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Quantitative assessment of the ventricular trabeculation by fractal dimension (FD) involves complex processing steps which may impact the results. We optimised the automated processing workflow for a reliable assessment of the left and right ventricles at end-diastole and end-systole which is suitable for the automated analysis of large-scale cohorts. Ventricular trabeculae and blood were segmented using a level-set method optimised to exclude pixels outside the heart on short-axis cardiac MRI. FD was derived by box-counting the trabeculae/blood boundary while investigating the impact of box size, sampling and rotation. Alternative non-fractal measures – the convexity related boundary length ratio (BLR) and the trabeculated mass ratio (TMR) – were also investigated.FD values with and without optimisation showed a strong linear correlation (R2 = 0.81) and narrow agreement limit (1.96·SD = 0.063) only for the end-diastolic left ventricle. Linear correlation and agreement was good between the optimised FD and BLR values for both ventricles and cardiac phases (R2 = 0.70–0.92, 1.96·SD = 0.037–0.064) but not for TMR (R2 = 0–0.37, 1.96·SD = 0.16–1.4). FD, BLR and TMR differed significantly (p < 0.001) between end-diastole and end-systole with lower FD (-0.07 ± 0.06) but higher BLR (0.31 ± 0.25) and TMR (0.26 ± 0.13) values at end-systole.The previously used fractal analysis is suboptimal except for assessing the end-diastole left ventricle. The optimised fractal analysis is suitable for the left and right ventricle at end-diastole and end-systole. The easy to compute non-fractal BLR gives equivalent information like FD. The volume-based TMR, on the other hand, captures different features of the trabeculation.
In most animals, a small number of descending neurons (DNs) connect the brain to circuits and motor neurons (MNs) in the nerve cord. To understand how brain signals generate behavior, it is critical to understand the organization of the neural pathways linking DNs to MNs. In companion papers, we introduced a densely reconstructed connectome of the Drosophila Male Adult Nerve Cord (MANC; Takemura et al., 2024), including cell types and developmental lineages (Marin et al., 2024), which provides complete connectivity of the ventral nerve cord (VNC) at synaptic resolution. Here, we present a first look at the organization of the networks connecting DNs to MNs. We first proofread and curated all DNs and MNs, then systematically matched their morphology to light microscopy data. We report both broad organizational patterns of the entire network and fine-scale analysis of selected circuits of interest. We discover that direct DN-MN connections are infrequent and identify neuron communities putatively linked to control of different motor systems, including walking, flight steering and power generation, and coordinated action of wings and legs. Our analyses generate hypotheses for future functional experiments and empowers others to investigate these and other circuits of the VNC in richer mechanistic detail.
We previously developed REXER (Replicon EXcision Enhanced Recombination); this method enables the replacement of >100 kb of the Escherichia coli genome with synthetic DNA in a single step and allows the rapid identification of non-viable or otherwise problematic sequences with nucleotide resolution. Iterative repetition of REXER (GENESIS, GENomE Stepwise Interchange Synthesis) enables stepwise replacement of longer contiguous sections of genomic DNA with synthetic DNA, and even the replacement of the entire E. coli genome with synthetic DNA. Here we detail protocols for REXER and GENESIS. A standard REXER protocol typically takes 7-10 days to complete. Our description encompasses (i) synthetic DNA design, (ii) assembly of synthetic DNA constructs, (iii) utilization of CRISPR-Cas9 coupled to lambda-red recombination and positive/negative selection to enable the high-fidelity replacement of genomic DNA with synthetic DNA (or insertion of synthetic DNA), (iv) evaluation of the success of the integration and replacement and (v) identification of non-tolerated synthetic DNA sequences with nucleotide resolution. This protocol provides a set of precise genome engineering methods to create custom synthetic E. coli genomes.
The assembly of tau into amyloid filaments is associated with more than 20 neurodegenerative diseases, collectively termed tauopathies. Electron cryo-microscopy (cryo-EM) structures of brain-derived tau filaments revealed that specific structures define different diseases, triggering a quest for the development of experimental model systems that replicate the structures of disease. Here, we describe 12 phosphomimetic serine/threonine-to-aspartate mutations in tau, which we term PAD12, that collectively induce the in vitro assembly of full-length three-repeat tau into filaments with the same structure as paired helical filaments extracted from the brains of individuals with Alzheimer’s disease. Solution-state nuclear magnetic resonance spectroscopy suggests that phosphomimetic mutations in the carboxy-terminal domain of tau may facilitate filament formation by disrupting an intramolecular interaction between two IVYK motifs. PAD12 tau can be used for both nucleation-dependent and multiple rounds of seeded assembly in vitro, as well as for the seeding of tau biosensor cells. PAD12 tau can be assembled into paired helical filaments under various shaking conditions, with the resulting filaments being stable for extended periods of time. They can be labelled with fluorophores and biotin. Tau filaments extracted from the brains of individuals with Alzheimer’s disease have been known to be made of hyperphosphorylated and abnormally phosphorylated full-length tau, but it was not known if the presence of this post-translational modification is more than a mere correlation. Our findings suggest that hyperphosphorylation of tau may be sufficient for the formation of the Alzheimer tau fold. PAD12 tau will be a useful tool for the study of molecular mechanisms of neurodegeneration.
Microelectrode arrays (MEAs) are increasingly used to profile the development of synchronized activity in neural organoids, yet no organoid study has reported on the consistency of electrophysiological development across cell lines. Here, we used dissociated neural organoids derived from six cell lines on MEAs to characterize functional synapse development using multiple parameters across time. The dissociated organoids demonstrated increasing functional connectivity and network activity over time across all cell lines and plasticity in response to synaptic-like stimulation. Like the organoids they were derived from, dissociated organoid cultures contained a diverse mixture of cell types. These results demonstrate that dissociated cerebral organoids can generate functional neurons, akin to primary neuronal cultures from brain tissue, providing a scalable model for studies of neurodevelopment and synaptic function. Consistent with unguided differentiation, we observed variability in activity parameters linked to donor cell line and batch effects, which must be considered in experimental design.
