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

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

A two-sample model for the comparison of radiation doses

The t-test is one of the most well known parametric statistical procedures that can be applied to the problem of two-sample comparison. However, it relies on several assumptions that might not be satisfied in practice and therefore alternative methods are called for. The contribution of this article is to present a relatively new technique for the comparison of two samples in the context of semiparametric statistical inference and apply the new method to the comparison of external radiation doses in the region of Cyprus. Accordingly, without specifying the parametric form of the distribution of the two samples, it is assumed that their log likelihood ratio is linear in some parameters. This in turn leads to empirical likelihood estimation and comparison. Real data analysis shows that the external dose rate does not vary upon the type of rock formation—a fact which does not hold for the terrestrial radiation

Genetic diversity and metabolic profile of Salvia officinalis populations: implications for advanced breeding strategies

As a result of this work, we were able to characterize seven indigenous to GreeceSalvia officinalispopulations using genetic and metabolomic tools. These tools can be used to select the most promising genotypes, capable to design future breeding programs for high valuable varieties. An initial investigation was carried out to compare the genetic and metabolic diversity in S. officinalis grown in Greece and to discern the relationship between the two sets of data. Analysis of inter-simple sequence repeats (ISSR) revealed significant genetic differences among seven sage populations, which were grouped into three main clusters according to an UPGMA ISSR data-based dendrogram and Principle Coordinate Analysis. 80 loci were scored of which up to 90% were polymorphic at species level. According to the composition of their essential oil, the populations were classified into two chemotypes: 1.8 cineole/α-thujone and α-thujone/1.8 cineole. Additionally, a targeted ultra performance liquid chromatography (UPLC–MS/MS) method was used to qualify and quantify phenolic compounds in methanolic extracts of the seven sage genotypes according to which they were districted in six clusters among the sage populations. The main compounds characterizing the seven genotypes were rosmarinic acid and carnosol, followed by apigenin-7-O-glucoside (Ap7glc), and luteolin-7-O-glucoside (Lu7glc). The correlation between matrices obtained from ISSR data and metabolic profiles was non-significant. However, based on the differences in metabolic fingerprint, we aimed to define populations using as main selection criteria the high polyphenol content and desired essential oil composition, using state to the art analytical tools for the identification of parent lines for breeding program

Metabolic mechanisms underpinning vegetative bud dormancy release and shoot development in sweet cherry

Few studies have focused on the metabolic characterization of bud dormancy and shoot growth in temperate fruit species, although this is an intresting framework to anticipate adaptation in global climate changes. To examine this issue, two experimental approaches were applied, using sweet cherry (Prunus avium L. cv ‘Grace Star’) bud and shoot tissues. Initially, annual shoots containing vegetative buds that collected at endodormancy and ecodormancy stages were used to compare changes in shoots- and buds-specific metabolic profiles under chamber-controlled conditions. Detailed analysis suggested that primary metabolites, such as arabitol, fucitol and tryptophan were modified in buds from endo- to eco-dormancy. Differences between buds and shoots metabolic fingerprints were also found in various secondary metabolites, including quercetin, glucosides and osmotic-associated metabolites. In order to investigate the mechanism underlying shoot developmental during bud dormancy break, metabolic analysis was also conducted in annual shoots, that were sampled at five distinct bud-related vegetative stages from ecodormancy to fully developed leaf stage under natural orchard conditions. Several amino acids (ornithine, alanine, isoleucine, GABA, asparagine and tryptophan) and classes of secondary metabolites, including anthocyanidins (peonidin-3-O-galactoside), flavonoids (apigenin, isorhamnetin, chrysin and trilobatin) and lignin-related compounds (sinapyl and coniferyl alcohols) were altered across developmental stages. Additionally, nutrient homeostasis was altered during shoot development, as N, P, Ca, Mg, B steady-state level as well as Ca/Mg + K and N/P stoichiometry were significantly changed. This study provides a bud- and shoot-based metabolic framework at different conditions and dormancy stages, thereby helps to understand dormancy release and bud-break in temperate fruit trees. © 2018 Elsevier B.V

High throughput pre-breeding evaluation of Greek oregano (Origanum vulgare L. subsp. hirtum) reveals multi-purpose genotypes for different industrial uses

Origanum vulgare L. subsp. hirtum (commonly known as Greek oregano) is a perennial species of the Lamiaceae family that counts numerous applications in the food and pharmaceutical industry. The exploitation of Greek oregano germplasm lies in its unique diversity in secondary metabolites, which are accumulated in the aerial parts of the plant. For the industrial exploitation of this crop the starting germplasm is of high importance in terms of valorizing well characterized chemotypes with desired and stable agronomic and phytochemical traits. For this reason, a pre-breeding evaluation of twenty-one accessions collected and preserved under the same environmental conditions was performed in order to develop a descriptive database containing: a) morphometric traits, b) the genetic structure, c) the extensive metabolic fingerprint in terms of volatile composition, polyphenolic and triterpenoid content, and d) the ‘bio-activity’ on the embryonic development of Ascaridia galli eggs for highly divergent accessions. This study revealed the existence of essential oil rich chemotypes superior in morphometric characteristics, containing high carvacrol content (> 75%) and traces of thymol (< 5%), according to the requested international standards. Apart from genotypes considered as ‘high yield essential oil-producers’ (> 6%), accessions of highly productivity of ‘green’ raw materials rich in polyphenols and triterpenoids were also identified. According to their rosmarinic acid content, Greek oregano accessions were classified in three groups; 1) those with significantly high concentration (1640 - 2726 mg 100 g-1 dry weight), 2) those expressing intermediate content (1103 - 1200 mg 100 g-1 dry weight), and 3) the ones with significantly low content (< 873 mg 100 g-1 dry weight). A total of 118 polymorphic alleles were identified using sequence-related amplified polymorphism (SRAPs) and simple sequence repeat (SSRs) markers, while 2 genetic clusters based on a single data matrix with SRAPs and SSRs alleles, were identified. The presence of private bands in some accessions was also observed, which can be used in future breeding programs for germplasm authentication purposes. As a result this study provides a comprehensive knowledge on metabolic and genetic diversity of Greek oregano accessions native to Greece, as well as powerful tools for the selection of starting genetic materials for subsequent breeding studies to create superior cultivars. The multifaceted approach was developed to exploit the genetic resources of Greek oregano in further breeding programs for subsequent selection of genotypes with desired trait

Fruit quality trait discovery and metabolic profiling in sweet cherry genebank collection in Greece

The current study characterizes the physicochemical, sensory and bioactive compound traits of twenty-two sweet cherry accessions, namely breeding lines, landraces and modern cultivars, embodying the majority of Greek germplasm. The evaluated accessions differ in several quality traits including colour parameters and textural properties as well as sensory attributes, such as taste intensity and overall acceptance. Significant differences in primary metabolites, including fructose, glucose, sorbitol, malic acid were recorded among tested accessions. All genotypes were rich in polyphenols, primarily in quercetin-3,4-O-diglucoside, esculetin, rutin and neochlorogenic acid. An anthocyanins-related discrimination among accessions was also obtained based on cyanidin-3-O-rutinoside and peonidin glycosides content. Overall, the cultivars ‘Tsolakeika’ and ‘Bakirtzeika’ exhibited the higher consumer acceptance while the cultivars ‘Vasiliadi’ and ‘Tragana Edessis-Naousis’ and especially the breeding line ‘TxAg33’ contained high polyphenol levels. These results represent a valuable resource for future breeding efforts for sweet cherry cultivars with improved nutritional quality trait

Interspecies spread of Staphylococcus aureus clones among companion animals and human close contacts in a veterinary teaching hospital. A cross-sectional study in Greece

Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) prevalence among companion animals and veterinary personnel (VP) was investigated. Strains' molecular characteristics were evaluated in order to assess S. aureus transmission.Specimens (224) from colonized and infected sites of 102 animals (92 dogs, 10 cats) and 18 VP were collected during 2012 and 2013. Antibiotic susceptibility was performed by the disk diffusion method and Etest. mecA, mecC, tst (toxic shock syndrome toxin) and lukF/lukS-PV (Panton-Valentine leukocidin, PVL) genes were investigated by PCR. Genotypes were identified by Multi Locus Sequence Typing (MLST), Staphylococcal Cassette Chromosome mec (SCCmec), accessory gene regulator group (agr), spa and Pulsed Field Gel Electrophoresis (PFGE). S. aureus prevalence among pets and VP was 36.3% (37/102) and 38.9% (7/18), respectively. Younger companion animals, those living in rural areas, having a disease upon admission or Coagulase-negative staphylococci co-carriage showed significantly higher prevalence of S. aureus isolation (p < 0.05). Twenty-six pets and five VP carried PVL-positive S. aureus. In total, 60 S. aureus strains were recovered (53 from pets, seven from VP) of which 16 were MRSA (26.7%), 12 mecA- and four mecC-positive. MRSA showed higher resistance rates against other antimicrobials as compared to methicillin-susceptible ones. Strains were classified by MLST in 13 STs, with the predominance of ST80 and ST15. In MRSA, SCCmec types II, IV and XI were identified. The most frequent spa types were t5559 and t7558. Fifty-six strains were classified into 15 PFGE types.Comparison of genetic markers shows that identical or very similar strains disseminate among animals and VP. Companion animals harbor PVL-positive clones constituting a possible source for transmission to humans. © 2016 Elsevier B.V