Paragon

‘Mercia’ and ‘Mercia(Chinese Spring 2B)’ lines were from the John Innes Centre Germplasm Resources Unit. ‘Paragon(Récital Ppd-B1a)’ lines were developed as follows. ‘Récital’ carries Ppd-B1a and Ppd-D1a alleles [12] (link). Sixty-four ‘Paragon’בRécital’ BC₁F₂ plants were assayed for Ppd-D1a as described in [5] (link) and Ppd-D1a carriers discarded. The remainder were grown in short days (10 h natural light). Early flowering plants would have Ppd-B1a and these were backcrossed to ‘Paragon’. BC₃F₂ families in short days showed 3∶1 segregation for flowering time and early flowering plants had higher Ppd-B1 copy number, showing that Ppd-B1a was the major determinant of flowering time. BC₃F₃ families that were all early flowering in short days (10 h natural light) were selected. BC₃F₄ plants were compared to ‘Paragon’ and previously described ‘Paragon’ BC₄F₄ introgression lines homozygous for the ‘GS-100’ Ppd-A1a, ‘Chinese Spring’ Ppd-B1a, ‘Sonora64’ Ppd-B1a or ‘Sonora64’ Ppd-D1a alleles [27] . Plants were grown under summer conditions using a glasshouse with moving benches that travelled into a closed (dark) room after 10 h in the light.

Viral RNA was extracted directly from viral transport media using QIAamp Viral RNA Mini kit (Germany, Qiagen), according to the manufacturer's instructions. A total of 100 ng of extracted RNA was reverse transcribed with N8 random hexamers. Second-strand synthesis, PCR amplification, and full virus genome sequencing were done using CleanPlex SARS-CoV-2 Research and Surveillance Panels provided by Paragon Genomics (Shenzhen, China). Indexed libraries were purified with Agencourt AMPure XP beads (Beckman Coulter, USA), quantified using Qubit 4.0 Fluorometer (Invitrogen, USA), and pooled to a final concentration of 8 pM. Pooled libraries were then loaded in a 300-cycle sequencing cartridge. Sequencing was performed on an Illumina MiSeq platform, which generated about six GB of data. The mean count of paired sequencing reads per sample was 1.5 × 10⁶ (minimum 0.5 × 10⁶ to maximum 3.5 × 10⁶).
The generated sequencing reads were first trimmed to remove adaptors using the cutadapt software (https://cutadapt.readthedocs.io/en/stable/). Additional filtration steps also included the removal of short reads (<40 nucleotides) and low-quality reads (<30 Phred score). High-quality sequencing reads were then aligned to the reference genome (accession number: MN908947) using Burrows-Wheeler Alignment (BWA) (Li and Durbin, 2009 (link)). Consensus sequences were constructed after trimming the PCR primer sequences using fgbio software package (https://github.com/fulcrumgenomics/fgbio). We were able to generate near-full genomes (from nucleotide 10 to nucleotide 29,700) from all samples with an average coverage of 98%. For variant calling, gatk tools were used for variant detection at both consensus and sub-consensus levels (https://gatk.broadinstitute.org). Only variants with Phred scores of more than 35 and depths of more than 100× were called. For low-frequency variant calling (frequency >10%), reads were realigned to consensus sequences reconstructed from each sample. A phylogenetic tree was generated employing the general-time reversible (GTR+G) nucleotide substitution model with 1,000 bootstrap replicates and was displayed using FigTree (version 1.4.4) (Yang, 1994 (link)). Lineages were assigned to sequences based on the lineage nomenclature system proposed by Rambaut et al. (2020 (link)). Statistical analysis was performed using Prism7 software.

From a technical perspective, present endoscopes are bending beams with a rather stiff “body,” and a “neck” with underactuated rotatory DoFs (degrees of freedom) around two orthogonal bending axes each. This principle biologically roughly mirrors a motion segment [23 (link)] of a vertebral column (without the rotatory DoF around the longitudinal axis, which leads to torsion of the spine), or a mechanically coupled (not independently movable) series of motion segments. The desirable extreme to optimize the versatility and adaptability of the endoscope to changing moving paths is a continuum, which is actuated locally leading to a DoF of theoretically up to infinity. The more obvious biological paragon, which realizes this principle nearly perfectly, is obvious: the elephant’s trunk. Structures like that are mesoscale or microscale, and at present are not realizable. Biomimetics in its technical part for present customer demands must find technologically realizable solutions, and biomimetics is no one-way system from biology to engineering. One must first present a realizable step in the wanted direction for endo devices, possibly the (phylogenetically back-) step from the structure of the vertebral column (which very roughly transferred underlies the present constructions) to that of its historical precursor Chorda dorsalis as a biomimetic paragon, as illustrated in Figure 2.
Figure 2, left, represents the working principle of conventional endoscopes. Bending is realized by a series of rigid bodies (forming “segments”), rotating around defined joint axes. The motion of all segments is coupled by the application of the same force via a Bowden drive. The principle of bending a vertebrate’s spine is represented in the center. This is comparable to the principle used in conventional endoscopes, with the exception that rotation in all segments may be different since forces are provided by intersegmental muscles. In the real animal, the rotation axis is slightly moving during motion, and additional longer muscles allow controlled coupled bending as well.
On the right, the principle of the Chorda dorsalis system is illustrated. The support structure is a compliant beam (in the natural paragon with changing geometry over length), which due to the action of small muscles can be bent locally with different curvatures along the length. For reasons of clarity, motion is shown around one axis in one direction; in technology, as in nature, actuation in up to three rotational DoFs in an antagonistic manner may provide six overlaid rotational directions. While the spine may be seen as a series of rigid segments, connected by “real” joints (diarthroses), actively moved by muscles bridging the gaps, Chorda dorsalis form a continuous bending beam, to which local bending is imprinted by local muscle fibers. These allow the controlled application of bending moments and axial (compressive) forces.
Transverse forces in a bending beam are coupled in a fixed manner to the bending moment around the transversal axis, thus they may also be controlled in a coupled manner as one DoF. The chorda is by no means a faulty construction; it has fulfilled its tasks for half a billion years and still fulfills them in recent animals. Beneath other factors, growing body masses and thus gravitational and inertial forces in combination with the utilization of torsional movements led to the evolution of the spinal column, which we as humans anthropocentrically consider to be the better solution. However, both the chorda and the spine are adapted to their respective tasks, and the chorda, with its lower load-bearing capacity but higher local bending, due to flexibility in combination with a monolithic structure when torsion is not required, corresponds better in its functional description to the requirements of an endoscope than a spine, so it is the more suitable biological paragon.
Derived from that (simplified) model of a chorda, we realized a demonstrator in mesoscale with a very limited number of synergistic “muscle pairs” on a continuous flexible beam (due to the lack of a yet not industrialized precise process presently feasible), which was formed by segments (technically in modular design), which are rigidly coupled to resemble a monolithic structure.

Outcome data for dapagliflozin and sacubitril–valsartan were extracted from the DELIVER trial and pooled analysis of the PARAGLIDE-HF and PARAGON-HF trials, respectively (Solomon et al., 2022 (link); Vaduganathan et al., 2023 (link)).