Species-specific secondary metabolites from Primula veris subsp. veris obtained In Vitro adventitious root cultures: an alternative for sustainable production

Primula veris subsp. veris L. is a perennial herbaceous and medicinal plant species the roots and flowers of which are a source of valuable pharmaceutical raw materials. The plant tissues are used to produce expectorant and diuretic drugs due to their high content of triterpene saponins and phenolic glycosides. Underground roots of P. veris can be obtained only through a destructive process during the plant’s harvesting. In the present study, an in vitro adventitious root production protocol was developed as an alternative way of production, focused on four species-specific secondary metabolites. Root explants were cultured in Murashing & Skoog liquid medium supplemented with 5.4 µM α-naphthaleneacetic acid, 0.5 µM kinetin, L-proline 100 mg/L, and 30 g/L sucrose, in the dark and under agitation. The effect of temperature (10, 15 and 22 ◦C) on biomass production was investigated. The content of two flavonoid compounds (primeverin and primulaverin), and two main triterpene saponins (primulic acid I and II) were determined after 60 days of culture and compared with 1.5-year-old soil-grown plants. The accumulated content (mg/g DW) of bioactive compounds of in vitro adventitious roots cultured under 22 ◦C was significantly higher than the other two temperatures of the study, being 9.71 mg/g DW in primulaverin, 0.09 mg/g DW in primeverin, 6.09 mg/g DW in primulic acid I, and 0.51 mg/g DW in primulic acid II. Compared to the soil-grown roots (10.23 mg/g DW primulaverin, 0.28 mg/g DW primeverin, 17.01 mg/g DW primulic acid I, 0.09 mg/g DW primulic acid II), the in vitro grown roots at 22 ◦C exhibited a 5.67-fold higher content in primulic acid II. However, primulic acid I and primeverin content were approximately three-fold higher in soil-grown roots, while primulaverin content were at similar levels for both in vitro at 22 ◦C and soil-grown roots. From our results, tissue culture of P. veris subsp. veris could serve not only for propagation but also for production of species-specific secondary metabolites such as primulic acid II through adventitious root cultures. This would therefore limit the uncontrolled collection of this plant from its natural environment and provide natural products free from pesticides in a sustainable wa

Protein hydrolysates supplement in the nutrient solution of soilless grown fresh peppermint and spearmint as a tool for improving product quality

7openInternationalInternational coauthor/editorThe present study investigated the potential of fresh peppermint (Mentha × piperita L.) and spearmint (Mentha spicata L.) production on a floating raft system combined with a commercial protein hydrolysate supplement (Amino16®) in a nutrient solution aiming to improve plant product quality. Three levels of the protein hydrolysate solution (0, 0.25 and 0.50%) were added in the nutrient solution, and the plants were harvested after twenty-four days. Plant growth characteristics were recorded, and nutritional, essential oil and polyphenolic composition were determined in fresh tissue. The addition of protein hydrolysates did not affect the fresh or dry weight but reduced plant height. Nitrate content significantly decreased, while total chlorophyll and essential oil content increased in both species. Moreover, the protein hydrolysate solution further increased total antioxidant capacity, total soluble phenol and carotenoid contents in spearmint plants, while it did not affect the essential oil and polyphenolic composition in both species. In conclusion, protein hydrolysates solution may be added in the nutrient solution, to improve the quality of peppermint and spearmint grown in a floating system, without adverse effects on crop yield or the essential oil and polyphenolic profile.openAktsoglou, D.; Kasampalis, D.; Sarrou, E.; Tsouvaltzis, P.; Chatzopoulou, P.; Martens, S.; Siomos, A.Aktsoglou, D.; Kasampalis, D.; Sarrou, E.; Tsouvaltzis, P.; Chatzopoulou, P.; Martens, S.; Siomos, A

Crystal structures of native cytochrome c 6 from Thermosynechococcus elongatus in two different space groups and implications for its oligomerization

Native cytochrome c (6) was purified from an extract of strain BP-1 of the thermophilic cyanobacterium Thermosynechococcus elongatus. The protein was crystallized, and with only slight modifications of the buffer and vapour-diffusion conditions two different space groups were observed, namely H3 and C2. Both crystal structures were solved; they contained three and six molecules per asymmetric unit and were refined to 1.7 and 2.25 Å resolution, respectively. To date, the structure of native cytochrome c (6) from T. elongatus has only been reported as a monomer using NMR spectroscopy, i.e. without addressing putative oligomerization, and related structures have only previously been solved using X-ray crystallography after recombinant gene overexpression in Escherichia coli. The reported space groups of related cyanobacterial cytochrome c (6) structures differ from those reported here. Interestingly, the protein–protein interfaces that were observed utilizing X-ray crystallography could also explain homo-oligomerization in solution; specifically, trimerization is indicated by infra-red dynamic light scattering and blue native gel electrophoresis in solution. Trimers were also detected by mass spectrometry. Furthermore, there is an indication of post-translational methylation in the crystal structure. Additionally, the possibility of modifying the crystal size and the redox activity in the context of photosynthesis is shaping the investigated cytochrome as a highly suitable model protein for advanced serial crystallography at highly brilliant X-ray free-electron laser sources

Foliar calcium effects on quality and primary and secondary metabolites of white-fleshed ‘Lemonato’ peaches

‘Lemonato’ is a Greek peach melting-flesh white-flesh cultivar with high nutritional value highly appreciated by the consumers. This study aimed to evaluate the effect of pre-harvest foliar calcium application on fruit quality, primary metabolite profile, antioxidant activity, total phenolic content, and phenolic profile of the ‘Lemonato’ peach, clone ‘Stamatis’. The experiment was conducted for two years, 2019 and 2020, in two commercial orchards at Kato Lehonia and Agios Vlasios regions, central Greece, where the ‘Lemonato’ clone ‘Stamatis’ is traditionally cultivated. The treatments were organic calcium (Ca), calcium–silicate in nanoparticles (Ca–Si), and calcium chloride (CaCl2). Foliar application of the different Ca formulations, commonly used as a horticultural practice, were not effective at improving the fruit quality characteristics in this clone, which is characterized by fruit softening during ripening. The study revealed the sugars and organic acid composition and phenolic profile of the ‘Lemonato’ peach, clone ‘Stamatis’. Peach fruit quality, primary metabolites, and phenolic compounds of the two orchards showed a different response to organic Ca and Ca–Si, indicating that genetic or environmental factors may also be involved. A higher concentration of organic Ca and CaCl2 increased the peach fruit phenolic compounds content and the total antioxidant activity, improving the fruit nutritional qualit

Chemical and Biological Characterization of the Anticancer Potency of \u3ci\u3eSalvia fruticosa\u3c/i\u3e in a Model of Human Malignant Melanoma

Malignant melanoma is one of the most aggressive types of skin cancer with an increasing incidence worldwide. Thus, the development of innovative therapeutic approaches is of great importance. Salvia fruticosa (SF) is known for its anticancer properties and in this context, we aimed to investigate its potential anti-melanoma activity in an in vitro model of human malignant melanoma. Cytotoxicity was assessed through a colorimetric-based sulforhodamine-B (SRB) assay in primary malignant melanoma (A375), non-malignant melanoma epidermoid carcinoma (A431) and non-tumorigenic melanocyte neighbouring keratinocyte (HaCaT) cells. Among eight (8) different fractions of S. fruticosa extracts (SF1-SF8) tested, SF3 was found to possess significant cytotoxic activity against A375 cells, while A431 and HaCaT cells remained relatively resistant or exerted no cytotoxicity, respectively. In addition, the total phenolic (Folin–Ciocalteu assay) and total flavonoid content of SF extracts was estimated, whereas the antioxidant capacity was measured via the inhibition of tert-butyl hydroperoxide-induced lipid peroxidation and protein oxidation levels. Finally, apoptotic cell death was assessed by utilizing a commercially available kit for the activation of caspases - 3, - 8 and - 9. In conclusion, the anti-melanoma properties of SF3 involve the induction of both extrinsic and intrinsic apoptotic pathway(s), as evidenced by the increased activity levels of caspases - 8, and - 9, respectively

Efficient Genome Editing of Mouse Hematopoietic Cells Using Crispr/Cas9 Technology

Background: A number of techniques have been used to study hematopoietic malignancies including viral overexpression of human oncogenes, RNA interference (RNAi) and transgenic animals. Each of these have their caveats for cell specific targeting, particularly RNAi approaches that are characterised by incomplete genetic inactivation without changing the genetic code. Genome editing technologies, including clustered regularly-interspaced short palindromic repeats (CRISPR), have been used to stably modulate the genome of several model organisms. The genome engineering of hematopoietic cells is complicated mainly due to the low efficiency of the non-viral delivery methods. Aims: To generate efficient and stable genome edited murine hematopoietic models using CRISPR/Cas9 technology. Methods: Two different guideRNAs (gRNAs) targeting either Mcm6 (gRNA5 and 8) or Sirt1 (gRNA3 and 9) were designed and cloned into the pLCRISPR.EFS.GFP lentiviral vector. These genes were chosen as they are involved in hematopoietic progenitor and stem cell (HSPC) properties. GFP+ 32D Cl3 mouse hematopoietic cells were FACS sorted at day 5 post-transduction. Knockout efficiency was confirmed by both western blotting and qRT-PCR. Functional analysis was performed by the SURVEYOR assay and Sanger sequencing. Murine HSPCs were isolated from 5FU treated C57BL/6 mice (CD45.2+), transduced with the most efficient gRNA for either Sirt1 or Mcm6 and injected into B6.SJL mice. Engraftment was measured by FACS analysis of blood sampling at 4 weeks post-transplant. Results: CRISPR/Cas9 efficiency was first tested on the mouse 32D cell line in vitro. Decreased protein and mRNA levels of Sirt1 were detected in 32D GFP+ bulk population transduced with gRNA9 (32D+gRNA9) at day 5 which was not evident with gRNA3. SURVEYOR assay revealed that Cas9-mediated cleavage efficiency was higher in 32D+gRNA9 cells (16.3% indels) compared to 32D+gRNA3 cells (12.5% indels). 32D+gRNA9 cells were further expanded and FACS sorted for single clones. 5/20 clones showed complete absence of Sirt1 at the protein level (25% of Cas9 efficiency). Efficient clones were further used for genotype analysis by Sanger sequencing to confirm the type of indels. On the other hand, we saw complete absence of Mcm6 protein expression in 32D GFP+ bulk cells transduced with gRNA5 or gRNA8, and >2-4-fold mRNA reduction was observed when transduced with gRNA5 and 8, respectively. Our CRISPR/Cas9 system was further tested in vivo using primary cells. Murine CD45.2+ HSPCs were isolated, transduced with equal viral titre of empty vector, Mcm6-gRNA8 or Sirt1-gRNA9 and transplanted into CD45.1+ mice. Blood sampling revealed high levels of engraftment as determined by CD45.2% and CRISPR transduction efficiency determined by the GFP levels at 4 weeks posttransplant. Our results show that CD45.2+GFP+ cells engrafted at 4 weeks showing effective CRISPR/Cas9 mediated transduction and transplantation of Mcm6 and Sirt1 knockout primary HSPCs. Summary/Conclusions: In this study we successfully generated in vitro and in vivo CRISPR/Cas9 models for efficient genome editing on hematopoietic cells. Our findings demonstrate a complete gene knockout in vitro but with variable Cas9-mediated cleavage which was further confirmed by our clonal analysis. This suggests that Cas9 function might depend on both the targeted gene and/or the gRNA. Also, transduction of HSPCs was sufficient to ensure engraftment of GFP+ cells. Long-term serial analysis will be further conducted to ensure stable expression and test Cas9-mediated cleavage in viv

Metabolite profiling and antioxidative activity of Sage (Salvia fruticosa Mill.) under the influence of genotype and harvesting period

Two cultivated accessions of Salvia fruticosa Mill. were investigated and evaluated for their essential oil, phenolic composition and antioxidant activity, during different harvesting time. The essential oil and its major compound 1.8 cineole, presented their higher yields during the early summer harvesting. The advanced analytical LC–MS/MS method applied in this work led to the identification of thirty five compounds with rosmarinic acid, the diterpene artefact carnosol and several flavones and flavonols as the main phenolic constituents, the concentration of which varied largely from spring to autumn. The antioxidant activity of respective methanolic extracts was determined using the Ferric Reducing Ability of Plasma (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assays, as a quality control tool. High positive correlations were observed between FRAP and ABTS antioxidant activities and total phenolic/flavonoid content, and particular phenolic constituent

Identification and characterization of genetic structures coding for carbapenemases in enterobacteria from Central Greece

The dissemination of carbapenemases among different species of Enterobacteriaceae was investigated in the University Hospital of Larissa, Central Greece. The presence of the isoform (Tn4401a) of the transponson carrying bla(KPC-2) and 5 divergent bla(VIM)-carrying class I integrons, including a novel structure, suggests interspecies transfer of these mobile elements and underscores their ongoing evolution. (C) 2015 Elsevier Inc. All rights reserved