
The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis.
This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.
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John, K. Riji. "Characterization of reovirus-like agents associated with snakehead fish and cell culture". Thesis, University of Stirling, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244645.
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Gignac, Sarah Jane. "Characterization of Fundulus heteroclitus embryonic cell lines and their applications to fish health". Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/46430.
Abstract (sommario):
Common killifish, or mummichogs (Fundulus heteroclitus), are a species of estuarine teleost that are widely used in comparative physiology, toxicology and embryology. Their ability to withstand extreme environmental conditions, widespread distribution, and relatively sedentary nature, makes them ideal as sentinel species of estuarine health. However, the lack of cell lines derived from F. heteroclitus places limitations on the utility of this species in environmental research. In contrast, cell cultures derived from other model organisms have assisted and facilitated our understanding of the effects that environmental contaminants have on organisms in vitro. The development and use of novel F. heteroclitus cell lines for toxicological and parasitological applications is reported here. Continuous proliferating cells were derived from pre-hatch embryos of killifish and have been maintained for 3 years. Three stable cell lines were obtained from the head and body tissues of F. heteroclitus; these stable cell lines have been dubbed KilliFish Embryo 1, 3, and 5 (KFE-1, KFE-3, KFE-5). All three cell lines have been characterized for origin and functionality, as well as for applications in toxicology, studying effects of model chemical pollutants, and in parasitology to evaluate a cod-infecting microsporidia that has been an emerging pathogen of concern, for their ability to infect and grow in cell lines derived from sentinel species. KFE-1 has characteristics of neuroepithelial cells, whereas KFE-3 are possibly liver derived cells, and KFE-5 are distinctly myogenic, as this line has cells that appear to be striated muscle cells. Like intact F. heteroclitus, these cell lines can withstand a wide temperature range from 4°C to 37°C. Mechanisms of thermotolerance and ability to withstand salinity and hypoxia as well as chemical toxicity tolerance could be readily studied with these new cell lines.
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Fleurbaix, Emmanuel. "Évaluation écotoxicologique des éléments terres-rares : approches cellulaires chez différentes espèces aquatiques". Electronic Thesis or Diss., Université de Lorraine, 2021. http://www.theses.fr/2021LORR0324.
Abstract (sommario):
Depuis 30 ans, l’utilisation croissante des Lanthanides dans les nouvelles technologies a entraîné des rejets importants de ces métaux vers les écosystèmes aquatiques. Dans une politique mondiale de développement durable visant à préserver la qualité des écosystèmes, la question de l’impact des Lanthanides sur les organismes aquatiques s’est naturellement posée. Néanmoins, les études restent peu nombreuses et aucun consensus ne subsiste concernant la toxicité des Lanthanides. Dans ce contexte, nous avons étudié la toxicité cellulaire des Lanthanides individuellement et en mélanges. Les effets toxiques ont été mis en évidence en mesurant la viabilité de cellules fibroblastiques (ZF4 ; ATCC®, CRL-2050™) et hépatiques (ZFL ; ATCC®, CRL-2643™) de poisson zèbre (Danio rerio), de cellules branchiales (RTgill-W1 ; ATCC®, CRL-2523™) de la truite arc-en-ciel (Oncorynchus mykiss), et des cellules primaires de glandes digestives de corbicule (Corbicula fluminea) exposées à ces métaux. Les résultats ont montré que les Lanthanides sont responsables d’effets toxiques directs sur nos modèles cellulaires. Concernant la toxicité des Lanthanides en mélanges, des effets synergiques ont été observés sur les 3 lignées cellulaires de poissons. Nous nous sommes également intéressés aux mécanismes de détoxification des Lanthanides dans les cellules ZF4 de Danio rerio. Nous avons décidé d’étudier ces acteurs en raison de leurs rôles respectifs dans les phases II et III de la détoxification cellulaire de métaux chez les bivalves et les poissons. Pour cela, la viabilité des cellules ZF4 a été mesurée après des expositions aux Lanthanides en présence d’inhibiteurs spécifiques des glutathion-S-transférases (acide éthacrynique) et des protéines MRP (MK571 et probénécide). Les résultats ont montré que les protéines MRP sensibles au MK571 jouent un rôle dans la détoxification des Lanthanides dans les cellules ZF4. Globalement, les résultats obtenus pour cette recherche ont confirmé que les effets toxiques des Lanthanides à l’échelle cellulaire sont pertinents pour prédire les effets in vivo, dans le cadre d’une évaluation de la toxicité de ces métauxSince 30 years ago, the growing use of Lanthanides in new technologies has contributed to important releases of these metals into aquatic ecosystems. In a global sustainable development policy aimed at preserving the quality of ecosystems, the impact of Lanthanides on aquatic organisms has naturally been questioned. However, studies on the aquatic ecotoxicology of Lanthanides are incomplete, and no consensus is established yet. In this context, we studied the cellular toxicity of Lanthanides individually and in mixtures. To determine these toxic effects, cell viability was measured on Danio rerio fibroblast-like cells (ZF4; ATCC®, CRL-2050™), Danio rerio hepatic cells (ZFL; ATCC®, CRL-2643™), Oncorhynchus mykiss epithelial cells (RTgill-W1; ATCC®, CRL-2523™), and primary culture of Corbicula fluminea digestive glands exposed to Lanthanides. Direct toxicity of Lanthanides has been observed on all cellular models. Concerning the toxicity of Lanthanides in mixtures, synergistic effects have been underlined on the three fish cell lines. In this research, we focused on the mechanisms of the detoxification of Lanthanides in the case of ZF4 cells from Danio rerio. The effects of Lanthanides were assessed in the presence of specific inhibitors of glutathione-S-transferases (ethacrynic acid) and MRP-like (MK571 and probenecid), by cell viability measurements. We decided to study these actors of the cellular detoxification due to their respective roles in phases II and III of the cellular detoxification of metals in fishes and bivalves. Regarding the results, MRP-like proteins are effectively involved in the detoxification of Lanthanides in ZF4 cells. Overall, our results highlighted the relevance of the toxic effects of Lanthanides at the cellular level for the risk assessment of these metals
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Wiersinga-Post, Jenny Esther Clasine. "Mechanophysiology of cupulae and hair cells in the lateral line of fish and pitch perception of complex sounds in humans". [S.l. : [Groningen] : s.n.] ; [University Library Groningen] [Host], 1997. http://irs.ub.rug.nl/ppn/164044949.
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Flemming, Leonard. "Comparative proteomic and genomic analysis of Flavobacterium johnsoniae-like biofilm, planktonic and agar surface-associated cells". Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/4020.
Abstract (sommario):
Thesis (PhD(Microbiology))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: Pathogenic Flavobacterium spp. cause serious disease outbreaks in a variety of farmed fish, which lead to large economic losses in the aquaculture industry on an annual basis. The ability of Flavobacterium johnsoniae-like isolates to grow as surface-associated communities (biofilms) in aquaculture systems poses a threat to fish health over extended periods of time. The biofilmforming ability of 28 F. johnsoniae-like isolates obtained from diseased fish were correlated with their chitin-degrading abilities and extracellular carbohydrate complexes (ECC) and their pulsed-field gel electrophoresis (PFGE) genotypes. Physiological changes in the proteome of 5 day planktonic, biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were analyzed by two-dimensional (2-D) gel electrophoresis and 17 differentially expressed and 14 uniquely expressed proteins were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Thirty-two differentially expressed genes in 5 day biofilm and agar surface-associated cultures of F. johnsoniae-like isolates YO12 and YO64 were identified using suppression subtractive hybridization (SSH). Significant negative correlations were observed between the chitin-degrading abilities and ECC and the biofilmforming capacity of 24 h biofilm cultures of F. johnsoniae-like isolates. Genetic heterogeneity was displayed by the F. johnsoniae-like isolates following PFGE. A significant positive correlation was observed between PFGE types and fish host species. Differentially and uniquely expressed proteins identified in planktonic, biofilm and agar surface-associated phases by 2-D/MS as well as differentially expressed genes identified in the biofilm and agar surface-associated phases by SSH were categorized as being involved in adaptation/protection, metabolic processes, membrane/transport/ motility and transcription/ translation. As far as we know, this is the first report on the characterization of differentially expressed genes and gene products of F. johnsoniae-like isolates obtained from diseased fish in South Africa.
AFRIKAANSE OPSOMMING: Patogene Flavobacterium spp. veroorsaak ernstige infeksie uitbrake in ’n verskeidenheid gekweekte vissoorte, wat jaarliks tot groot ekonomiese verliese in die akwakultuur bedryf lei. Die vermoë van Flavobacterium johnsoniae-tipe isolate om as oppervlak-gehegde gemeenskappe (biofilms) in akwakultuur sisteme te groei bedreig visgesondheid oor verlengde periodes. Die vermoë van 28 F. johnsoniae-tipe isolate om biofilms te vorm is vergelyk met hul vermoë om chitien te degradeer, die profiel van hul ekstrasellulêre koolhidraat komplekse (EKK) en bandpatrone verkry met puls-veld jel elektroforese (PVJE). Fisiologiese veranderinge in die proteoom van 5-dagoue planktoniese-, biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 is met twee-dimensionele (2-D) jel elektroforese geanaliseer. Sewentien differensieël uitgedrukte en 14 uniek uitgedrukte proteïene is deur middel van matriks-geassisteerde laser desorpsie ioniserings-tyd van vlug-massa spektrometrie (MGLDI-TVV MS) geïdentifiseer. Twee-en-dertig differensieël uitgedrukte gene in 5-dag-oue biofilm- en agar oppervlak-geassosieerde kulture van F. johnsoniae-tipe isolate YO12 en YO64 was deur middel van suppressie afgetrokke hibridisasie (SAH) geïdentifiseer. Beduidende negatiewe korrelasies is tussen die chitin-degraderings vermoë en EKK en die biofilm-vormings kapasiteit van 24-uur-oue biofilm kulture van F. johnsoniae-tipe isolate waargeneem. Resultate verkry met PVJE het die heterogene samestelling van F. johnsoniae-tipe isolate uitgewys. ‘n Beduidende positiewe korrelasie is tussen PVJE groeperings en vis gasheer spesie waargeneem. Differensieël en uniek uitgedrukte gene geidentifiseer in die planktoniese-, biofilm- en agar oppervlak-geassosieerde fases is deur middel van 2-D/MS asook differensieël uitgedrukte gene geïdentifiseer in die biofilm en agar oppervlakgeassosieerde fases deur middel van SAH was as betrokke by aanpassing/beskerming, metaboliese prosesse, membraan/vervoer/ beweeglikheid en transkripsie/translasie gekategoriseer. Sover bekend is hierdie die eerste beskrywing van differensieël uitgedrukte gene en geenprodukte van F. johnsoniae-tipe isolate afkomstig van geinfekteerde vis in Suid Afrika.
9
Privitera, Giovanna. "Studio di nuovi approcci farmacologici in grado di inibire l'attivazione dei recettori del fattore di crescita epidermico (EGF) in linee cellulari di carcinoma polmonare non a piccole cellule (NSCLC)". Doctoral thesis, Università di Catania, 2013. http://hdl.handle.net/10761/1436.
Abstract (sommario):
Il carcinoma del polmone rappresenta attualmente la neoplasia più frequentemente diagnosticata e costituisce la principale causa di morte per tumore solido al mondo. E una malattia multifattoriale che riconosce nella cancerogenesi cause ambientali e cause genetiche. L istotipo più frequente è il carcinoma non a piccole cellule (NSCLC) che rappresenta circa il 75-80% delle diagnosi. Il principale approccio terapeutico consiste nella resezione chirurgica (eventualmente preceduta e/o seguita da chemioterapia) limitata però ai soli stadi iniziali.
Studi precedenti hanno dimostrato che l iper-espressione o l attivazione costitutiva dei recettori del fattore di crescita epidermico (EGF) è coinvolta nella crescita dei carcinomi umani, incluso quello polmonare. In questo lavoro sperimentale è stato studiato l effetto del blocco di due recettori dell EGF, EGFR e HER-2, sulla proliferazione delle cellule A549 e NCI-H226 di carcinoma polmonare. Sono stati utilizzati, a tale scopo, cetuximab, anticorpo monoclonale diretto contro l EGFR e trastuzumab, anticorpo monoclonale diretto contro l HER-2, a diverse concentrazioni per 72 h. Mediante il saggio di proliferazione MTT (3,(4,5-dimethylthiazol-2)2,5 difeniltetrazolium bromide) è stato osservato il 91,4% di inibizione della proliferazione delle cellule A549 trattate con una combinazione di cetuximab e trastuzumab alla concentrazione di 40 ug/ml, mentre le cellule NCI-H226 hanno mostrato una resistenza allo stesso trattamento combinato. Sono state successivamente valutate le eventuali variazioni di espressione dei livelli di mRNA dei recettori EGFR e HER-2 di entrambe le linee cellulari. Nelle cellule A549 è stato osservato un significativo incremento dei livelli di mRNA di EGFR dopo 30 h dal trattamento farmacologico, spiegabile considerando il meccanismo di azione del cetuximab, che determina internalizzazione e degradazione del recettore in tempi brevi. A questa segue quindi nuova sintesi di proteine recettoriali. Questi livelli di mRNA diminuiscono dopo 48 h e 72 h dal trattamento per avvicinarsi poi ai valori normali. Un comportamento analogo è stato osservato nei livelli di mRNA del recettore HER-2, in cui l internalizzazione del recettore, conseguente all interazione con il trastuzumab, determina già a 18 h di trattamento un incremento dell espressione di tale recettore.
Le cellule NCI-H226 hanno mostrato invece un aumento di circa 3,5 volte dei livelli di mRNA dell EGFR dopo 48 h dal trattamento e il mantenimento di alti livelli anche dopo 72 h. Il recettore HER-2 non sembra essere invece influenzato dal trattamento farmacologico e, almeno nei primi tempi della somministrazione, i livelli di mRNA non vengono alterati. Solo dopo 72 h di trattamento è stato osservato un aumento di tali livelli, che vengono circa raddoppiati, ad indicare un possibile coinvolgimento tardivo di tale recettore nella risposta al trattamento combinato. L iniziale scarso coinvolgimento del recettore HER-2 potrebbe quindi essere responsabile della resistenza delle cellule NCI-H226. E pertanto nostro obiettivo futuro prolungare il tempo di trattamento delle cellule NCI-H226 con cetuximab e trastuzumab allo scopo di valutare un effetto antiproliferativo più tardivo.
La differenza di sensibilità al cetuximab e al trastuzumab, nelle due linee cellulari prese in esame, non dipende invece dal numero di copie del gene, in quanto, mediante la tecnica di ibridazione in situ fluorescente (FISH), abbiamo osservato lo stesso incremento del numero di copie dei geni EGFR e HER-2 e quindi una condizione di polisomia, in entrambe le linee cellulari.
La strategia di agire su entrambi i recettori potrebbe quindi migliorare il trattamento del carcinoma polmonare, aumentare la sopravvivenza e ridurre gli effetti collaterali della terapia, garantendo così al paziente un sensibile miglioramento della qualità di vita.
10
Kienzler, Aude. "Intérêt des lignées cellulaires de poisson en écotoxicologie pour l'étude de nouveaux biomarqueurs de génotoxicité". Phd thesis, INSA de Lyon, 2013. http://tel.archives-ouvertes.fr/tel-00952888.
Abstract (sommario):
Un contexte réglementaire de plus en plus strict en évaluation du risque écotoxicologique des milieux aquatiques exige de renforcer les outils d'évaluation. A ce titre, l'étude des biomarqueurs de génotoxicité doit être privilégiée, compte tenu du rôle central de l'ADN dans le fonctionnement du vivant. L'exposition à des agents génotoxiques peut générer des dommages par interaction directe avec l'ADN, mais aussi indirectement, en modulant l'efficacité des mécanismes de réparation de l'ADN, ou la régulation épigénétique de l'expression des gènes. Aujourd'hui, la plupart des biomarqueurs de génotoxicité visent les dommages primaires à l'ADN ou la mutagènicité mais les effets indirects sur sa fonctionnalité sont encore peu étudiés. Dans ce contexte, à l'issue d'une analyse bibliographique comprenant la rédaction d'une revue sur les mécanismes de réparation des dommages à l'ADN chez le poisson, ce travail visait au développement méthodologique de plusieurs biomarqueurs de génotoxicité à l'aide de trois lignées cellulaires pisciaires (RTL-W1, RTGill-W1 et PLHC-1). Pour ce faire, l'essai des comètes en conditions alcalines a été décliné sous plusieurs versions dans l'objectif d'utiliser une technique de base unique permettant la mesure complémentaire de plusieurs biomarqueurs de génotoxicité : les dommages primaires à l'ADN, les activités de réparation et le niveau de méthylation des cytosines du génome. Les résultats soulignent l'intérêt des trois lignées en évaluation de la génotoxicité 1) pour détecter in vitro de manière sensible des atteintes primaires à l'ADN de natures variées à de faibles concentrations grâce à un essai des comètes modifié par une étape de digestion enzymatique avec une glycosylase (Fpg), 2) pour évaluer l'influence des contaminants sur l'activité de réparation par excision de bases (BER) via la mesure de la capacité d'incision d'un ADN substrat porteur de lésions de type 8-oxoGua par des extraits cellulaires (essai BERc). Le niveau de méthylation des cytosines (5-meCyt) des lignées RTgill-W1et RTL-W1 a été mesuré par HPLC-MS-MS, leur valeur élevée permet d'envisager le paramètre méthylation comme biomarqueur potentiel. Ce volet nécessitera cependant des étapes de validations ultérieures car il n'a pas été techniquement possible de mettre au point un essai des comètes modifié pour la mesure du niveau de méthylation de l'ADN. Plusieurs activités de réparation des lignées RTgill-W1 et RTL-W1 ont été caractérisées et révèlent de bonnes aptitudes de réparation de type " Base Excision Repair " (BER) et " Photo Enzymatic Repair " (PER) et une plus faible capacité au " Nucleotide Excision Repair " (NER), soit un profil proche de celui décrit in vivo et sans différence marquée entre les deux lignées. Les biomarqueurs développés sur les lignées cellulaires de poisson au cours de ce travail ont également été appliqués à la mesure des effets génotoxiques d'effluents issus du lessivage de revêtements routiers.
Libri sul tema "Fish cell line":
1
India) National Workshop on Fish Cell Line : Development & Storage (2012 National Bureau of Fish Genetic Resources. National Workshop on Fish Cell Line: Development & storage : proceedings. Lucknow, India: National Bureau of Fish Genetic Resources, Indian Council of Agricultural Research, 2012.
2
Liehr, Thomas. Fluorescence In Situ Hybridization (FISH) — Application Guide. Berlin, Heidelberg: Springer Berlin Heidelberg, 2009.
3
International Conference on Invertebrate and Fish Tissue Culture (7th 1987 Ohito, Japan). Invertebrate and fish tissue culture: Proceedings of the Seventh International Conference on Invertebrate and Fish Tissue Culture, Japan, 1987. Tokyo: Japan Scientific Societies Press, 1988.
4
Test No. 249: Fish Cell Line Acute Toxicity - The RTgill-W1 cell line assay. OECD, 2021. http://dx.doi.org/10.1787/c66d5190-en.
5
Mokhtar, Doaa M. Fish Histology: From Cells to Organs. Apple Academic Press, Incorporated, 2017.
6
Mokhtar, Doaa M. Fish Histology: From Cells to Organs. Apple Academic Press, Incorporated, 2021.
7
Mokhtar, Doaa M. Fish Histology: From Cells to Organs. Taylor & Francis Group, 2018.
8
Mokhtar, Doaa M. Fish Histology: From Cells to Organs. Apple Academic Press, Incorporated, 2017.
9
Mokhtar, Doaa M. Fish Histology: From Cells to Organs. Apple Academic Press, Incorporated, 2021.
10
Mokhtar, Doaa M. Fish Histology: From Cells to Organs. Apple Academic Press, Incorporated, 2017.
Capitoli di libri sul tema "Fish cell line":
1
Inoue, H., K. Taniai, T. Tamura e T. Kanda. "Infection of Silkworm Cell Line with an Insect Virus". In Invertebrate and Fish Tissue Culture, 163–65. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_39.
2
Suyama, I., e H. Etoh. "Establishment of a Cell Line from Umbra Limi (Umbridae; Pisces)". In Invertebrate and Fish Tissue Culture, 270–73. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_64.
3
Chen, Q., e X. Dai. "Replication of Pieris Rapae Granulosis Virus in an Insect Cell Line". In Invertebrate and Fish Tissue Culture, 170–72. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_41.
4
Chen, Q., L. Li, Z. Yu e J. Peng. "Establishment of Cell Line from Embryos of the Silkworm, Bombyx Mori". In Invertebrate and Fish Tissue Culture, 259–61. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_61.
5
Watanabe, T., e T. Moritomo. "Establishment and Properties of a Cell Line Derived from Salmonid Fish Liver". In Invertebrate and Fish Tissue Culture, 199–202. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_48.
6
Baeze-Squiban, A., M. Best-Belpomme e F. Marano. "Effects of Deltamethrin on a Drosophila Melanogaster Cell Line In Vitro: Cytotoxicity, Accumulation and Metabolism". In Invertebrate and Fish Tissue Culture, 58–59. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_15.
7
Romano, H., E. Kasama, Y. Nakanishi, K. Matsuyama, K. Ando, Y. Nagasawa e S. Natori. "Synthesis of Sarcophaga Lectin and Sarcotoxins in Nih-Sape-4, an Embryonic Cell Line of Sarcophaga Peregrina". In Invertebrate and Fish Tissue Culture, 75–78. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_19.
8
Ferkovich, S. M., e H. Oberlander. "Isolation of a Morphogen That Induces Vesicle Formation in a Cell Line Derived from Imaginal Wing Discs of Trichoplusia Ni". In Invertebrate and Fish Tissue Culture, 79–84. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_20.
9
Segner, H. "Fish cell lines as a tool in aquatic toxicology". In Fish Ecotoxicology, 1–38. Basel: Birkhäuser Basel, 1998. http://dx.doi.org/10.1007/978-3-0348-8853-0_1.
10
Yoshimizu, M., M. Kamei, S. Dirakbusarakom e T. Kimura. "Fish Cell Lines : Susceptibility to Salmonid Viruses". In Invertebrate and Fish Tissue Culture, 207–10. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73626-1_50.
Atti di convegni sul tema "Fish cell line":
1
Dykina, N. V., e S. L. Rudakova. "CONDITIONS FOR PASSAGING AND MAINTAINING A CONTINUOUS EPC FISH CELL LINE FOR A LONG TIME WITHOUT CRYOPRESERVATION". In OpenBio-2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-245.
Abstract (sommario):
One of the common ways of long-term maintenance of cell lines is cryopreservation. However, this method is far from always applicable. Is it possible to preserve the fish cell line Epithelioma papulosum cyprini (EPC) for a long time without freezing? This will be discussed in the current work.
2
Torrissen, Martina, Astrid Nilsson, Binh Minh Trinh, Elisabeth Ytteborg, Gerd Marit Berge, Harald Svenson, Iren Stoknes e Marta Bou Mira. "Novel n-3 very-long-chain polyunsaturated fatty acids and their potential role in skin tissue". In 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/nkdk5807.
Abstract (sommario):
Very-long-chain fatty acids (VLC-FA) have a chain length of ≥24 carbon atoms. They are generally not provided through dietary sources but generated endogenously, involving chain elongation of LC-FA by ELOVL4. There is emerging substantial evidence suggesting they play important roles in tissues where they are naturally found, including retina, skin, testis, and brain. Mutations in ELOVL4 have been associated with several tissue-specific conditions, suggesting these FA may be involved in the disease pathology. A lack of availability of these fatty acids for dietary interventions has however, until recently, made them difficult to investigate. After identifying VLC-FA in fish oil and developing a method for concentrating n-3 VLC-PUFAs in kg scale, our research team have conducted feeding trials to determine if they are taken up directly from diet through supplementation, and their effect on development and maturation of skin tissue. Salmon fed different dietary levels of the concentrate were analysed for tissue fatty acid composition by GC and histology by H&E and Von Kossa staining. After establishing a clear tissue-specific uptake, we conducted in-vitro trials where we observed promising effects by incubating skin cells from human and Atlantic salmon with n-3 VLC-PUFA concentrate in scratch assay and cell migration trials. The in-vitro results show improved cell migration, which is in line with our in-vivo findings and demonstrates a promising effect on skin tissue development, maturation, and skin cell migration. Here we will present our data and discuss the relevance of this in skin biology. As VLC-FA potentially play a critical role in skin barrier function and skin biology, understanding these FAs may lead to improvements in treatment of dermatological diseases and conditions.
3
Zhang, Lelin, Hongkai Xiong, Yang Zhao, Kai Zhang, Xiaobo Zhou, Tuan D. Pham e Xiaobo Zhou. "Computational Graph Model for 3D Cells Tracking in Zebra Fish Datasets". In COMPUTATIONAL MODELS FOR LIFE SCIENCES/CMLS '07. AIP, 2007. http://dx.doi.org/10.1063/1.2816615.
4
Ge, Xiaowei, Fatima C. Pereira, Meng Zhang, Peng Lin, Michael Wagner e Ji-Xin Cheng. "SRS-FISH: high-throughput bridging of phenotype and genotype at single cell level". In Advanced Chemical Microscopy for Life Science and Translational Medicine 2021, a cura di Garth J. Simpson, Ji-Xin Cheng e Wei Min. SPIE, 2021. http://dx.doi.org/10.1117/12.2577969.
5
Tamaddoni, Nima, e Andy Sarles. "Fabrication and Characterization of a Membrane Based Hair Cell Sensor That Features Soft Hydrogel Materials". In ASME 2012 Conference on Smart Materials, Adaptive Structures and Intelligent Systems. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/smasis2012-8067.
Abstract (sommario):
One of the most common sensory structures in nature is the hair cell. Examples of hair cells include the inner and outer hair cells in the inner ears of vertebrates, external sensory hairs on the legs of spiders, and neuromasts found along the lateral lines of fish. Recent work by Sarles and Leo demonstrated that self-assembly methods could be used to construct a membrane-based hair cell that responds to a physical disturbance of the hair. An artificial cell membrane (or lipid bilayer) formed at the interface of two lipid-encased hydrogel volumes, serves as the transduction element in the device. In this study, a revised sensor embodiment is presented in which the hair is fixed at its base by the encapsulating polymeric substrate. In addition, a highly elastic, photo-polymerizable aqueous gel (PEGDA, 6000g/mole) is used to further increase the resiliency of the hair and to provide a compliant cushion for the bilayer. These changes yield a considerably more durable hair cell sensor. We perform a series of experimental tests to characterize the transduction element (i.e. the bilayer) and the sensing current produced by free vibration of the hair, and we study the directional sensitivity of this hair cell embodiment by perturbing the hair in three directions. These tests demonstrate that the magnitude of the sensing current (30–300pA) is significantly affected by direction of perturbation, where the largest signals result from motion of the hair in a direction perpendicular to the plane of the bilayer.
6
Kitazawa, Daisuke, Hiroki Shimizu e Yoichi Mizukami. "Tank Model Testing on the Fish Cage Installed in Variable Depths in Current and Waves". In ASME 2013 32nd International Conference on Ocean, Offshore and Arctic Engineering. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/omae2013-11239.
Abstract (sommario):
A fish cage should be submerged to reduce hydrodynamic forces from high waves if the fish cage is installed in an exposed sea area. Usually, the submergible fish cage is suspended from the framework at a fixed depth. The framework is set by floats and anchors at the middle position between water surface and the top surface of the submergible fish cage. The submergible fish cage will be used not only for reduction of hydrodynamic forces but for the other purposes such as choosing the best environment for cultured fishes in the vertical direction, and escaping from the flood with high-level nitrogen or turbidity, harmful algal blooming, and floating ices. In such cases, it is useful for the fish cage to be installed in variable depths. The purpose of the present study is to examine the safety of the fish cage installed in variable depths in current and waves by means of tank model testing. The mooring system consists of a fish cage and four floats. The vertical position of the fish cage is variable by adjusting the buoyancy of these floats. First, the drag of the fish cage was examined by towing test, and the results were compared with the drag estimated by the existing studies. The effects of interaction among twines, the angle of attack, wake, and the top and bottom nets were discussed. Then the fish cage was moored in the water tank, which has the length of 50 m and the width of 10 m. The tank model has a scale of 1/100 of the full-scale model of the fish cage used for tuna farming. The model was made according to Tauti’s similarity law. The water depth was set at 0.68 m by adjusting the position of the variable floor. The motion of the fish cage and four floats, and the tension of the mooring lines between the fish cage, floats, and anchors were measured by the underwater video camera and load cells, respectively. As a result, the drag of the fish cage could be estimated from the experimental results of the drag of a plane net since the results include the effect of interaction among twines. The effects of the angle of attack and the reduction in water current velocity inside the cage were also taken into account. The drag of the fish cage could be estimated well by the above method, while it was underestimated by 10% in comparison with the experimental data. In the water tank testing of the mooring system, the tension of the mooring line increased rapidly with the increase in water current velocity since the drag of the fish cage was proportional to the 1.8th power of water current velocity and increased due to the inclination of the fish cage. The increase in the tension due to wave-induced forces to the fish cage could be negligible when the fish cage was submerged. The safety and the design guideline of the mooring system should be assessed by the simulations using a numerical model, which is being developed by the authors. The experimental data obtained in the present study will be useful for the validation of the numerical model.
7
Park, Daniel S., Robert Egnatchik, Hali Bordelon, Terrence R. Tiersch e W. Todd Monroe. "A Microfluidic Mixer to Activate Sperm Cells of Aquatic Species for Standardization of Computer-Assisted Motion Analysis". In ASME 2011 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2011. http://dx.doi.org/10.1115/sbc2011-53839.
Abstract (sommario):
The objective of this paper is to develop a microfluidic device to: 1) activate a small volume of aquatic species sperm by rapid mixing with diluent, and 2) position sperm in a viewing chamber for motility evaluation using computer-assisted sperm analysis (CASA). Analysis of aquatic species sperm is becoming more important as the use of fish as biomedical models expands. Because it is more efficient to maintain frozen stocks of genetic material rather than thousands of research lines of adult fish, there has been increased study on cryopreservation for model fish. The analysis of fish gametes is challenging due to small sample size, short motility duration, and inconsistent activation (motility induction). For many aquatic species, sperm motility is initiated through the manual alteration of the medium osmolality, typically accomplished through manual dilution and mixing by hand. Manual methods limit control over the activation process and therefore viability analysis. The short lifespan of these cells makes CASA challenging due to the limitations in capturing and processing data rapidly enough to monitor the peak motility, as CASA systems were designed for mammalian sperm which have a longer motility duration.
8
Sharif, Montassar Aidi, Matthew J. McHenry e Xiaobo Tan. "Modeling of a Bio-Inspired Canal-Type Lateral Line System". In ASME 2017 Conference on Smart Materials, Adaptive Structures and Intelligent Systems. American Society of Mechanical Engineers, 2017. http://dx.doi.org/10.1115/smasis2017-3756.
Abstract (sommario):
It is of interest to exploit the insight from the lateral line system of fish for flow sensing applications. The lateral line consists of arrays of flow sensors, known as neuromasts, with hair cells encased within a gel-like structure called cupula. There are two types of neuromasts, superficial neuromasts, which reside on the surface, and canal neuromasts, which are recessed within a channel with its ends open at the body’s surface. In this work we investigate the modeling of a canal-type artificial lateral line system. The canal is filled with viscous fluid to emulate its biological counterpart. The artificial neuromast consists of an ionic polymer-metal composite (IPMC) sensor embedded within a soft molded cupula structure. The displacement of the cupula structure and the resulting short-circuit current of the IPMC sensor under an oscillatory flow are modeled and solved with finite-element methods. The Poisson-Nernst-Planck (PNP) model is used to describe the fundamental physics within the IPMC, where the bending stimulus due to the cupula displacement is coupled to the PNP model through the cation convective flux term. Comparison of the numerically computed cupula displacement with an analytical approximation is conducted. The effects of material stiffness and and device size on the device sensitivity are further explored.
9
Scherr, Thomas F., Gerald Knapp, Terrence Tiersch, W. Todd Monroe e Krishnaswamy Nandakumar. "The Activation of Zebrafish Sperm Cells in a Micromixer". In ASME 2013 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/sbc2013-14734.
Abstract (sommario):
The freshwater fish, Danio rerio (zebrafish), have become widely used as a model organism for vertebrate development, DNA mutation, and human disease studies [1]. Maintaining live colonies of the numerous developed strains of zebrafish under investigation can be prohibitively costly. As such, there is a growing need to catalog their reproductive cells and have them available on demand [2]. Thus cryopreservation of model strain gametes has become an important endeavor, where evaluation of freezing and thawing techniques is currently a bottleneck to these procedures.
10
Norbach, Alexandra, Kotryna Bedrovaite Fjetland, Gina Vikum Hestetun e Thomas J. Impelluso. "Gyroscopic Wave Energy Generator for Fish Farms and Rigs". In ASME 2018 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2018. http://dx.doi.org/10.1115/imece2018-86188.
Abstract (sommario):
Norway has an opportunity to harvest ocean wave energy through gyroscopic precession as an alternative source of renewable energy, within practical limitations. This research assesses the energy extracted by gyroscopic wave energy generators and their use to provide supplementary power to fish farms and lighting on oilrigs. This project implements the Moving Frame Method (MFM) in dynamics to model the extracted power from a gyroscopic wave energy generator. The MFM leverages Lie Group Theory, Cartan’s moving frames and a new notation from the discipline of geometrical physics. Continuing, the Principle of Virtual Work extracts the equations of motion from the structure of the Special Orthogonal Group. However, the MFM supplements its analysis with a novel application of the restriction on the variations of the angular velocities. This research extends previous work as follows: it accounts for motor torques, it opens a placeholder for buoyancy, and it solves the full 3D set of equations (without assuming negligible yaw). After showing how to obtain the suite of descriptive equations of motion, this project integrates them, however with a relatively simple integration scheme. To complete each step in the analysis, the rotation matrix is updated using the Cayley Hamilton Theorem and the Rodriguez formula. Finally, the results are displayed using the Web Graphics Library such that the actual numerical analysis and display happens on cell phones.
Rapporti di organizzazioni sul tema "Fish cell line":
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Funkenstein, Bruria, e Shaojun (Jim) Du. Interactions Between the GH-IGF axis and Myostatin in Regulating Muscle Growth in Sparus aurata. United States Department of Agriculture, marzo 2009. http://dx.doi.org/10.32747/2009.7696530.bard.
Abstract (sommario):
Growth rate of cultured fish from hatching to commercial size is a major factor in the success of aquaculture. The normal stimulus for muscle growth in growing fish is not well understood and understanding the regulation of muscle growth in fish is of particular importance for aquaculture. Fish meat constitutes mostly of skeletal muscles and provides high value proteins in most people's diet. Unlike mammals, fish continue to grow throughout their lives, although the size fish attain, as adults, is species specific. Evidence indicates that muscle growth is regulated positively and negatively by a variety of growth and transcription factors that control both muscle cell proliferation and differentiation. In particular, growth hormone (GH), fibroblast growth factors (FGFs), insulin-like growth factors (IGFs) and transforming growth factor-13 (TGF-13) play critical roles in myogenesis during animal growth. An important advance in our understanding of muscle growth was provided by the recent discovery of the crucial functions of myostatin (MSTN) in controlling muscle growth. MSTN is a member of the TGF-13 superfamily and functions as a negative regulator of skeletal muscle growth in mammals. Studies in mammals also provided evidence for possible interactions between GH, IGFs, MSTN and the musclespecific transcription factor My oD with regards to muscle development and growth. The goal of our project was to try to clarify the role of MSTNs in Sparus aurata muscle growth and in particular determine the possible interaction between the GH-IGFaxis and MSTN in regulating muscle growth in fish. The steps to achieve this goal included: i) Determining possible relationship between changes in the expression of growth-related genes, MSTN and MyoD in muscle from slow and fast growing sea bream progeny of full-sib families and that of growth rate; ii) Testing the possible effect of over-expressing GH, IGF-I and IGF-Il on the expression of MSTN and MyoD in skeletal muscle both in vivo and in vitro; iii) Studying the regulation of the two S. aurata MSTN promoters and investigating the possible role of MyoD in this regulation. The major findings of our research can be summarized as follows: 1) Two MSTN promoters (saMSTN-1 and saMSTN-2) were isolated and characterized from S. aurata and were found to direct reporter gene activity in A204 cells. Studies were initiated to decipher the regulation of fish MSTN expression in vitro using the cloned promoters; 2) The gene coding for saMSTN-2 was cloned. Both the promoter and the first intron were found to be polymorphic. The first intron zygosity appears to be associated with growth rate; 3) Full length cDNA coding for S. aurata growth differentiation factor-l I (GDF-II), a closely related growth factor to MSTN, was cloned from S. aurata brain, and the mature peptide (C-terminal) was found to be highly conserved throughout evolution. GDF-II transcript was detected by RT -PCR analysis throughout development in S. aurata embryos and larvae, suggesting that this mRNA is the product of the embryonic genome. Transcripts for GDF-Il were detected by RT-PCR in brain, eye and spleen with highest level found in brain; 4) A novel member of the TGF-Bsuperfamily was partially cloned from S. aurata. It is highly homologous to an unidentified protein (TGF-B-like) from Tetraodon nigroviridisand is expressed in various tissues, including muscle; 5) Recombinant S. aurata GH was produced in bacteria, refolded and purified and was used in in vitro and in vivo experiments. Generally, the results of gene expression in response to GH administration in vivo depended on the nutritional state (starvation or feeding) and the time at which the fish were sacrificed after GH administration. In vitro, recombinantsaGH activated signal transduction in two fish cell lines: RTHI49 and SAFI; 6) A fibroblastic-like cell line from S. aurata (SAF-I) was characterized for its gene expression and was found to be a suitable experimental system for studies on GH-IGF and MSTN interactions; 7) The gene of the muscle-specific transcription factor Myogenin was cloned from S. aurata, its expression and promoter activity were characterized; 8) Three genes important to myofibrillogenesis were cloned from zebrafish: SmyDl, Hsp90al and skNAC. Our data suggests the existence of an interaction between the GH-IGFaxis and MSTN. This project yielded a great number of experimental tools, both DNA constructs and in vitro systems that will enable further studies on the regulation of MSTN expression and on the interactions between members of the GHIGFaxis and MSTN in regulating muscle growth in S. aurata.
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Noga, Edward J., Angelo Colorni, Michael G. Levy e Ramy Avtalion. Importance of Endobiotics in Defense against Protozoan Ectoparasites of Fish. United States Department of Agriculture, settembre 2003. http://dx.doi.org/10.32747/2003.7586463.bard.
Abstract (sommario):
Infectious disease is one of the most serious causes of economic loss in all sectors of aquaculture. There is a critical need to understand the molecular basis for protection against infectious disease so that safer, more reliable and more cost-effective strategies can be designed for their control. As part of this effort, the major goal of our BARD project was to determine the importance of endobiotics as a defense against protozoan ectoparasites in fish. Endobiotics, or antimicrobial polypeptides, are peptides and small proteins that are increasingly recognized as having a vital role in the innate defense of virtually all animals. One objective of our BARD project was to determine the antiparasitic potency of one specific group of endobiotics that were isolated from hybrid striped bass (Morone saxatilis x M chrysops). We found that these endobiotics, which we had previously named histone-like proteins (HLPs), exhibited potent activity against Amyloodinium and that the putative levels of HLPs in the skin were well within the levels that we found to be lethal to the parasite in vitro. We also found evidence for the presence of similar antibiotics in sea bream (Sparus aurata) and Mediterranean sea bass (Dicentrarchus labrax). We also examined the effect of chronic stress on the expression of HLP in fish and found that HLP levels were dramatically decreased after only one week of a crowding/high ammonia sublethal stress. We also began to explore the feasibility of upregulating endobiotics via immunostimulation. However, we did not pursue this objective as fully as we originally intended because we spent a much larger effort than originally anticipated on the last objective, the attempted isolation of novel endobiotics from hybrid striped bass. In this regard, we purified and identified four new peptide endobiotics. These endobiotics, which we have named piscidins (from "Pisces" meaning fish), have potent, broad-spectrum activity against a number of both fish and human pathogens. This includes not only parasites but also bacteria. We also demonstrated that these peptides are present in the mast cell. This was the first time that the mast cell, the most common tissue granulocyte in vertebrates, was shown to possess any type of endobiotic. This finding has important implications in explaining the possible function of mast cells in the immune response of vertebrates. In summary, the research we have accomplished in this BARD project has demonstrated that endobiotics in fish have potent activity against many serious pathogens in aquaculture and that there is considerable potential to use these compounds as stress indicators in aquaculture. There is also considerable potential to use some of these compounds in other areas of medicine, including treatment of serious infectious diseases of humans and animals.
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Kotler, Moshe, Larry Hanson e Shane Burgess. Replication Defective Cyprinid Herpes Virus-3 (CyHV-3) as a Combined Prophylactic Vaccine in Carps. United States Department of Agriculture, dicembre 2010. http://dx.doi.org/10.32747/2010.7697104.bard.
Abstract (sommario):
Aquacultured koi and common carp fish (Cyprinus carpio) are intensively bred as ornamental and food fish in many countries worldwide. Hatcheries of carp and koi have recently suffered massive financial damages due to two viral diseases caused by the Cyprinid herpesvirus-3 (CyHV-3), previously designated as Carp Interstitial Nephritis and Gill Necrosis Virus (CNGV) and Koi herpesvirus (KHV), and by the Spring Viremia of Carp Virus (SVCV). CyHV-3 is a large dsDNA virus, which is infectious mostly to koi and common carp, while SVCV is a rhabdovirus with a relatively broad host range. Both viruses induce contagious disease with mortality rate up to 90%. Strategies for the control of viral infection in fish are of limited use. While efforts to prevent introduction of infectious agents into culture facilities are desirable, such exclusion strategies are far from fail-safe. Extensive vaccination methods that are useful for use in aquaculture facilities produce weak immunity, when used with proteins or inactivated viruses. Methods to overcome this obstacle are to vaccinate the fish with large amounts of antigen and/or use adjuvant and immune modulators over a long period. These techniques usually require individual handling of the fish. On the other hand, live attenuated virus is efficient and economical when used as an immersionvaccine. However, this technique poses certain environmental risks and thus may be difficult to license and scale up. Another option is a vaccine based on the replication defective virus (RDV) (pseudovirus), which can infect cells, but is unable to produce infectious particles. This vaccine may circumvent many of the problems related to attenuated-live vaccine (e.g., inadvertent infection and reversion to the virulent strain).
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Bacharach, Eran, W. Ian Lipkin e Avigdor Eldar. Identification of the etiological agent of tilapia disease in the Lake of Galillee. United States Department of Agriculture, gennaio 2013. http://dx.doi.org/10.32747/2013.7597932.bard.
Abstract (sommario):
Background to the topic. Tilapines serve as the second most important group of farmed fish worldwide. Massive mortality of wild and cultured tilapia has been observed recently in Israel but the pathogen of this disease has not been identified. We proposed to identify the agent responsible for disease. Major conclusions, solutions, achievements. We characterized the lesions in diseased fish and found that the brain was one of the affected organs. We found conditions to isolate from brains of diseased fish the etiological agent of the tilapia disease and to propagate it in cell culture. This led to the identification of the pathogen as a novel RNA virus, which we named Tilapia Lake Virus (TiLV). Electron microscopy of TiLV revealed virion-like particles and ether/chloroform-sensitivity assays demonstrated that TiLV is enveloped. Low passage TiLV, injected intra-peritoneally to tilapia, induced a disease with over 80% mortality. Cohabitation of healthy with diseased fish demonstrated that the disease is contagious, and that mortalities occur within few days. Fish surviving initial mortality were immune to further TiLV infections, suggesting the mounting of protective immune response. Screening cDNA libraries and high throughput sequencing determined the sequence of TiLV genome. This demonstrated that TiLV is indeed a novel virus and allowed the design of a PCRbased diagnostic test. Implications, both scientific and agricultural. The characterization of a novel, emerging RNA virus that imposes major threat to the tilapia industry, enables the specific identification of the virus in tilapines. This allows prompt screening and surveillance of TiLV, epidemiological studies, and disease containment. This also potentially opens the way for the development of vaccines against TiLV.
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Breewood, Helen, e Tara Garnett. Meat, metrics and mindsets: Exploring debates on the role of livestock and alternatives in diets and farming. TABLE, marzo 2023. http://dx.doi.org/10.56661/2caf9b92.
Abstract (sommario):
Should we eat meat, eggs, dairy and other animal-sourced foods? If so, how should we produce them and how much should we eat? If not, what should we eat instead? These are just some of the more contentious debates about the future of food systems. This short briefing paper summarises some of the key debates about livestock and its alternatives and describes both the arguments and the evidence underpinning different points of view. We look both at foodstuffs (meat, fish, plants and new foods based on cells grown in bioreactors) and farming methods (both intensive and extensive) with regards to discussions about their environmental, health and social impacts. In so doing, we explore the assumptions and values that often lead stakeholders to differing conclusions about what a sustainable food system looks like.
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Eldar, Avigdor, e Donald L. Evans. Streptococcus iniae Infections in Trout and Tilapia: Host-Pathogen Interactions, the Immune Response Toward the Pathogen and Vaccine Formulation. United States Department of Agriculture, dicembre 2000. http://dx.doi.org/10.32747/2000.7575286.bard.
Abstract (sommario):
In Israel and in the U.S., Streptococcus iniae is responsible for considerable losses in various fish species. Poor understanding of its virulence factors and limited know-how-to of vaccine formulation and administration are the main reasons for the limited efficacy of vaccines. Our strategy was that in order to Improve control measures, both aspects should be equally addressed. Our proposal included the following objectives: (i) construction of host-pathogen interaction models; (ii) characterization of virulence factors and immunodominant antigens, with assessment of their relative importance in terms of protection and (iii) genetic identification of virulence factors and genes, with evaluation of the protective effect of recombinant proteins. We have shown that two different serotypes are involved. Their capsular polysaccharides (CPS) were characterized, and proved to play an important role in immune evasion and in other consequences of the infection. This is an innovative finding in fish bacteriology and resembles what, in other fields, has become apparent in the recent years: S. iniae alters surface antigens. By so doing, the pathogen escapes immune destruction. Immunological assays (agar-gel immunodiffusion and antibody titers) confirmed that only limited cross recognition between the two types occurs and that capsular polysaccharides are immunodominant. Vaccination with purified CPS (as an acellular vaccine) results in protection. In vitro and ex-vivo models have allowed us to unravel additional insights of the host-pathogen interactions. S. iniae 173 (type II) produced DNA fragmentation of TMB-8 cells characteristic of cellular necrosis; the same isolate also prevented the development of apoptosis in NCC. This was determined by finding reduced expression of phosphotidylserine (PS) on the outer membrane leaflet of NCC. NCC treated with this isolate had very high levels of cellular necrosis compared to all other isolates. This cellular pathology was confirmed by observing reduced DNA laddering in these same treated cells. Transmission EM also showed characteristic necrotic cellular changes in treated cells. To determine if the (in vitro) PCD/apoptosis protective effects of #173 correlated with any in vivo activity, tilapia were injected IV with #173 and #164 (an Israeli type I strain). Following injection, purified NCC were tested (in vitro) for cytotoxicity against HL-60 target cells. Four significant observations were made : (i) fish injected with #173 had 100-400% increased cytotoxicity compared to #164 (ii) in vivo activation occurred within 5 minutes of injection; (iii) activation occurred only within the peripheral blood compartment; and (iv) the isolate that protected NCC from apoptosis in vitro caused in vivo activation of cytotoxicity. The levels of in vivo cytotoxicity responses are associated with certain pathogens (pathogen associated molecular patterns/PAMP) and with the tissue of origin of NCC. NCC from different tissue (i.e. PBL, anterior kidney, spleen) exist in different states of differentiation. Random amplified polymorphic DNA (RAPD) analysis revealed the "adaptation" of the bacterium to the vaccinated environment, suggesting a "Darwinian-like" evolution of any bacterium. Due to the selective pressure which has occurred in the vaccinated environment, type II strains, able to evade the protective response elicited by the vaccine, have evolved from type I strains. The increased virulence through the appropriation of a novel antigenic composition conforms with pathogenic mechanisms described for other streptococci. Vaccine efficacy was improved: water-in-oil formulations were found effective in inducing protection that lasted for a period of (at least) 6 months. Protection was evaluated by functional tests - the protective effect, and immunological parameters - elicitation of T- and B-cells proliferation. Vaccinated fish were found to be resistant to the disease for (at least) six months; protection was accompanied by activation of the cellular and the humoral branches.
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Liu, Zhanjiang John, Rex Dunham e Boaz Moav. Developmental and Evaluation of Advanced Expression Vectors with Both Enhanced Integration and Stable Expression for Transgenic Farmed Fish. United States Department of Agriculture, dicembre 2001. http://dx.doi.org/10.32747/2001.7585196.bard.
Abstract (sommario):
The objectives of the project were to develop expression vectors using the Sleeping Beauty transposon technology and the genetic border elements to provide both enhanced integration rate and stable transgene expression, and to evaluate the application of such vectors in farmed fish such as catfish and carp. The panel recommended adding the objective of evaluating the endogenous transposable elements, particularly in catfish, in order to evaluate the applicability of the expression vectors while reduc1ng efforts in real production of transgenic fish considering the focus of the project was to develop the vector and evaluation of its applicability, not producing transgenic fish. Efficient production of transgenic farmed fish is hindered by two major problems: mosaicism due to delayed integration after single-cell stage, and silencing of transgene expression. In this project, we proposed to combat these problems by coupling the Sleeping Beauty transposon technology that can enhance integration rate and the border elements that can insulate transgene from position effect. Our major objective was to develop a new generation of expression vector that contains both of these elements. We have developed expression vectors containing both the Sleeping Beauty transposon signals, inverted repeats and direct repeats (IR and DR, respectively), and the border elements, scs and scs'. Growth hormone minigene has been cloned into this vector for applications of such vectors in growth enhancement. Luc reporter gene has been also cloned into this vector cascades for relative easy evaluation of transgene expression. Transgenic fish have been produced using these expression vectors in both catfish (US) and carp (Israel). Much effort was also devoted to evaluation of the endogenous transposable elements in catfish as recommended by the BARD grant panel. Multiple families of Tcl-like transposons were identified from catfish. Surprisingly, many Tc I-related transcripts were identified. Among these transcripts, both the sense and antisense transcripts were present. Some of the transcripts may be useful for development of novel transposase-based technology for aquaculture applications in the future. This project has both scientific and aquaculture implications. First, to develop expression vectors containing both IR/DR and scs/scs' repeated elements have been reported being extremely technically difficult due to excision of the repeated sequences by the E. coli host during cloning processes. We have successfully constructed this advanced vector that contained very complex cascades for both gene integration and gene regulation. We have produced transgenic fish using such vectors. This advanced expression vector should be useful for production of transgenic fish. By simply replacing the growth hormone gene, any gene of interest can be readily inserted in this vector. Thus this vector should provide technological possibility for early integration and stable expression of any economically important genes in aquaculture. We have also evaluated the applications of the Sleeping Beauty-based vectors in terms of the impact of gene size and found that the size of trans gene drastically affects transposition. The system will be only useful for transferring genes smaller than 5.6 kb. We have also identified novel transposase-related transcripts that may be useful for the development of novel transposase-based technologies for general scientific research and for aquaculture applications.
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Gothilf, Yoav, Roger Cone, Berta Levavi-Sivan e Sheenan Harpaz. Genetic manipulations of MC4R for increased growth and feed efficiency in fish. United States Department of Agriculture, gennaio 2016. http://dx.doi.org/10.32747/2016.7600043.bard.
Abstract (sommario):
The hypothalamic melanocortin system plays a central role in the regulation of food consumption and energy homeostasis in mammals. Accordingly, our working hypothesis in this project was that genetic editing of the mc4r gene, encoding Melanocortin Receptor 4 (MC4R), will enhance food consumption, feed efficiency and growth in fish. To test this hypothesis and to assess the utility of mc4r editing for the enhancement of feed efficiency and growth in fish, the following objectives were set: Test the effect of the mc4r-null allele on feeding behavior, growth, metabolism and survival in zebrafish. Generate mc4r-null alleles in tilapia and examine the consequences for growth and survival, feed efficiency and body composition. Generate and examine the effect of naturally-occurring mc4r alleles found in swordfish on feeding behavior, growth and survival in zebrafish. Define the MC4R-mediated and MC4R-independent effects of AgRP by crossing mc4r- null strains with fish lacking AgRP neurons or the agrpgene. Our results in zebrafish did not support our hypothesis. While knockout of the agrpgene or genetic ablation of hypothalamic AgRP neurons led to reduced food intake in zebrafish larvae, knockout (KO) of the mc4r gene not only did not increase the rate of food intake but even reduced it. Since Melanocortin Receptor 3 (MC3R) has also been proposed to be involved in hypothalamic control of food intake, we also tested the effectofmc3r gene KO. Again, contrary to our hypothesis, the rate of food intake decreased. The next step was to generate a double mutant lucking both functional MC3R and MC4R. Again, the double KO exhibited reduced food intake. Thus, the only manipulation within the melanocortin system that affected food intake in consistent with the expected role of the system was seen in zebrafish larvae upon agrpKO. Interestingly, despite the apparent reduced food intake in the larval stage, these fish grow to be of the same size as wildtype fish at the adult stage. Altogether, it seems that there is a compensatory mechanism that overrides the effect of genetic manipulations of the melanocortin system in zebrafish. Under Aim 3, we introduced the Xna1, XnB1l, and XnB2A mutations from the Xiphophorus MC4R alleles into the zebrafish MC4R gene. We hypothesized that these MC4R mutations would act as dominant negative alleles to increase growth by suppressing endogenous MC4R activity. When we examined the activity of the three mutant alleles, we were unable to document any inhibition of a co-transfected wild type MC4R allele, hence we did not introduce these alleles into zebrafish. Since teleost fish possess two agrpgenes we also tested the effect of KO of the agrp2 gene and ablation of the AgRP2 cells. We found that the AgRP2 system does not affect food consumption but may rather be involved in modulating the stress response. To try to apply genetic editing in farmed fish species we turned to tilapia. Injection of exogenous AgRP in adult tilapia induced significant changes in the expression of pituitary hormones. Genetic editing in tilapia is far more complicated than in zebrafish. Nevertheless, we managed to generate one mutant fish carrying a mutation in mc4r. That individual died before reaching sexual maturity. Thus, our attempt to generate an mc4r-mutant tilapia line was almost successful and indicate out non-obvious capability to generate mutant tilapia.
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Ohad, Nir, e Robert Fischer. Regulation of Fertilization-Independent Endosperm Development by Polycomb Proteins. United States Department of Agriculture, gennaio 2004. http://dx.doi.org/10.32747/2004.7695869.bard.
Abstract (sommario):
Arabidopsis mutants that we have isolated, encode for fertilization-independent endosperm (fie), fertilization-independent seed2 (fis2) and medea (mea) genes, act in the female gametophyte and allow endosperm to develop without fertilization when mutated. We cloned the FIE and MEA genes and showed that they encode WD and SET domain polycomb (Pc G) proteins, respectively. Homologous proteins of FIE and MEA in other organisms are known to regulate gene transcription by modulating chromatin structure. Based on our results, we proposed a model whereby both FIE and MEA interact to suppress transcription of regulatory genes. These genes are transcribed only at proper developmental stages, as in the central cell of the female gametophyte after fertilization, thus activating endosperm development. To test our model, the following questions were addressed: What is the Composition and Function of the Polycomb Complex? Molecular, biochemical, genetic and genomic approaches were offered to identify members of the complex, analyze their interactions, and understand their function. What is the Temporal and Spatial Pattern of Polycomb Proteins Accumulation? The use of transgenic plants expressing tagged FIE and MEA polypeptides as well as specific antibodies were proposed to localize the endogenous polycomb complex. How is Polycomb Protein Activity Controlled? To understand the molecular mechanism controlling the accumulation of FIE protein, transgenic plants as well as molecular approaches were proposed to determine whether FIE is regulated at the translational or posttranslational levels. The objectives of our research program have been accomplished and the results obtained exceeded our expectation. Our results reveal that fie and mea mutations cause parent-of-origin effects on seed development by distinct mechanisms (Publication 1). Moreover our data show that FIE has additional functions besides controlling the development of the female gametophyte. Using transgenic lines in which FIE was not expressed or the protein level was reduced during different developmental stages enabled us for the first time to explore FIE function during sporophyte development (Publication 2 and 3). Our results are consistent with the hypothesis that FIE, a single copy gene in the Arabidopsis genome, represses multiple developmental pathways (i.e., endosperm, embryogenesis, shot formation and flowering). Furthermore, we identified FIE target genes, including key transcription factors known to promote flowering (AG and LFY) as well as shoot and leaf formation (KNAT1) (Publication 2 and 3), thus demonstrating that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Using the Yeast two hybrid system and pull-down assays we demonstrated that FIE protein interact with MEA via the N-terminal region (Publication 1). Moreover, CURLY LEAF protein, an additional member of the SET domain family interacts with FIE as well. The overlapping expression patterns of FIE, with ether MEA or CLF and their common mutant phenotypes, demonstrate the versatility of FIE function. FIE association with different SET domain polycomb proteins, results in differential regulation of gene expression throughout the plant life cycle (Publication 3). In vitro interaction assays we have recently performed demonstrated that FIE interacts with the cell cycle regulatory component Retinobalsoma protein (pRb) (Publication 4). These results illuminate the potential mechanism by which FIE may restrain embryo sac central cell division, at least partly, through interaction with, and suppression of pRb-regulated genes. The results of this program generated new information about the initiation of reproductive development and expanded our understanding of how PcG proteins regulate developmental programs along the plant life cycle. The tools and information obtained in this program will lead to novel strategies which will allow to mange crop plants and to increase crop production.
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Funkenstein, Bruria, e Cunming Duan. GH-IGF Axis in Sparus aurata: Possible Applications to Genetic Selection. United States Department of Agriculture, novembre 2000. http://dx.doi.org/10.32747/2000.7580665.bard.
Abstract (sommario):
Many factors affect growth rate in fish: environmental, nutritional, genetics and endogenous (physiological) factors. Endogenous control of growth is very complex and many hormone systems are involved. Nevertheless, it is well accepted that growth hormone (GH) plays a major role in stimulating somatic growth. Although it is now clear that most, if not all, components of the GH-IGF axis exist in fish, we are still far from understanding how fish grow. In our project we used as the experimental system a marine fish, the gilthead sea bream (Sparus aurata), which inhabits lagoons along the Mediterranean and Atlantic coasts of Europe, and represents one of the most important fish species used in the mariculture industry in the Mediterranean region, including Israel. Production of Sparus is rapidly growing, however, in order for this production to stay competitive, the farming of this fish species has to intensify and become more efficient. One drawback, still, in Sparus extensive culture is that it grows relatively slow. In addition, it is now clear that growth and reproduction are physiological interrelated processes that affect each other. In particular sexual maturation (puberty) is known to be closely related to growth rate in fish as it is in mammals, indicating interactions between the somatotropic and gonadotropic axes. The goal of our project was to try to identify the rate-limiting components(s) in Sparus aurata GH-IGF system which might explain its slow growth by studying the ontogeny of growth-related genes: GH, GH receptor, IGF-I, IGF-II, IGF receptor, IGF-binding proteins (IGFBPs) and Pit-1 during early stages of development of Sparus aurata larvae from slow and fast growing lines. Our project was a continuation of a previous BARD project and could be divided into five major parts: i) obtaining additional tools to those obtained in the previous project that are necessary to carry out the developmental study; ii) the developmental expression of growth-related genes and their cellular localization; iii) tissue-specific expression and effect of GH on expression of growth-related genes; iv) possible relationship between GH gene structure, growth rate and genetic selection; v) the possible role of the IGF system in gonadal development. The major findings of our research can be summarized as follows: 1) The cDNAs (complete or partial) coding for Sparus IGFBP-2, GH receptor and Pit-1 were cloned. Sequence comparison reveals that the primary structure of IGFBP-2 protein is 43-49% identical to that of zebrafish and other vertebrates. Intensive efforts resulted in cloning a fragment of 138 nucleotides, coding for 46 amino acids in the proximal end of the intracellular domain of GH receptor. This is the first fish GH receptor cDNA that had been cloned to date. The cloned fragment will enable us to complete the GH - receptor cloning. 2) IGF-I, IGF-II, IGFBP-2, and IGF receptor transcripts were detected by RT-PCR method throughout development in unfertilized eggs, embryos, and larvae suggesting that these mRNAs are products of both the maternal and the embryonic genomes. Preliminary RT-PCR analysis suggest that GH receptor transcript is present in post-hatching larvae already on day 1. 3) IGF-1R transcripts were detected in all tissues tested by RT-PCR with highest levels in gill cartilage, skin, kidney, heart, pyloric caeca, and brain. Northern blot analysis detected IGF receptor only in gonads, brain and gill cartilage but not in muscle; GH increased slightly brain and gill cartilage IGF-1R mRNA levels. 4) IGFBP-2 transcript were detected only in liver and gonads, when analyzed by Northern blots; RT-PCR analysis revealed expression in all tissues studied, with the highest levels found in liver, skin, gonad and pyloric caeca. 5) Expression of IGF-I, IGF-II, IGF-1R and IGFBP-2 was analyzed during gonadal development. High levels of IGF-I and IGFBP-2 expression were found in bisexual young gonads, which decreased during gonadal development. Regardless of maturational stage, IGF-II levels were higher than those of IGF-L 6) The GH gene was cloned and its structure was characterized. It contains minisatellites of tandem repeats in the first and third introns that result in high level of genetic polymorphism. 7) Analysis of the presence of IGF-I and two types of IGF receptor by immunohistochemistry revealed tissue- and stage-specific expression during larval development. Immunohistochemistry also showed that IGF-I and its receptors are present in both testicular and ovarian cells. Although at this stage we are not able to pinpoint which is the rate-limiting step causing the slow growth of Sparus aurata, our project (together with the previous BARD) yielded a great number of experimental tools both DNA probes and antibodies that will enable further studies on the factors regulating growth in Sparus aurata. Our expression studies and cellular localization shed new light on the tissue and developmental expression of growth-related genes in fish.