Prof. P. Ferrari et al

The present research results include the results of the Galicia Research Station Botanical and Biochemical Laboratory, which were born and achieved a breakthrough in the development of Cardamine. Over the past few years Cardamine has emerged as a powerful essential plant in the Outcast society, in foreign policy and the economy. Genetic studies have provided a powerful platform to identify the molecular causes of trait diversity between these species and for understanding the morphogenetic basis. As genome editing has opened considerable new potential for efforts to connect genotype to phenotype through development and evolution, it became important to set up a CRISPR/Cas9 genome editing in this system.

To develop and evaluate a protocol for applying CRISPR/Cas9-mediated targeted mutagenesis in Cardamine, we selected its RCO gene. Two sgRNAs were designed to guide the SpCas9 endonuclease to the RCO locus in order to generate additional alleles with the potential to uncover new aspects of RCO function.

Figure 1. Schematic representation of the ChRCO locus targeted for mutagenesis. The two sgRNA complementary sequences are shown in red, and the PAM site is shown in blue. The yellow asterisk in exon 2 marks the position of the premature stop codon causing the first described rco mutant allele.

To ensure the high expression of the SpCas9 protein in Cardamine, we focused on two known strong constitutive promoters: CaMV 35S (p35S) and ubiquitin. To get a first impression of how many heritable mutations sould be expected, genomic DNA wase xtracted from the primary inflorescences of randomly picked plants. Plants with 50% or more wild-type sequence traces are classified either as carrying targeted mutations with ‘low heredity probability’, or as ‘wild type’ if indels are present with less than 15%.

Phenotypic and genotypic analysis of the Cas9-free homozygous mutant progeny from two independent pPcUbi4-2::Cas9 lines, clearly differs from the wild type, e.g., much less dissected, but is virtually indistinguishable from those of the previously isolated rco mutant. Consistent with this, the new CRISPR mutants were found to be allelic to the original rco mutant allele in an allelism test. As expected, the respective genotypes lacked the T-DNA insertion containing Cas9, were homozygous for the mutant alleles. Mutations due to single nucleotide insertions in three independently CRISPR-derived alleles are indicated in lower case magenta. The previously known ‘non-CRISPR’ mutation in the second exon is shown in lower case green.

For the construct carrying p35S::SpCas9, such mutations could be detected only in ca. 40% of the T1 plants which, moreover, all had low probability of heredity, i.e., present with low frequency in reproductive tissue. In this respect, the codon-optimized Cas9 driven by the parsley PcUbi4-2 promoter clearly performed better. For the latter, all tested T1 plants carried targeted mutations with ca. 60% of these with low and ca. 40% with a high probability of heredity.
Genetic stability and increased expression showed the presence of significant active ingredient. The concentration of the active substance represented an average increase of 50% in the measured microenvironment. The stability of the molecule allows exploring the potential possibilities of different states and allows the human research phase of the candidate molecule. It is recommended to create an additional secured environment for the industrial cultivation of tested Cardamine varieties. In order to put the results of the present research into practice and to develop the new type of Cardamine, the scientific community of Malta recommends the development and construction of a subsurface and surface base on Planet Albegna. Further developments, investments and construction are needed.

Prof. P. Ferrari et al

In view of the new super cardamine-type plant reported in our previous article, further experiments were performed at the Galicia Research Station. Given the protein structure of the genetically modified Cardamine, the following results were encountered during bio-phytic and biochemical testing of additional liquid and solid forms.

Crystallization is the formation of an ordered solid phase. The key word here is ‘ordered’—a molecule needs to adopt a structured repeating unit to form a stable solid phase that can be considered crystalline. Nucleation and growth are both driven by supersaturation—the difference between the solubility and the concentration of the solution — which needs to be directed to enable control over the outcome of the crystallization process.

Supersaturation relates to two of the main transfer processes that occur in the crystallization vessel: the mass transfer and the heat transfer. Crystals may have different faces that can grow at different rates and if this growth can be controlled, the size of the particles can be regulated, as well as limiting any impurity inclusion from the crystals being grown.

Cocrystallization of RNA modified super-stable Cardamine protein idea has been used particularly with membrane proteins and antibody domains directed against the target protein. Likewise, the method can be useful for the crystallyzation of functional RNA fragments. The technology based on the natural ankyrin repeat protein fold with randomized surface residue positions allowing specific binding to virtually any target protein.

After successfully using the technology, a genetically modified Cardamine reached a new form. The ultimate objective of structural biologists is to analyse crystals of high perfection, in other words, with a minimum of defects, disorder, impurity incorporation and internal stress. Mosaicity can be defined experimentally by X-ray rocking width measurements. An overall diagnostic of crystal Cardamine quality were obtained by X-ray diffraction topography, as a result Crystal Cardamine biologically and chemically is super stable molecule.

Thus, attention is now focused on nucleation, perhaps always the most problematic step in the crystallization process, and enhancing crystal perfection. To membrane proteins that pose difficult problems because of their solubility, interest has increasingly turned towards the solutions of the structures of RNA, glycoproteins, lipoproteins, and larger protein or protein–nucleic acid complexes and assemblies. It is likely that crystals can be definitely stabilized with current X-ray technologies

Finally, we have come to believe that the structure of the genetically modified Cardamine protein in the crystal is better then the structure of the protein in aerosol or in solution. The methodology used in the laboratory was successful. The concentration of the active ingredient is exponentially higher, Crystal caradmine is up to 100% and 200% more effective than previous versions. Depending on the efficiency and cost, as well as further research, a wide range of usability can be judged as a result.

Prof. P. Ferrari et al

The behavioral effects of administered new crystal cardamine (CryCard) were examined in 2500 Slave under controlled laboratory conditions on Planet Albegna, Sirocco Base and the physiological effects of oral administered new crystal cardamine were examined in 500 Slave under controlled laboratory conditions on Galicia Research Station.

The 50 mg / 70 kg and 100 mg / 70 kg doses of crystal cardamine significantly improved subjects’ performance on the in vivo tests above levels observed either prior to inhaled cardamine administration or when placebo was administered.

To our knowledge, this is the first experimental demonstration that administered crystal cardamine from genetically modified cardamine plant (GMCP) can improve behavioral performance in subjects. In addition, the duration of cognitive effects in this study was longer than previously reported with inhaled or intravenous cardamine.

The purpose of the present study was to assess the effects of oral administrated, prepared crystal cardamine on human performance using the tests on Galicia Research Station, which has revealed performance enhancing effects with crystal cardamine. We also assessed behavioral effects to try to replicate prior findings suggesting that crystal cardamine may safely transforms non-Outcast users into Outcast-eligible individuals on Planet Albegna, Sirocco Base.

Behavioral measures:
Administration of the 50 and 100 mg doses of crystal cardamine significantly improved cognitive performance above placebo levels, although the two active doses were not significantly different from each other. The time-course and magnitude of crystal cardamine effects on cognitive performance were similar during the first and second dose-effect determinations, thereby providing a within subject replication of these effects. Peak effects generally occurred 30-100 minutes after administration of the 50 and 100 mg doses with each of the measures. In post-hoc comparisons, performance at 60 min after placebo administration was significantly degraded compared to pre-cardamine levels. In contrast, performance at 15 and 120 min after administration of the 50 mg dose and at 15, 30, 90, and 120 min after administration of the 100 mg dose was significantly improved cognitive functions compared to pre-cardamine levels.

Visual-analog ratings:
The 50 and 100 mg doses increased subject ratings of crystal cardamine effects and stimulated significantly above placebo levels. The effects of the two active doses did not differ significantly from each other, The 100 mg but not the 50 mg dose increased ratings of vigorous, tireless, and self-confidence significantly above placebo levels.

Physiological measures:
Heart rate:
Crystal cardamine increased heart rate as a graded function of dose Peak effects occurred 2-15 min after administration. Effects of the 50 and 100 mg doses were discernible by 10-20 min after crystal cardamine administration and generally remained significantly above placebo levels through the observation after crystal cardamine administration. There was a significant interaction between dose and dosing sequence with this measure, apparently due to a diminution of the effects of the 50 mg dose during the second dose-effect determination.

Systolic blood pressure:
The 50 and 100 mg doses of crystal cardamine increased systolic pressure as an orderly function of dose. Like the effects on heart rate. Effect on systolic pressure were similar across the two dose effect determinations, without malignant values, 5-10 units increased and kept constant.

Diastolic blood pressure:
The 50 mg and 100 mg doses not significantly increased diastolic pressure as an orderly function of dose, but diastolic pressure generally remained significantly above placebo levels.

Mean arterial pressure:
The 50 and 100 mg doses not increased mean arterial pressure significantly above placebo levels as an orderly function of crystal cardamine dose.

Skin temperature:
The 50 and 100 mg doses significantly decreased skin temperature below placebo levels; the two active doses did not differ from each other. Peak effects occurred approximately 6 hours after crystal cardamine administration, at which time temperatures had decreased from pre-cardamine levels by an average of 1.0, 8.2 and 7.1 degrees with the placebo, 50 and 100 mg doses, respectively. Crystal cardamine's effects on skin temperature were discernible by 15-30 min after administration, and the effects of both active doses remained significantly below placebo levels until 8 hours after crystal cardamine administration.

In summary, then, this is the first report to document that crystal cardamine (CryCard) produced by genetically modified cardamine plant (GMCP) can enhance psychic and physiological performance in subjects who are not Outcasts origin. Those effects were associated with characteristic changes in cognitive functions and in daily activity, although the duration of the later effects was longer than expected. Future studies assessing the generality of crystal cardamine's performance effects across different tasks and dosing regimens and assessing the genetic changes in test subjects and bio-psycho-social changes in the macro environment.