Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

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Structural variations (SVs) are a major contributor of genetic diversity and phenotypic variations, however their prevalence and functions in domestic animals are largely unexplored. Here, we assembled 26 haplotype-resolved genome assemblies from 13 genetically diverse sheep breeds using PacBio HiFi sequencing. We then constructed an ovine graph pan-genome and demonstrated its advantage in discovering 142,593 biallelic SVs (Insertions and deletions), 7,028 divergent alleles and 13,419 multiallelic variations with high accuracy and sensitivity. To link the SVs to genotypes, we genotyped the SVs in 687 resequenced individuals of domestic and wild sheep using a graph-based approach and identified numerous population-stratified variants, of which expression-associated SVs were detected by integrating RNA-seq data. Taking the varying sheep tail morphology as example, we located a putative causative insertion in HOXB13 gene responsible for the long tail and reported multiple large SVs associated with the fat tail. Beyond generating a benchmark resource for ovine structural variants, our study also highlighted that the population genetics analysis based on graph pan-genome rather than reference genome will greatly benefit the animal genetic research.

Characterization of genetic circuits and biosynthetic pathways in different hosts always requires promoter substitution and redesigning. Here, a strong, broad-spectrum promoter, P, for Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae was constructed, and it was incorporated into the minimal E. coli-B. subtilis-S. cerevisiae shuttle plasmid pEBS (5.8 kb). By applying a random mutation strategy, three broad-spectrum promoters P, P, and P, with different strengths were generated and characterized. These broad-spectrum promoters will expand the synthetic biology toolbox for E. coli, B. subtilis, and S. cerevisiae.

MiR-208a stimulates cardiomyocyte hypertrophy, fibrosis and β-MHC (β-myosin heavy chain) expression, being involved in cardiovascular diseases. Although miR-208a is known to play a role in cardiovascular diseases, its role in cancer and cancer stem cells (CSCs) remains uncertain. We identified an inverse relationship between miR-208a and let-7a in breast cancer specimens, and found that SOX2, β-catenin and LIN28 are highly expressed in patients with advanced breast cancer opposed to lesser grades. Further, we isolated ALDH1+ CSCs from ZR75–1 and MDA-MB-231 (MM-231) breast cancer cell lines to test the role of miR-208a in breast CSCs (BrCSCs). Our studies showed that overexpression of miR-208a in these cells strongly promoted the proportion of ALDH1+ BrCSCs and continuously stimulated the self-renewal ability of BrCSCs. By using siRNAs of SOX2 and/or β-catenin, we found that miR-208a increased LIN28 through stimulation of both SOX2 and β-catenin. The knockdown of either SOX2 or β-catenin only partially attenuated the functions of miR-208a. Let-7a expression was strongly inhibited in miR-208a overexpressed cancer cells, which was achieved by miR-208a induction of LIN28, and the restoration of let-7a significantly inhibited the miR-208a induction of the number of ALDH1+ cells, inhibiting the propagations of BrCSCs. In let-7a overexpressed ZR75–1 and MM-231 cells, DICER1 activity was significantly inhibited with decreased miR-208a. Let-7a failed to decrease miR-208a expression in ZR75–1 and MM-231 cells with DICER1 knockdown. Our research revealed the mechanisms through which miR-208a functioned in breast cancer and BrCSCs, and identified the miR-208a-SOX2/β-catenin-LIN28-let-7a-DICER1 regulatory feedback loop in regulations of stem cells renewal.

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