Biology subjects

Bouchard, C.

Publications and source records attributed to Bouchard, C..

3 recordsLinked to original sources

bioRxiv2025

Split Cas12a protospacer engineering enables ultra-specific, PAM-free detection

ABSTRACTCRISPR-Cas12a is a programmable, RNA-guided endonuclease that has revolutionized biotechnology, with applications in genome engineering and diagnostics. To induce nuclease activity, Cas12a must first interact with the target dsDNA duplex by associating with a short protospacer adjacent motif (PAM) in the sequence. In this study we have split this target duplex to create PAM-proximal and PAM-distal duplex regions, which has allowed us to regulate trans-cleavage activity when these regions are included in combination or separately. These observations on Cas12a activity led to hypotheses into the related functional mechanisms, which we have tested and that have highlighted DNA/protein interactions during Cas12a complex assembly that were not otherwise apparent. Selective destabilization of the nucleic acid complexes appears to drive greater reliance on the Cas12a protein for complex stability. We have exploited this to provide significant improvements in both structural selectivity and nucleotide specificity in PAM-proximal and PAM-distal duplex regions, respectively. The result is an architecture that shows promise as a PAM-free ultra-specific platform to resolve single nucleotide polymorphisms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/667643v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b1771dorg.highwire.dtl.DTLVardef@103197aorg.highwire.dtl.DTLVardef@7dd393org.highwire.dtl.DTLVardef@cd812d_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Task-Assisted GAN for Resolution Enhancement and Modality Translation in Fluorescence Microscopy

AbstractWe introduce a deep learning model that predicts super-resolved versions of diffraction-limited microscopy images. Our model, named Task- Assisted Generative Adversarial Network (TA-GAN), incorporates an auxiliary task (e.g. segmentation, localization) closely related to the observed biological nanostructures characterization. We evaluate how TA-GAN improves generative accuracy over unassisted methods using images acquired with different modalities such as confocal, brightfield (diffraction-limited), super-resolved stimulated emission depletion, and structured illumination microscopy. The generated synthetic resolution enhanced images show an accurate distribution of the F-actin nanostructures, replicate the nanoscale synaptic cluster morphology, allow to identify dividing S. aureus bacterial cell boundaries, and localize nanodomains in simulated images of dendritic spines. We expand the applicability of the TA-GAN to different modalities, auxiliary tasks, and online imaging assistance. Incorporated directly into the acquisition pipeline of the microscope, the TA-GAN informs the user on the nanometric content of the field of view without requiring the acquisition of a super-resolved image. This information is used to optimize the acquisition sequence, and reduce light exposure. The TA-GAN also enables the creation of domain-adapted labeled datasets requiring minimal manual annotation, and assists microscopy users by taking online decisions regarding the choice of imaging modality and regions of interest.

bioinformatics↗

bioRxiv2018

The dynamics of spawning acts by a semelparous fish and its associated energetic expenses

AO_SCPLOWBSTRACTC_SCPLOWO_LIDuring the reproductive season, animals have to manage both their energetic budget and gamete stock. In particular, for semelparous capital breeders with determinate fecundity and no parental care other than gametic investment, the depletion of energetic stock must match the depletion of gametic stock, so that individuals get exhausted just after their last egg is laid and fertilized. Although these budgets are managed continuously, monitoring the dynamics of mating acts and energy expenditure at a fine temporal scale in the wild is challenging. C_LIO_LIThis study aimed to quantify the individual dynamics of spawning acts and the concomitant energy expenditure of female Allis shad (Alosa alosa) throughout their mating season. C_LIO_LIUsing eight individual-borne accelerometers for one month, we collected tri-axial acceleration, temperature, and pressure data that we analysed to i) detect the timing of spawning acts, ii) estimate energy expenditure from tail beat frequency and water temperature, and iii) monitor changes in body roundness from the position of the dorsally-mounted tag relative to the vertical plane. C_LIO_LIFemale shad had a higher probability to spawn during warmer nights, and their spawning acts were synchronized (both individually and inter-individually) within each active night. They experienced warmer temperature, remained deeper, swan more slowly and spent less energy during daytime than night time. Over one month of spawning, they performed on average 15.75 spawning acts, spent on average 6 277 kJ and died with a significant portion of residual oocytes. The acceleration-based indicator of body roundness was correlated to condition coefficient measured at capture, and globally decreased through the spawning season, although the indicator was noisy and was not correlated to changes in estimated energy expenditure. C_LIO_LIDespite significant individual variability, our results indicate that female shad exhausted their energetic stock faster than their egg stock. Water warming will increase the rate of energy expenditure, which might increase the risk that shad die with a large stock of unspent eggs. Although perfectible, the three complementary analyses of acceleration data are promising for in situ monitoring of energy expenditure related to specific behaviour. C_LI

ecology↗