Biology subjects

Silva, N.

Publications and source records attributed to Silva, N..

3 recordsLinked to original sources

bioRxiv2018

Computational fluid dynamic analysis reveals the underlying physical forces playing a role in 3D multiplex brain organoid cultures

Organoid cultivation in suspension culture requires agitation at low shear stress to allow for nutrient diffusion, which preserves tissue structure. Multiplex systems for organoid cultivation have been proposed, but whether they meet similar shear stress parameters as the regularly used spinner flask and its correlation with the successful generation of brain organoids, has not been determined. Herein, we used computational fluid dynamics (CFD) analysis to compare two multiplex culture conditions: steering plates on an orbital shaker and the use of a previously described bioreactor. The bioreactor had low speed and high shear stress regions that may affect cell aggregate growth, depending on volume, whereas the CFD parameters of the steering plates were closest to the parameters of the spinning flask. Our protocol improves the initial steps of the standard brain organoid formation, and organoids produced therefrom displayed regionalized brain structures, including retinal pigmented cells. Overall, we conclude that suspension culture on orbital steering plates is a cost-effective practical alternative to previously described platforms for the cultivation of brain organoids for research and multiplex testing.\n\nHighlightsO_LIImprovements to organoid preparation protocol\nC_LIO_LIMultiplex suspension culture protocol successfully generate brain organoids\nC_LIO_LIComputational fluid dynamics (CFD) reveals emerging properties of suspension cultures\nC_LIO_LICFD of steering plates is equivalent to that of spinner flask cultures\nC_LI

bioengineering↗

bioRxiv2018

The BRCA1-BARD1 complex associates with the synaptonemal complex and pro-crossover factors and influences RAD-51 dynamics during Caenorhabditis elegans meiosis

During meiosis, the maternal and paternal homologous chromosomes must align along their entire length and recombine to achieve faithful segregation in the gametes. Meiotic recombination is accomplished through the formation of DNA double-strand breaks, a subset of which can mature into crossovers to link the parental homologous chromosomes and promote their segregation. Breast and ovarian cancer susceptibility protein BRCA1 and its heterodimeric partner BARD1 play a pivotal role in DNA repair in mitotic cells; however, their functions in gametogenesis are less well understood. Here we show that localization of BRC-1 and BRD-1 (Caenorhabditis elegans orthologues of BRCA1 and BARD1) is dynamic during meiotic prophase I; they ultimately becoming concentrated at regions surrounding the presumptive crossover sites, co-localizing with the pro-crossover factors COSA-1, MSH-5 and ZHP-3. The synaptonemal complex is essential for BRC-1 loading onto chromosomes but recombination is not. BRC-1 forms an in vivo complex with the synaptonemal complex component SYP-3 and the crossover-promoting factor MSH-5. Furthermore, BRC-1 is essential for efficient stage-specific recruitment of the RAD-51 recombinase to DNA damage sites when synapsis is impaired and upon induction of exogenous DNA double-strand breaks. Taken together, our data provide new insights into the localization and meiotic function of the BRC-1-BRD-1 complex and highlight their essential role in DNA double-strand break repair during gametogenesis.\n\nAuthor summarySexually reproducing species rely on meiosis to transmit their genetic information across generations. Parental chromosomes (homologues) undergo many distinctive processes in their complex journey from attachment to segregation. The physiological induction of DNA double strand breaks is crucial for promoting correct chromosome segregation: they are needed to activate the DNA repair machinery responsible for creating physical connections, or crossovers (COs), between the homologues. In turn, crossovers promote the accurate segregation of the chromosomes in daughter cells. The BRCA1-BARD1 complex has a pivotal role during DNA repair in somatic cells and is exclusively located on unaligned chromosomal regions during mammalian meiosis. We show that in Caenorhabditis elegans, BRCA1 and BARD1 localize to chromosomes at all stages of meiotic prophase I and are enriched at presumptive crossover sites. We found that BRCA1 promotes DNA loading of the repair factor RAD-51 in specific mutant backgrounds and upon exogenous damage induction. Our data provide evidence for a direct physical association between BRCA1 and pro-crossover factors (including the synaptonemal complex) and identify an important role for BRCA1 in stimulating meiotic DNA repair. Further studies are necessary to identify the substrates acted upon by BRCA1-BARD1 complex to maintain genome stability in the gametes.

genetics↗

bioRxiv2017

The conserved LEM-3/Ankle1 nuclease is involved in the combinatorial regulation of meiotic recombination repair and chromosome segregation in Caenorhabditis elegans

Homologous recombination is essential for crossover (CO) formation and accurate chromosome segregation during meiosis. It is of considerable importance to work out how recombination intermediates are processed leading to CO and non-crossover (NCO) outcome. Genetic analysis in budding yeast and Caenorhabditis elegans indicates that the processing of meiotic recombination intermediates involves a combination of nucleases and DNA repair enzymes. We previously reported that in C. elegans meiotic Holiday junction resolution is mediated by two redundant pathways, conferred by the SLX-1 and MUS-81 nucleases, and by the HIM-6 Blooms helicase in conjunction with the XPF-1 endonucleases, respectively. Both pathways require the scaffold protein SLX-4. However, in the absence of all these enzymes residual processing of meiotic recombination intermediates still occurs and CO formation is reduced but not abolished. Here we show that the LEM-3 nuclease, mutation of which by itself does not have an overt meiotic phenotype, genetically interacts with slx-1 and mus-81 mutants, the respective double mutants leading to 100% embryonic lethality. LEM-3 and MUS-81 act redundantly, their combined loss leading to a reduced number of early meiotic recombination intermediates, to a delayed disassembly of foci associated with CO designated sites, and to the formation of univalents linked by SPO-11 dependent chromatin bridges (dissociated bivalents). However, LEM-3 foci do not co-localize with ZHP-3 a marker that congresses into CO designated sites. In addition, neither CO frequency nor distribution is altered in lem-3 single mutants or in combination with mus-81 or slx-4 mutations, indicating that LEM-3 drives NCO outcome. Finally, we found persistent chromatin bridges during meiotic divisions in lem-3; slx-4 double mutants. Supported by the localization of LEM-3 between dividing meiotic nuclei, this data suggests that LEM-3 is able to process erroneous recombination intermediates that persist into the second meiotic divisions.\n\nAuthor SummaryMeiotic recombination is required for genetic diversity and for the proper chromosome segregation. Recombination intermediates, such as Holliday junctions (HJs), are generated and eventually resolved to produce crossover (CO) and non-crossover (NCO). While an excess of meiotic double-strand breaks is generated, most breaks are repaired without leading to a CO outcome and usually only one break for each chromosome pair matures into a CO-designated site in Caenorhabditis elegans. Resolution of meiotic recombination intermediates and CO formation have been reported to be highly regulated by several structure-specific endonucleases and the Bloom helicase. However, little is known about enzymes involved in the NCO recombination intermediate resolution. In this study, we found that a conserved nuclease LEM-3/Ankle1 acts in parallel to the SLX-1/MUS-81 pathway to process meiotic recombination intermediates. Mutation of lem-3 has no effect on CO frequency and distribution, indicating LEM-3 functions as a nuclease promoting NCO outcome. Interestingly, a prominent localization of LEM-3 is found between dividing meiotic nuclei. We provide evidence that LEM-3 is also involved in processing remaining, erroneous recombination intermediates during meiotic divisions.

cell biology↗