Molecular cloning, characterization, genomic organization and promoter analysis of the α1,6-fucosyltransferase gene (fut8) expressed in the rat hybridoma cell line YB2/0

<p>Abstract</p> <p>Background</p> <p>The rat hybridoma cell line YB2/0 appears a good candidate for the large-scale production of low fucose recombinant mAbs due to its lower expression of <it>fut8 </it>gene than other commonly used rodent cell lines. However, important variations of the fucose content of recombinant mAbs are observed in production culture conditions. To improve our knowledge on the YB2/0 fucosylation capacity, we have cloned and characterized the rat <it>fut8 </it>gene.</p> <p>Results</p> <p>The cDNAs encoding the rat α1,6-fucosyltransferase (FucT VIII) were cloned from YB2/0 cells by polymerase chain reaction-based and 5' RNA-Ligase-Mediated RACE methods. The cDNAs contain an open reading frame of 1728 bp encoding a 575 amino acid sequence showing 94% and 88% identity to human and pig orthologs, respectively. The recombinant protein expressed in COS-7 cells exhibits a α1,6-fucosyltransferase activity toward human asialo-agalacto-apotransferrin. The rat <it>fut8 </it>gene is located on chromosome 6 q and spans over 140 kbp. It contains 9 coding exons and four 5'-untranslated exons. FISH analysis shows a heterogeneous copy number of <it>fut8 </it>in YB2/0 nuclei with 2.8 ± 1.4 mean copy number. The YB2/0 <it>fut8 </it>gene is expressed as two main transcripts that differ in the first untranslated exon by the usage of distinct promoters and alternative splicing. Luciferase assays allow defining the minimal promoting regions governing the initiation of the two transcripts, which are differentially expressed in YB2/0 as shown by duplex Taqman QPCR analysis. Bioinformatics analysis of the minimal promoter regions upstream exons E-2 and E-3, governing the transcription of T1 and T2 transcripts, respectively, evidenced several consensus sequences for potential transcriptional repressors. Transient transfections of Rat2 cells with transcription factor expression vectors allowed identifying KLF15 as a putative repressor of T1 transcript in Rat2 cells.</p> <p>Conclusion</p> <p>Altogether, these data contribute to a better knowledge of <it>fut8 </it>expression in YB2/0 that will be useful to better control the fucosylation of recombinant mAbs produced in these cells.</p

Management for sustainable cephalopod fisheries in Europe: review and recommendations

Although cephalopod fisheries are of world-wide importance, in Europe catching cephalopods is managed only in small-scale fisheries, at national level, and few stocks are formally assessed. Because cephalopods are not quota species under the EU’s Common Fisheries Policy, there is currently no requirement for assessment or management at European level. Given increasing interest in targeting cephalopods in Europe, there is a risk that they will be fished unsustainably. Although there have been recent review papers on progress in stock assessment and fishery forecasting for commercially fished cephalopods there has been no recent review of cephalopod fishery management. We aim to fill this gap, with a particular focus on European cephalopod fisheries.We review potential barriers to sustainable fishing and reasons why management of cephalopod fisheries differs from that for finfish fisheries, e.g. due to the high inherent volatility and the possibly cyclic nature of year-to-year variation in cephalopod abundance, reflecting their short lifespan, rapid growth and high sensitivity to environmental conditions. We review fishery management approaches in important cephalopod fisheries worldwide (e.g. in the USA, Japan, Falklands, South Africa, Australia and Russia) and current management of small-scale cephalopod fisheries in Europe. We identify knowledge gaps and limitations to current monitoring programmes and stock assessments and discuss the options available for cephalopod fishery management in Europe, considering the suitability or otherwise of catch and effort limits, use of closed areas and seasons, restrictions on sizes caught and types of fishing gear, and the ole of market-based sustainability pathways.info:eu-repo/semantics/publishedVersio

Working group on cephalopod fisheries and life history (Wgceph; outputs from 2022 meeting)

Rapports Scientifiques du CIEM. Volume 5, nº 1WGCEPH worked on six Terms of Reference. These involved reporting on the status of stocks; reviewing advances in stock identification, assessment for fisheries management and for the Ma- rine Strategy Framework Directive (MSFD), including some exploratory stock assessments; re- viewing impacts of human activities on cephalopods; developing identification guides and rec- ommendations for fishery data collection; describing the value chain and evaluating market driv- ers; and reviewing advances in research on environmental tolerance of cephalopods. ToR A is supported by an annual data call for fishery and survey data. During 2019–2021, com- pared to 1990–2020, cuttlefish remained the most important cephalopod group in terms of weight landed along the European North Atlantic coast, while loliginid squid overtook octopus as the second most important group. Short-finned squid remained the least important group in land- ings although their relative importance was almost double in 2019–2022 compared to 1992–2020. Total cephalopod landings have been fairly stable since 1992. Cuttlefish landings are towards the low end of the recent range, part of a general downward trend since 2004. Loliginid squid landings in 2019 were close to the maximum seen during the last 20 years but totals for 2020 and 2021 were lower. Annual ommastrephid squid landings are more variable than those of the other two groups and close to the maximum seen during 1992– 2021. Octopod landings have generally declined since 2002 but the amount landed in 2021 was higher than in the previous four years. Under ToR B we illustrate that the combination of genetic analysis and statolith shape analysis is a promising method to provide some stock structure information for L. forbsii. With the sum- mary of cephalopod assessments, we could illustrate that many cephalopod species could al- ready be included into the MSFD. We further provide material from two reviews in preparation, covering stock assessment methods and challenges faced for cephalopod fisheries management. Finally, we summarise trends in abundance indices, noting evidence of recent declines in cuttle- fish and some octopuses of the genus Eledone. Under ToR C, we describe progress on the reviews of (i) anthropogenic impacts on cephalopods and (ii) life history and ecology. In relation to life history, new information on Eledone cirrhosa from Portugal is included. Under ToR D we provide an update on identification guides, discuss best practice in fishery data collection in relation to maturity determination and sampling intensity for fishery monitoring. Among others, we recommend i) to include the sampling of cephalopods in any fishery that (a) targets cephalopods, (b) targets both cephalopods and demersal fishes or (c) takes cephalopods as an important bycatch, ii) Size-distribution sampling, iii) the use of standardized sampling pro- tocols, iv) an increased sampling effort in cephalopod. Work under ToR E on value chains and market drivers, in conjunction with the Cephs & Chefs INTERREG project, has resulted in two papers being submitted. Abstracts of these are in the report. Finally, progress under ToR F on environmental tolerance limits of cephalopods and climate en- velope models is discussed, noting the need to continue this work during the next cycle.info:eu-repo/semantics/publishedVersio

Management for sustainable cephalopod fisheries in Europe: review and recommendations

Cephalopod International Advisory Council Conference, Cephalopods in the Anthropocene: Multiple Challenges in a Changing Ocean, April 2-8, 2022, Sesimbra, PortugalAlthough cephalopod fisheries are of world-wide importance, in Europe catching cephalopods is managed only in small-scale fisheries, at national level, and few stocks are formally assessed. Because cephalopod are not quota species under the EU’s Common Fisheries Policy, there is currently no requirement for assessment or management at European level. Given increasing interest in targeting cephalopods in Europe, there is a risk that they will be fished unsustainably. Although there have been recent review papers on progress in stock assessment and fishery forecasting for commercially fished cephalopods there has been no recent review of cephalopod fishery management. We aim to fill this gap, with a particular focus on European cephalopod fisheries.We review potential barriers to sustainable fishing and reasons why management of cephalopod fisheries differs from that for finfish fisheries, e.g. due to the high inherent volatility and the possibly cyclic nature of year-to-year variation in cephalopod abundance, reflecting their short lifespan, rapid growth and high sensitivity to environmental conditions. We review fishery management approaches in important cephalopod fisheries worldwide (e.g. in the USA, Japan, Falklands, South Africa, Australia and Russia) and current management of small-scale cephalopod fisheries in Europe. We identify knowledge gaps and limitations to current monitoring programmes and stock assessments and discuss the options available for cephalopod fishery management in Europe, considering the suitability or otherwise of catch and effort limits, use of closed areas and seasons, restrictions on sizes caught and types of fishing gear, and the ole of market-based sustainability pathwaysN

Tumour-associated carbohydrate antigens in breast cancer

Glycosylation changes that occur in cancer often lead to the expression of tumour-associated carbohydrate antigens. In breast cancer, these antigens are usually associated with a poor prognosis and a reduced overall survival. Cellular models have shown the implication of these antigens in cell adhesion, migration, proliferation and tumour growth. The present review summarizes our current knowledge of glycosylation changes (structures, biosynthesis and occurrence) in breast cancer cell lines and primary tumours, and the consequences on disease progression and aggressiveness. The therapeutic strategies attempted to target tumour-associated carbohydrate antigens in breast cancer are also discussed

Transcriptionnal regulation and role of GD3 synthase, a key enzyme for the biosynthesis of gangliosides, in breast cancer

La GD3 synthétase (GD3S) est une enzyme capable de synthétiser le ganglioside GD3, contrôlant ainsi la biosynthèse des gangliosides des séries b et c. Ces gangliosides sont principalement exprimés au cours de l’embryogenèse, dans le cerveau adulte, ainsi que dans certaines tumeurs. En particulier, le GD3 et le GD2 sont des marqueurs du mélanome et du neuroblastome, où ils jouent un rôle clef dans la progression tumorale. Une surexpression du gène codant la GD3S a également été observée dans les tumeurs de sein négatives pour le récepteur aux œstrogènes (ER-). Afin de déterminer le rôle de la GD3S dans la progression du cancer du sein, un modèle de cellules de cancer du sein MDA-MB-231 surexprimant la GD3S a été créé au laboratoire. Ces cellules se caractérisent par une prolifération et une migration accrues, dues à l’activation du récepteur c-Met. Dans le cadre de ma thèse, nous avons d’abord montré que le ganglioside GD2 induisait spécifiquement l’activation de c-Met et la prolifération des cellules GD3S+. Ensuite, nous avons étudié les mécanismes moléculaires responsables de la surexpression de ST8SIA1 dans les tumeurs ER-. Nous avons caractérisé le promoteur core essentiel à la transcription de ST8SIA1 dans les cellules de cancer du sein et nous avons montré que l’œstradiol réprime à la fois l’expression des ARNm de la GD3S et le promoteur core uniquement dans les cellules de cancer du sein exprimant le récepteur ERα. Ces résultats suggèrent que la forte expression de ST8SIA1 dans les tumeurs de sein ER-, suite à la perte de signalisation par ERα, pourrait augmenter l’expression des gangliosides tels que le GD2, et ainsi contribuer à l’agressivité de ce sous-type de tumeur.The GD3 synthase (GD3S) is an enzyme that synthesizes the ganglioside GD3 from GM3. It is therefore the key enzyme for the biosynthesis of b- and c-series gangliosides. Essentially expressed during embryogenesis and in the adult brain, the expression of these gangliosides increases in several diseases including neuro-ectoderm derived cancer. In particular, GD3 and GD2 are oncofetal markers in melanoma and neuroblastoma, where they play a key role in tumor progression. Clinical studies have also showed a high expression of GD3S in Estrogen Receptor negative (ER-) breast tumors. In order to determine the role of GD3S in breast cancer progression, we have previously induced GD3S over-expression in MDA-MB-231 breast cancer cell line. The resulting GD3S+ cells displayed an increased migration and proliferative phenotype that directly proceed from the constitutive activation of c-Met receptor. During my thesis, we first demonstrated that GD2 is directly involved in the constitutive activation of c-Met in absence of the ligand HGF/SF. Secondly, we have undertaken the study of the molecular mechanisms responsible for ST8SIA1 over-expression in ER- tumors. We characterized the core promoter, essential for ST8SIA1 transcription in breast cancer cells and we showed that estradiol represses both GD3S mRNA expression and the core promoter only in ERα expressing breast cancer cells. Altogether, these results suggest that high expression of GD3S in ER negative tumours, due to the loss of ERα signalling, could increase complex gangliosides such as GD2 at the cell surface, and possibly enhance the aggressiveness of this tumour subtype

Régulation transcriptionnelle et rôle de la GD3 synthétase, enzyme clef de la biosynthèse des gangliosides, dans le cancer du sein

La GD3 synthétase (GD3S) est une enzyme capable de synthétiser le ganglioside GD3, contrôlant ainsi la biosynthèse des gangliosides des séries b et c. Ces gangliosides sont principalement exprimés au cours de l embryogenèse, dans le cerveau adulte, ainsi que dans certaines tumeurs. En particulier, le GD3 et le GD2 sont des marqueurs du mélanome et du neuroblastome, où ils jouent un rôle clef dans la progression tumorale. Une surexpression du gène codant la GD3S a également été observée dans les tumeurs de sein négatives pour le récepteur aux œstrogènes (ER-). Afin de déterminer le rôle de la GD3S dans la progression du cancer du sein, un modèle de cellules de cancer du sein MDA-MB-231 surexprimant la GD3S a été créé au laboratoire. Ces cellules se caractérisent par une prolifération et une migration accrues, dues à l activation du récepteur c-Met. Dans le cadre de ma thèse, nous avons d abord montré que le ganglioside GD2 induisait spécifiquement l activation de c-Met et la prolifération des cellules GD3S+. Ensuite, nous avons étudié les mécanismes moléculaires responsables de la surexpression de ST8SIA1 dans les tumeurs ER-. Nous avons caractérisé le promoteur core essentiel à la transcription de ST8SIA1 dans les cellules de cancer du sein et nous avons montré que l œstradiol réprime à la fois l expression des ARNm de la GD3S et le promoteur core uniquement dans les cellules de cancer du sein exprimant le récepteur ERa. Ces résultats suggèrent que la forte expression de ST8SIA1 dans les tumeurs de sein ER-, suite à la perte de signalisation par ERa, pourrait augmenter l expression des gangliosides tels que le GD2, et ainsi contribuer à l agressivité de ce sous-type de tumeur.The GD3 synthase (GD3S) is an enzyme that synthesizes the ganglioside GD3 from GM3. It is therefore the key enzyme for the biosynthesis of b- and c-series gangliosides. Essentially expressed during embryogenesis and in the adult brain, the expression of these gangliosides increases in several diseases including neuro-ectoderm derived cancer. In particular, GD3 and GD2 are oncofetal markers in melanoma and neuroblastoma, where they play a key role in tumor progression. Clinical studies have also showed a high expression of GD3S in Estrogen Receptor negative (ER-) breast tumors. In order to determine the role of GD3S in breast cancer progression, we have previously induced GD3S over-expression in MDA-MB-231 breast cancer cell line. The resulting GD3S+ cells displayed an increased migration and proliferative phenotype that directly proceed from the constitutive activation of c-Met receptor. During my thesis, we first demonstrated that GD2 is directly involved in the constitutive activation of c-Met in absence of the ligand HGF/SF. Secondly, we have undertaken the study of the molecular mechanisms responsible for ST8SIA1 over-expression in ER- tumors. We characterized the core promoter, essential for ST8SIA1 transcription in breast cancer cells and we showed that estradiol represses both GD3S mRNA expression and the core promoter only in ERa expressing breast cancer cells. Altogether, these results suggest that high expression of GD3S in ER negative tumours, due to the loss of ERa signalling, could increase complex gangliosides such as GD2 at the cell surface, and possibly enhance the aggressiveness of this tumour subtype.LILLE1-Bib. Electronique (590099901) / SudocSudocFranceF

How Do Gangliosides Regulate RTKs Signaling?

Gangliosides, the glycosphingolipids carrying one or several sialic acid residues, are located on the outer leaflet of the plasma membrane in glycolipid-enriched microdomains, where they interact with molecules of signal transduction pathways including receptors tyrosine kinases (RTKs). The role of gangliosides in the regulation of signal transduction has been reported in many cases and in a large number of cell types. In this review, we summarize the current knowledge on the biosynthesis of gangliosides and the mechanism by which they regulate RTKs signaling

Retinoids Issued from Hepatic Stellate Cell Lipid Droplet Loss as Potential Signaling Molecules Orchestrating a Multicellular Liver Injury Response

Hepatic stellate cells (HSCs) serve as the main body storage compartment for vitamin A through retinyl ester (RE)-filled lipid droplets (LDs). Upon liver injury, HSCs adopt a myofibroblastic phenotype characterized by an elevated expression of extracellular matrix proteins and a concomitant loss of LDs. On the one hand, LD breakdown has been suggested to provide the energy required for HSC activation into myofibroblast-like cells. On the other hand, this process could mitigate HSC activation following the transformation of released REs into retinoic acids (RAs), ligands for nuclear receptors exerting antifibrotic transcriptional regulatory activities in HSCs. Importantly, RAs may also constitute a means for HSCs to orchestrate the liver response to injury by triggering transcriptional effects in multiple additional surrounding liver cell populations. We envision that new approaches, such as single-cell technologies, will allow to better define how RAs are issued from LD loss in HSCs exert a multicellular control of the liver (patho)physiology