Biology subjects

Carvalho, J. E.

Publications and source records attributed to Carvalho, J. E..

5 recordsLinked to original sources

bioRxiv2023

The Mediterranean mussel, Mytilus galloprovincialis, a novel model for developmental studies of mollusks

A model organism in developmental biology is defined by its experimental amenability as well as by resources created for the model system by the scientific community. For the most powerful models, the combination of both has already yielded a thorough understanding of development. However, the number of developmental model systems is still very limited, and their phylogenetic distribution is heavily biased. Members of one of the largest animal phyla, the mollusks, for example, have long been neglected as developmental model organisms. To remedy this shortcoming, we produced a detailed developmental transcriptome for the Mediterranean mussel Mytilus galloprovincialis, a bivalve mollusk, and expanded the list of experimental protocols available for this species. Our high-quality transcriptome allowed us to identify transcriptomic signatures of developmental transitions and to perform a first comparison with the Pacific oyster Crassostrea gigas that can be used in future multi-species analyses. To allow co-labelling studies, we optimized protocols for immunohistochemistry and hybridization chain reaction and combined both techniques to create high-resolution co-expression maps of developmental genes. The resources and protocols we describe here thus represent an enormous boost for the establishment of the Mediterranean mussel as a laboratory model in developmental biology. Summary statementResources and techniques are described for the Mediterranean mussel Mytilus galloprovincialis, which, together, establish a novel model system for studying mollusk development and animal evolution.

developmental biology↗

bioRxiv2023

Optimized husbandry and targeted gene-editing for the cnidarian Nematostella vectensis

Optimized laboratory conditions for research models are crucial for the success of scientific projects. This includes the control of the entire life cycle, access to all developmental stages and maintaining stable physiological conditions. Reducing the life cycle of a research model can also enhance the access to biological material and speed up genetic tool development. Thus, we optimized the rearing conditions for the sea anemone Nematostella vectensis, a cnidarian research model to study embryonic and post-metamorphic processes, such as regeneration. We adopted a semi-automated aquaculture system for N. vectensis and developed a dietary protocol optimized for the different life stages. Thereby, we increased spawning efficiencies and post-spawning survival rates, and considerably reduced the overall life cycle down to two months. To further improve the obtention of CRISPR-Cas9 mutants, we optimized the design of sgRNAs leading to full KO animals in F0 polyps using a single sgRNA. Finally, we show that NHEJ-mediated transgene insertion is possible in N. vectensis. In sum our study provides additional resources for the scientific community that uses or will use N. vectensis as a research model. Summary statementOptimized life cycle, in combination with efficient gene-editing approaches facilitates the establishment of genetic tools in N. vectensis, an emerging model for environmental stress response, regeneration, and longevity.

developmental biology↗

bioRxiv2020

Structural insights into the cooperative interaction of the intrinsically disordered co-activator TIF2 with retinoic acid receptor heterodimer (RXR/RAR)

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved -helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a cooperative binding of the three NR-boxes as well as an active role of their flanking regions in the interaction.

biophysics↗

bioRxiv2020

An updated staging system for cephalochordate development: one table suits them all

Chordates are divided into three subphyla: Vertebrata, Tunicata and Cephalochordata. Phylogenetically, the Cephalochordata, more commonly known as lancelets or amphioxus, constitute the sister group of Vertebrata and Tunicata. Lancelets are small, benthic, marine filter feeders, and their roughly three dozen described species are divided into three genera: Branchiostoma, Epigonichthys and Asymmetron. Due to their phylogenetic position and their stereotypical chordate morphology and genome architecture, lancelets are key models for understanding the evolutionary history of chordates. Lancelets have thus been studied by generations of scientists, with the first descriptions of adult anatomy and developmental morphology dating back to the 19th century. Today, several different lancelet species are used as laboratory models, predominantly for developmental, molecular and genomic studies. Surprisingly, however, a universal staging system and an unambiguous nomenclature for developing lancelets have not yet been adopted by the scientific community. In this work, we characterized the development of the European amphioxus (Branchiostoma lanceolatum) using confocal microscopy and compiled a streamlined developmental staging system, from fertilization through larval life, including an unambiguous stage nomenclature. By tracing growth curves of the European amphioxus reared at different temperatures, we were able to show that our staging system permitted an easy conversion of any developmental time into a specific stage name. Furthermore, comparisons of embryos and larvae from the European lancelet (B. lanceolatum), the Florida lancelet (B. floridae), the Chinese lancelet (B. belcheri), the Japanese lancelet (B. japonicum) and the Bahamas lancelet (Asymmetron lucayanum) demonstrated that our staging system could readily be applied to other lancelet species. Although the detailed staging description was carried out on developing B. lanceolatum, the comparisons with other lancelet species thus strongly suggested that both staging and nomenclature are applicable to all extant lancelets. We conclude that this description of embryonic and larval development will be of great use for the scientific community and that it should be adopted as the new standard for defining and naming developing lancelets. More generally, we anticipate that this work will facilitate future studies comparing representatives from different chordate lineages.

developmental biology↗

bioRxiv2020

Cytotoxic Activity Of A New Isoform L-Amino Acid Oxidase (Balt-LAAO-II) From Bothrops alternatus (Urutu) Snake Venom In Human Leukemic HL60 Cells

In this work we describe the isolation of a new isoform L-amino acid oxidase (LAAO) referred to as Balt-LAAO-II from Bothrops alternatus snake venom, which was highly purified using a combination of molecular exclusion (Sephadex G-75) and RP-HPLC chromatographics steps. When analyzed by SDS-PAGE, the purified Balt-LAAO-II presented a molecular weight of [~]66 kDa. The N-terminal amino acid sequence and internal peptide sequences showed close structural homology to other snake venom L-amino acid oxidases. This enzyme induces in vitro cytotoxicity on cultured human leukemic HL60 cells. Cells were grown in RPMI medium and were incubated with isoform Balt-LAAO-II (1, 10 and 100 g/mL) for up to 72 h. All three concentrations of venom markedly decreased the cell viability from 6 h onwards based on the staining with propidium iodide, the reduction of 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and the uptake of neutral red. Flow cytometry showed that all isoform Balt-LAAO-II and whole venom concentrations induced apoptosis after 2-6 h of incubation. Morphological analysis of cells incubated with isoform Balt-LAAO-II and whole venom showed cell rounding and lysis that increased with the venom concentration and duration of incubation. These results show that isoform Balt-LAAO-II from venom Bothrops alternatus is cytotoxic to cultured HL60 cells and suggest that this damage may involve apoptotic and oxidative stress pathways.

pharmacology and toxicology↗